STUDIES ON VASCULAR INTRACELLULAR

pH IN HYPERTENSION

ASHLEY S IZZARD

A thesis submitted for the degree of

Doctor of Philosophy

to the University of Leicester

October 1990

UMI Number: U032337

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STUDIES ON VASCULAR INTRACELLULAR pH IN HYPERTENSION

Abstract

Changes in intracellular pH (pH^) in vascular smooth muscle cells

influence growth and contraction. It has been proposed that both

processes are critically dependent on cell alkalinisation due to increased

Na*/H* exchange; therefore pH^ may be an important mediator of the

vascular hypertrophy resulting in an increased peripheral resistance which

characterises established hypertension. Resistance artery morphology

followed by simultaneous recording of isometric contraction and pH were

measured using a myograph in conjunction with a pH sensitive fluorescent

probe and microscope capable of exciting the intracellular trapped dye at

appropriate wavebands and recording the fluorescence. Mesenteric

resistance arteries from the genetically hypertension prone spontaneously

hypertensive rat were more alkaline than the normotensive Wistar Kyoto

control. However secondary hypertension due to aortic coarctation did not

result in mesenteric artery alkalinisation, although pH^ regulation was

altered. In contrast pHj and its regulation were unchanged in

subcutaneous resistance arteries from the gluteal region from untreated

hypertensive patients and first degree offspring of hypertensive patients

compared to matched controls. Mesenteric arteries displayed anticipated

pH£ changes to manoeuvres which directly cause alkalinisation and

acidification. On the other hand contraction induced intracellular

acidification rather than alkalinisation as observed in cells grown in

tissue culture conditions. These measurements of pH^ carried out for the

first time in human resistance arteries highlight the limitations of using

animal models or surrogate cells when studying this potentially crippling

vascular disorder.

CONTENTS

CHAPTER ONE

INTRODUCTION : Intracellular pH - Its Measurements 1and Regulation

Historical Background 1

Invertebrate pHi Regulation 5

Vertebrate pHi Regulation 9

Na//H* Exchange 10

Bicarbonate dependent pHi regulation 13

Na^ dependent HCO^ /Cl exchange 14

Cl /HCO^ (Na* independent) exchange 14

Na*-HCOj cotransport 16

Intracellular Buffering 17

pHi and Cell Growth 20

pHi and Vascular Tone 25

Resistance Vessel pHi and Hypertension 28

Na*/H^ exchange and pHi in circulating Blood Cells 31

Aims of the Study 33

CHAPTER TWO

MATERIALS AND METHODS 34

The Myograph 34

Morphology Measurements 35

Normalisation Procedure 35

Expressions 36

Indirect Systolic Blood Pressure Measurements 37

Dissections 37

Solutions 38

Animals 38

Standard Start Procedure 39

Intracellular pH Measurement 39

Background Cell Autofluorescence 41

Dye Loading 41

Calibration 42

Fluorescence Changes 43

Intracellular pH and pHi Changes 44

Chemicals Used 44

Analysis of Data 44

CHAPTER THREE

RESISTANCE VESSEL pHi IN THE SPONTANEOUSLY HYPERTENSIVERAT COMPARED WITH THE NORMOTENSIVE WISTAR KYOTO CONTROL 50

Introduction 50

Methods 50

Results 51

Discussion 55

CHAPTER FOUR

RESISTANCE VESSEL pHi DURING EXPERIMENTAL HYPERTENSION 72

Introduction 72

Methods 73

Results 75

Discussion 76

CHAPTER FIVE

RESISTANCE VESSEL pHi IN HUMAN ESSENTIAL HYPERTENSION 91

Introduction 91

Methods 91

Results 92

Discussion 94

CHAPTER SIX

RESISTANCE VESSEL pHi FROM OFFSPRING OF ESSENTIAL HYPERTENSIVE PATIENTS 102

Introduction 102

Methods 102

Results 103

Discussion 104

CHAPTER SEVEN

GENERAL DISCUSSION 112

CONCLUSION 128

BIBLIOGRAPHY 132

ACKNOWLEDGEMENTS

I would like to thank Dr. AM Heagerty for the excellent supervision and

his primary role in organising and providing the biopsies and relevant

data for the human studies. In addition I would like to thank Dr. SJ

Bund for teaching me the myograph technique and Dr. DM Maclver for

performing the surgical procedures in the coarctation study. Finally

many thanks to Mrs R Aldwinckle for typing this manuscript.

CHAPTER ONE

INTRODUCTION: INTRACELLULAR pH; ITS MEASUREMENTS AND REGULATION

Historical Background

Intracellular pH (pHi) was not measured reliably until 15 years

ago; therefore data concerning the regulation of such a

fundamental parameter are relatively new (Roos and Boron I98I).

Until then whether or not H^ ions were distributed passively

across the plasma membrane was controversial (Robson et al.

1968). If H^ ions were passively distributed across the plasma

membrane then pHi could be predicted from the Donnan theory of

membrane equilibrium as proposed by Conway and co-workers (Boyle

and Conway 1941; Conway and Fearon 1944). According to this

theory, if a boundary between two solutions is impermeable to one

or more ionic species, then for the permeant ions the ratio of

their concentrations in the two solutions are the same for ions

of the same charge and valency. (Strictly speaking the term

activity should replace concentration which implies 'effective

concentration’). Based on the evidence that potassium ions are in

equilibrium across mammalian muscle cell membranes, Conway and

Fearon (1944) proposed that [H]^/[H]^ = [K]/^[K]^ (where o and i

are the extracellular and intracellular ions respectively) and

calculated an intracellular pH of 6.0.

However, the majority of experimental data around this period of

time gave values of pHi substantially higher than that predicted

by the passive distribution of H ions (see review by Caldwell

(1956)). A large amount of the data were based on the

dissociation of CO^ which derives pHi from the Henderson

Hasselbach equation: pHi = pK + log [HCO^]^

[total CO^]^

Total CO^ can be calculated by its release from the tissue under

test by the addition of strong acid. Because cell membranes are

highly permeable to CO^ it is assumed that the intracellular and

extracellular concentrations of dissolved CO^ are the same. Also

it is expected that the intracellular carbonic acid concentration

is negligible and that the dissociation constant for CO^ will not

differ intracellularly, thus [HCO^]^ = [total CC^]^ - [CO^]^.

Using this technique, a pHi of 6.9 was obtained from frog

sartorius muscle (Stella 1929). This technique was applied to

anaesthetised cats and the effects of intravenous and

intraperitoneal injections of acid and alkali were studied as

well as changes in the partial pressure of CO^ (PCO^) in

skeletal muscle. The results suggested that pHi was largely

influenced by the PCO^ and not primarily by extracellular pH;

again a pHi value of 6.9 was obtained (Wallace and Hastings

1942). These findings were also confirmed in vitro (Wallace and

Lowry 1942).

That weak acids and bases have a more profound effect on

intracellular pH than changes in extracellular pH alone was

originally demonstrated by Jacobs (1920,1922). Interested by the

finding that a solution gassed with CO^ and buffered to neutral

or slightly alkaline pH was as toxic to tadpoles as an unbuffered

solution gassed with CO^, Jacobs proposed that the carbonic acid

or CO^, but not bicarbonate, could freely enter cells, and that

3

the dissociation of carbonic acid would result in intracellular

acidity. This effect of CO^ on pHi was demonstrated regardless of

the pH of the solution using the flowers of the plant Symphytum

perequineum which contains a natural indicator which turns from

blue to pink on increasing acidity.

A potentially serious shortcoming in the use of the CO^ method

for the determination of pHi was that the CO^ released on

addition of strong acid to the tissue may not be derived solely

from intracellular bicarbonate and dissolved CO^. Experimental

evidence demonstrated that only a small proportion of the ’acid

labile’ CO^ was precipitated by barium chloride in alkaline

medium whereas addition of potassium bicarbonate to the alkaline

extract were precipitated on addition of barium chloride (Conway

and Fearon 1944). Thus Conway and Fearon (1944) subtracted the

’barium soluble’ fraction of CO^ from the ’acid labile’ fraction

when calculating pHi which gave a value of 6.0 in agreement with

a pHi determined from the Donnan principles of a membrane

permeable to K^, H^, Cl and HCO^ ions.

Attempts to use pH sensitive microelectrodes to measure pHi were

first undertaken by Caldwell (1954). The large size of the

electrodes used (50-100 um) necessitated the use of large (600 um

in length) single muscle fibres from the crab Cancinus maenas.

The pHi value obtained was 6.9; however, these electrodes did

cause considerable cell damage since when removed the muscle

fibres became opaque and contracted. Also during the pHi

measurements the membrane potential declined due to injury.

Nevertheless, the lack of pHi change with a declining membrane

potential argued against the passive distribution of H ions.

Studies on isolated frog muscle showed that they are capable of

contraction when placed in Ringers solution of pH 5.0. The

internal pH would be 3.8 according to a Donnan distribution of H^

ions; doubts were raised concerning the ability to contract at

such a low internal pH (Hill 1956).

The conclusions from these studies were challenged by Conway. He

believed that the degree of cell damage invalidated Caldwell's

findings. With regard to the finding of Hill, Conway argued that

attainment of equilibrium would be more than 15 days and that

after a few hours in a pH 5.0 Ringer solution pHi would be far

from equilibrium (Conway 1957).

More recently, the weak acid 5,-5 dimethyl-2,4-oxazalidinedione

(DM0). was used to determine the pHi of muscle in the

anaesthetised dog (Waddell and Butler 1959). This method is less

controversial when compared with the CO^ techniques. Resting pHi

was 7.04 and decreased with increasing tensions of CO^

irrespective of blood pH. Metabolic acidosis only marginally

reduced pH, and these results were in close agreement with the

findings of Wallace and Hastings (1942). Furthermore, it was

found that tissues possess a substance (probably protein) which

had an inhibitory action on the precipitation of barium carbonate

when the CO^ method was used. The nature of the substance was

unknown at that time but now the effect can be mimicked with 4%

bovine serum albumin (BSA) solution. This implies that

subtraction of the 'barium soluble' fraction, from the 'acid

labile' CO^ of the tissue as performed by Conway, led to

erroneously low values of total tissue CO^ concentration and

therefore a low calculated value of pHi (Butler et al. 1967).

Improvements in the design of pH sensitive microelectrodes,

(Thomas 1978) were used to measure pHi in the snail neurone

(Thomas 1974) and squid axon (Boron and De Weer 1976). These

microelectrodes opened the door for the measurement of pHi and

its regulation since pHi could now be continually monitored. The

technique based on the distribution of weak acid would give

reliable values of pHi (Hinke and Menard 1978) but only one

determination can be made because the assay requires destruction

of the tissue and in intact tissue calculations of the

extracellular space are prone to error.

Invertebrate pHi Regulation

The fact that intracellular pH is too high to be explained by the

passive distributions of H^ ions across the plamsa membrane means

that a 'proton pump' has to be proposed which transports H^ ions

out or OH + HCO^ ions into the cell against the prevailing

electrochemical gradient and therefore at the expense of

metabolic effort (Hill 1956; Caldwell 1956; Thomas 1974; Boron

and De Weer 1976).

The large size of invertebrate cells allows them to be impaled

with pH sensitive and membrane potential sensitive electrodes and

extensive work into the mechanisms of pH regulation was carried

out in the snail neurone and squid axon by Thomas and Boron and

their co-workers (for a review see Thomas 1984; and Boron 1983).

6

The classic method for investigating the mechanisms of the

'proton pump' is to stimulate it by inducing intracellular

acidification either by direct injection of acid (Thomas 1976),

increasing the pCO^ or the addition and subsequent washout of

ammonium chloride (Thomas 1974; Boron and De Weer 1976). The

latter method is commonly used to induce cell alkalinisation and

acidification. It was first shown by Jacobs (1922) that ammonium

chloride caused cytoplasmic alkalinisation using Rhododendron

flowers which contain a pigment which changes from red to blue

between pH 7-8. This alkalinisation is due to the high

permeability of cell membrane to the ammonia, which on entering

the cells of many of this species, will associate with a H^ ion

to form NH^* thus causing cell alkalinisation. Prolonged exposure

results in a slow fall after the initial pHi rise and washout of

ammonium chloride causes pHi to undershoot its original level

resulting in cell acidification. The reason for this is that cell

membranes have a finite permeability to NH^* which enters the

cell down its electrochemical gradient and a fraction of this

incoming species dissociates and consequently intracellular pH

falls. The excess ammonia formed will diffuse out of the cell

maintaining equilibrium: therefore the intracellular [NH^^] will

rise to levels much higher than if the cell were only permeable

to ammonia. On washout of external ammonium chloride almost all

the NH^* will shed a proton and leave as NH^ which is vastly more

membrane permeant and the result is an intracellular

acidification below its original pH value (Boron and De Weer

1976; Thomas 1984). During prolonged exposure to ammonia pHi may

also decline due to the action of plasma membrane alkali

extruding systems, (discussed below). If the cell membrane was

7

totally impermeable to and ions, exposure to ammonium

chloride would result in a steady state alkalinisation followed

by return to baseline on washout (De Weer 1978). Inducing cell

acidification by washout of ammonium chloride is frequently

termed the ammonium chloride pre-pulse method.

In invertebrate preparations recovery from an acid load is

dependent on a CO^ /HCO^ buffer, therefore recovery is largely

inhibited in bicarbonate free Hepes buffer gassed with oxygen

(Boron and De Weer 1976b; Thomas 1976). In addition, after acid

load intracellular pH recovery is accompanied by extracellular

acidification. Also, direct injection of HCl is repeatedly

followed by complete recovery of pHi, indicative of effective

acid extrusion across the cell membrane as opposed to internal

sequestration (Boron and De Weer 1976; Thomas 1976). Moreover it

has been shown that pHi recovery from an acid load is

electroneutral, blocked by the stilbene derivatives 4,4'-

diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) and 4-

acetamide-4'-isothioazono-stilbene-2,2'-disulphonic acid (SITS)

which are known inhibitors of erythrocyte Cl /HCO^ exchange and

unaffected by oubain,a sodium/potassium ATP-ase pump inhibitor

(Thomas 1976a). Dependence on intracellular chloride was

demonstrated and dependence on ATP was observed and so it was

proposed that acid recovery occurred via electroneutral exchange

of intracellular chloride for extracellular bicarbonate, these

movements being against their electrochemical gradients and

therefore driven by ATP (Russell and Boron 1976). Thus effective

acid extrusion occurs via metabolically driven HCO^ influx which

combines with H^ ions to produce 00^+ H^O which diffuse out of

the cell.

In a classic experiment on the snail neurone Thomas (1977) found

that metabolic inhibitors did not affect pHi regulation but

recovery from cell acidification was inhibited by removal of the

extracellular sodium. In addition intracellular sodium

concentration increased during pHi recovery and when pHi recovery

was blocked with DIDS so was the increase in intracellular

sodium. Thus on the basis that snail neurone pHi recovery was

dependent on external Na^ and HCO^ plus internal Cl an

electroneutral Na^-dependent HCO^ /Cl exchanger was proposed in

which one extracellular Na^ and HCO^ ion are exchanged for one

intracellular Cl ion and one proton, i.e. Na^-HCO^ /Cl -H*.

exchange. However, equally compatible with experimental

observations are Na*- 2HC0^ /Cl exchange or NaCO^ /Cl exchange.

Unlike the ATP dependent HCO^ /Cl exchanger proposed by Boron

and De Weer (1976) this system could be driven by the large

transmembrane sodium gradient which is maintained by the sodium

pump (Thomas 1984) and as a result may be regarded as a form of

secondary active transport (Boron 1983). Subsequently an absolute

requirement for extracellular sodium was demonstrated in the

squid axon (Boron and Russell 1983). Therefore the regulation of

squid axon pHi was extrapolated to the model proposed by Thomas

(1977) with the exception of its ATP dependence. Since there is

sufficient energy in the sodium gradient to account for the

movement of the other ions, ATP could be permissive by being

required for an essential phosphorylation. Another possibility is

that the ATP dependence provides a mechanism for turning off

9

dissipating reactions in energy starved cells because acid

extrusion leads indirectly to an increased sodium pump rate and

therefore an increased rate of ATP consumption (Boron 1983). The

sodium dependence of this transport system has been observed in

other invertebrate cells, e.g. barnacle muscle (Boron et al.

1981).

This acid extrusion system is ideally suited as a pHi regulator

because it becomes increasingly more active at more acidic pHi

and as pHi approaches normal resting levels acid extrusion is

curtailed (Thomas 1976; Boron et al. 1979). Besides displaying

marked sensitivity to pHi this system is also inhibited by a

reduction in extracellular pH which is not due purely to a

reduction in extracellular HCO^ (Boron et al. 1979;1981).

Vertebrate pHi Regulation

The majority of vertebrate cells are much smaller than the snail

neurone and squid axon and therefore less accessible to impaling

with pH and membrane potential sensitive electrodes, although

there are exceptions, such as cardiac purkinje fibres (Ellis and

Thomas 1976). Our knowledge of the mechanisms of pH regulation in

vertebrate cells has increased considerably with the development

of pH sensitive dyes derived from fluorecein. Rotman and

Papermaster (1966) discovered that fluorescein can readily enter

cell membranes when ester linked to acyl groups. Intracellular

esterase activity cleaves these bonds and a green fluorescence is

observed when viable cells are excited with light in the range

440-490 nm. The pH sensitivity of the fluorescence from

fluorescein and its derivatives has enabled pH transients to be

10

monitored in cellular organelles, namely the lysosome (Ohkuma and

Poole, 1978) and in the cytoplasm (Thomas et al. 1979). In

particular, the fluorescein derivative 2',7'-bis(carboxyethyl)-

5,6-carboxyfluorescein (BCECF) is especially suitable for

measuring intracellular pH because it is well retained within

cells, and has a pK near neutrality and hence maximal changes in

fluorescence are observed during pHi transients in the

physiological range (Rink et al. 1982). Nevertheless, in many

preparations, fluorescence intensity does change due to slow

leakage of the dye from cells and photodegradation. Such

artefactual changes can be adequately corrected by recording a

fluorescence intensity ratio (see materials and methods). As with

pH sensitive microelectrodes, pHi can be monitored continuously

and cytoplasmic pH is recorded (Thomas 1979; Chaillet and Boron

1985) unlike the weak acid technique which records whole cell pH

and which might be influenced by intracellular organelles (Roos

and Boron I98I). Also intracellular pH can be measured quasi-

continuously using nuclear magnetic resonance techniques (Spurway

and Wray I987), although the drawback to this is the timelag

necessary between readings whilst the spectrum is stabilised.

Na*/H* exchange

Investigations into pHi regulation in vertebrate cells

demonstrated an ionic mechanism different to most invertebrate

cells. In mouse soleus muscle the majority of pHi recovery from

an acid load was SITS insensitive but dependent on external

sodium (Aickin and Thomas 1977). However, pHi recovery was

largely inhibited by amiloride, an inhibitor of Na*/H* exchange,

in the sea urchin (Johnson et al. 1976). Amiloride also blocked

11

the rise in intracellular sodium observed during pHi recovery

(Aickin and Thomas 1977). Evidence of an Na*/H* exchange system

was demonstrated in sheep cardiac purkinje fibres: pHi recovery

from an acid load was independent of external bicarbonate and

blocked by amiloride (Deitmer and Ellis I98O). Direct evidence

for an electroneutral Na^/H^ exchange system was obtained from

brush border membrane vesicles. Acidification of the

extravesicular medium was observed on the addition of sodium, the

effect being resistant to the loss of membrane potential, which

demonstrated electroneutrality. Also, a proton gradient

stimulated sodium uptake into the intravesicular space (Murer et

al 1976).

A Na^/H^ exchanger which acts as a cell alkalinising mechanism

has been demonstrated in all vertebrate cells studied to date and

its operating characteristics are quite similar in all cases

(Mahnensmith and Aronson 1985; Seifter and Aronson 1986; Frelin

et al. 1988). The dependence of the transporter on external

sodium follows simple Michaelis Menton kinetics in fibroblasts

(Moolenaar et al. 1984a), thymocytes (Grinstein et al. 1985) and

vascular smooth muscle cells (Berk et al. 1987a; Weissberg et al.

1987) indicative of one external binding site. When the

transmembrane gradient for sodium is balanced by an equivalent

but opposite gradient, the exchanger is in equilibrium and

mediates no net flow of these ions: therefore under most

physiological conditions where Na^^/Na^^,> this antiport

mediates the uphill extrusion of ions coupled to an influx of

sodium down its electrochemical gradient.

12

In addition to Na^/H^ exchange this transporter will mediate

Na*/Na* exchange, Li*/H* exchange and exchange and these

systems can function in reverse mode (Kinsella and Aronson 1981;

Aronson et al. 1982; Burnham et al. 1982). No other cations have

yet been identified which have an appreciable affinity for this

transport system (Aronson 1985).

With regard to intracellular ions, the Na^/H^ exchanger does

not follow simple Michaelis Menton kinetics; antiport activity

increases at a lower pH more than predicted from an increase in

the available substrate, namely H^ ions. Since the transporter

has only one external binding site and is electroneutral this

finding suggests the presence of one or more 'modifier sites' to

which H^ ions can bind and activate the exchanger without being

transported. This is supported experimentally: when the

transporter is in Na^/Na^ exchange mode, it is more active at pH

6.9 than pH 7.47 and inhibited by amiloride (Aronson et al.

1982). This modifier site appears to be confined to the

cytoplasmic side of the exchanger because when the gradients for

sodium and H^ ions are reversed, sodium efflux is not

significantly increased on external acidification (Grinstein and

Rothstein 1986). The presence of the modifier site has important

consequences with regard to pHi regulation. First, it enables the

Na*/H* exchanger to respond to a fall in pHi promptly due to

protonation of its modifier site. Second, as pHi rises Na*/H*

decreases, eventually becoming inactive if the rate of acid

loading in the resting state is low (Moolenaar et al. 1984; Boron

1983). Thus the exchanger has a 'set point' at which the

exchanger becomes inactive due to deprotonation of the modifier

13site. This protects the cytoplasm from an excess alkaline load,

the sodium gradient being capable of the uphill extrusion of

ions upto pHi 8.0 if allowed to reach thermodynamic equilibrium

(Grinstein and Rothstein 1986). This can be demonstrated by

adding the Na*/H* exchange ionophore monensin which causes a

dramatic elevation in pHi (Moolenaar et al. 1984a).

In addition to the different ionic requirements for Na*/H*

exchange compared with Na^ dependent HCO^ /Cl exchange, they are

also distinguishable pharmacologically. The former is inhibited

by amiloride , as already described albeit rather poorly at

physiological sodium concentrations, this compound primarily

being an inhibitor of epithelial sodium channels which explains

its diuretic action (Benos 1982). Derivatives of amiloride have

been synthesised which are more potent inhibitors of the Na^/H^

exchange system although again not specific (Kleyman and Cragoe

1988).

Bicarbonate dependent pHi regulation

The important role of the bicarbonate ion in vertebrate pH

regulation has only been recognised widely in the last few years.

The reasons for this are that vertebrate cells can fully recover

from an acid load in bicarbonate free medium via activation of

the Na*/H* exchanger and the early reports in vertebrate cells

did not detect a significant role of the bicarbonate ion in pHi

regulation (Moolenaar et al. 1984). Nevertheless, three different

bicarbonate dependent pHi regulators have been described in

vertebrate cells to date, these being (1) Na^ dependent HCO^ /Cl

exchange (2) Cl /HCO^ exchange Na^-independent and (3) Na^-HCO^

14

cotransport.

Na dependent HCO^ /Cl exchange

In the vertebrate cell, L'Allemain et al. (1985) demonstrated a

Na^ - dependent HCO^ /Cl system in a mutant Chinese hamster

fibroblast line (PS120) in which Na*/H* exchange had been bred

out. As would be expected, these cells fail to recover from an

acid load in bicarbonate-free medium but in the presence of

CO^/HCO^ buffered medium the ability to effectively extrude acid

was restored. In addition in the non-mutant CCL39 cell line,

recovery from an acid load was observed in the presence of

amiloride in an CO^/HCO^ buffer. This bicarbonate-dependent pHi

regulator had an absolute requirement for extracellular sodium.

Sodium and chloride flux studies and DIDS sensitivity strongly

suggest the presence of a Na^ dependent HCO^ /Cl exchanger as

described by Thomas (1977). Independent evidence of this

transport system in the PS120 cell line has been reported (Cassel

et al. 1988). This transport system has now been observed in

several other cells including a leukaemic cell line (Ladoux et

al. 1988), frog skeletal muscle (Abercrombie et al. I983),

mesangial cells (Boyarsky et al. 1988a) and a smooth muscle-like

cell (Putnam 1988). Thus in the presence of bicarbonate, this

system acts in parallel with the Na^/H^ exchanger to promote

recovery from an acid load in several cell types.

Cl /HCO^ (Na independent) exchange

In the erythrocyte a Cl /HCO^ exchange system is well documented

(Cabantchik et al. 1978). Evidence that this system may enhance

recovery from an alkaline load was first obtained from the

15

purkinje fibre. In this cell type Vaughan-Jones (1982)

demonstrated that the exchange of external chloride for internal

HCO^ enabled the intracellular [Cl ] to rise to levels higher

than predicted by a passive distribution of these ions due to a

low membrane permeability to this ion which prevents Cl

accumulation from being instantly truncated. Absence of

extracellular HCO^ dramatically reduces Cl accumulation after

intracellular depletion. This system is independent of any other

ionic species and therefore easily distinguished from the Na^

dependent HCO^ /Cl exchanger, but is also electroneutral and

inhibited by stilbene derivatives. This system has been reported

in a number of cell types including guinea pig vas deferens

(Aickin and Brading 1984) rat type II epithelial cells (Nord et

al. 1988), mesangial cells (Boyarsky 1988a) and vascular smooth

muscle cells (Korbmacher et al. I988; Gerstheimer et al. 198?).

This sytem is controlled by the gradients for Cl and HCO^ and

therefore the exchange of external Cl for internal HCO^

increases on application of an alkaline load due to increase in

intracellular HCO^ , this alkali extrusion system will tend to

restore pHi. This system will be inhibited by an acidosis due to

the reduced intracellular [HCO^ ] thus the HCO^ ion will compete

less favourably with Cl ions for the internal binding site and

will mediate Cl /Cl exchange (Vaughan Jones 1982). In an extreme

acidosis the system may actually be reversed and function as an

acid extruder (Boron I983); this probably accounts for the small

sodium independent SITS sensitive acid extrusion observed in the

mouse soleus muscle after an acute acidosis (Aickin and Thomas

1977). Since this system is reversible a DIDS sensitive

alkalinisation is observed on removal of extracellular chloride

16

if this exchanger is present. In lymphocytes data have been

presented which suggest that Cl /HCO^ exchange is controlled by

a pH sensitive modifier site, activation occurring when pHi is

elevated independently of the intracellular HCO^ concentration

(Mason et al. 1989).

Although the main threat to steady state pHi is a tendency to

acidosis due to an influx of H^ ions down its electrochemical

gradient and possibly metabolic acid production (Busa and

Nuccitelli 1984) acid secreting cells (those cells which possess

an ATP driVen proton pump) are faced with a large base load. It

has been elegantly demonstrated that acid secreting oxyntic cells

have a higher rate of Cl /HCO^ exchange compared with

neighbouring chief cells. On the other hand chief cells are

challenged with a large inwardly directed H^ ion electrochemical

gradient and display higher Na*/H* exchange activity (Paradiso et

al. 1987).

Na - HCOg Cotransport

The existence of a Na^ - HCO^ cotransport system was first

described in the Salamander proximal tubule (Boron and Boulpaep

1983). This system being electrogenic, chloride independent and

mediating Na^ HCO^ and net negative charge across the basolateral

membrane. This implies a stochiometry of 2HC0^ :INa^ or greater.

This system has also been identified in cultured bovine corneal

endothelial cells a coupling ratio of 2HC0^~:lNa^ was observed

and the system was sensitive to DIDS (Jentsch et al. 1984).

However, in rabbit renal cortex basolateral membrane vesicles a

17

stochiometry of 3HC0^ :lNa* was reported for this transport

system (Soleimani et al.1987). In all the cases the uphill efflux

of sodium could well be driven by the outwardly directed HCO^

gradient.

In contrast to the above, inwardly directed Na^ - HCO^

cotransporters have been described, e.g. in monkey kidney

epithelial cells (Jentch et al. 1986), mouse oligodendrocytes

(Kettenmann and Schlue 1988) and in smooth muscle-like cells

(Putnam 1988). In the above cases the system was unaffected by

intracellular chloride depletion, ruling out possible confusion

with the Na^-dependent HCO^ /Cl . Contrary to the other

experiments, Ketternmann and Schlue (1988) and Aickin (1988) have

been unable to demonstrate any sensitivity of this to the

stilbene derivatives.

Intracellular Buffering

Since pHi is higher than expected due to passive distribution of

ions across the membrane, efficient acid extrusion systems

become imperative to maintain cellular integrity. Nevertheless it

is important to realise that much of the acid load (or base load

in certain circumstances) imposed on a cell is buffered. Only

when the buffering power of a cell is exhausted will pHi decline,

thus it is important to understand the concept of buffering.

Pioneering work in this field has been credited largely to Van

Slyke but should properly be afforded to Koppel and Spiro (Roos

and Boron 1980). Buffers are substances which by their presence

in a solution increase the amount of acid or alkali that must be

18

added to cause a unit pH change. Van Slyke (1922) defined buffer

value (B) as B

pH

in other words the amount of base added to a solution divided by

the resultant pH change. The definition of Koppel and Spiro was

essentially the same but includes the self buffering of water,

this occurs at the extreme end of the pH range and therefore is

not important physiologically (Roos and Boron I98O). Both workers

mathematically and experimentally demonstrated the following:

first the maximum buffer value of any monovalent weak acid or

weak base is the same. Second a weak acid or weak base exerts its

maximum buffer action when its pK is equal to the pH of the

solution; in other words when it is 50% dissociated. Thus for

monovalent weak acids and bases, they differ at the point along

the pH scale at which their buffering power is maximal but not a

maximal buffering power per se, which for a 1 molar solution is

0.575 MpH'l.

The nature of all the systems responsible for intracellular

buffering is likely to be complex, therefore it is sensible to

define intracellular buffering as all the processes which

minimise pHi shifts with the exclusion of ion transport across

thé membrane in active pHi regulation (Roos and Boron 1981).

It is important to realise the influence on an CO^/HCO^ buffer

can have an intracellular buffering. Extracellularly, a CO^/HCO^

buffer system has a molar buffering power four times greater than

a monovalent weak acid or base if PCO^ remains constant. (Woodbury

19

1965). Cell membranes are highly permeable to CO^, and therefore

this system contributes significantly to intracellular buffering

as demonstrated in the snail neurone (Thomas 1976). Since the

hydration of 00^ is a slow process, fast buffering requires the

presence of carbonic anhydrase (Thomas 1989).

Intracellular buffering can be estimated from the direct

injections of acid or alkali if cell volume can be estimated and

the resultant pH change noted. Alternatively, weak acids and

bases can be used: here the change in concentration of the

conjugate base or conjugate acid can be calculated from the

Henderson Hasselbalch equation, indicative of the degree of

imposed acid or alkaline load which is then divided by the pH

change observed. Theoretically small pH changes should be induced

if the buffering power at given pHi is required, since the value

obtained represents the mean buffering power over the pH range at

which the change occured (see Van Slyke (1922)). In the presence

of a CO^/HCO^ buffer these techniques derive the total

intracellular buffering power (B). In a bicarbonate free buffer

the intrinsic intracellular buffering power (Bi) is derived,

(when no other external weak acid or base is present). Thus BT =

Bi + BCO^ where BCO^ is the additional buffering observed is

CO^/HCO^ containing buffers (Roos and Boron 1981; Szwatkowski

and Thomas 1989), BCO^ = 2.3 [HCO^]^ (Woodbury 1965). Clearly,

BCO^ will increase when intracellular pHi is increased but will

be less effective during an intracellular acidosis.

Interestingly, several studies have demonstrated an increase in

Bi as pHi falls (Szwatkowski and Thomas 1989; Boyarsky et al.

1988) which would, to some extent, off-set the loss of BCO^ to

20

the total intracellular buffering power.

Clearly intracellular buffering is the first line of defence in

pH homeostasis. A knowledge of this parameter is essential if one

wishes to calculate the rate of net acid or base transport across

the cell membrane, which is the product of the total

intracellular buffering power and the rate of pHi recovery from

an acid or base load, this parameter being useful for instance if

the stochiometry of a coupled ion transport to acid or base

transport is to be calculated.

pHi and Cell Growth

Cellular metabolism involves numerous reactions, many of which

produce and consume protons (Busa and Nuccitelli 1984). The free

energy of hydrolysis of ATP is pH sensitive, although not

dramatically so; nevertheless small changes in pHi could have

profound effects on the unfavourable reactions which could be

energised by the hydrolysis of ATP or even on cellular metabolism

itself (Alberty 1968). The possibility that cells may utilise

changes in pHi as a means of controlling cell function was

proposed by Johnson et al. (1976). They observed an increase in

sea urchin egg pHi immediately after fertilisation, this pHi rise

was mediated by the Na^/H^ exchanger. Inhibition of Na^/H^

exchange via a reduction in external sodium or amiloride

attenuated subsequent cell division but this inhibition could be

overcome by the addition of ammonium chloride which results in

cytoplasmic alkalinisation. Cytoplasmic alkalinisation acting as

an important signal for mitosis was proposed by Gerson (1978) who

observed fluctuations in pHi throughout the growth cycle in slime

21

mould.

The addition of appropriate growth factors to quiescent cultured

cells initiates several early ionic events prior to entry into

the cell cycle including increased sodium entry into the cell and

consequently activation of the sodium pump and an elevation in

intracellular potassium (Burns and Rozengurt 1984). The

accelerated sodium influx occurs via activation of the Na^/H*

exchange system. Hence as for the fertilized sea urchin, an

increase in pHi maybe an important intracellular second messenger

for serum growth factors (Moolenaar et al. I98I). Evidence that

growth factor stimulation results in cell alkalinisation was

demonstrated in Swiss 3T3 cells (Schuldiner and Rozengurt 1982).

Using the pH sensitive dye BCECF it was also demonstrated that

serum growth factors and epidermal growth factor cause a

persistent elevation of pHi in fibroblasts. Kinetic analysis

suggests that this is due to an increased sensitivity of the

antiport to internal protons (Moolenaar et al. 1984). Further

work demonstrated that the effects of growth factors on pHi could

be mimicked by phorbol esters which are known to activate protein

kinase C (Moolenaar et al. 1984a). This suggests a strong link

between activation or resetting of the Na^/H* exchange and the

polyphosphoinositide cell signalling system since diacylglycerol

which is the physiological activator of protein kinase C

(Nishizuka 1984) is one of the products of the hydrolysis of the

membrane lipid phosphatidyinositol 4,5 bisphosphate (FTP^)

(Berridge 1984). The occupation of some cell receptors by their

agonists, including certain growth factors, activates

phospholipase C, this event being coupled via a G protein. It is

22

this enzyme which cleaves PIP2 to form inositol 1,4,5

trisphosphate which is hydrophylic and mobilises calcium from

intracellular stores and the lipid 1,2 diacylglycerol which

remains in the cell membrane (Berridge and Irvine 1984).

Alternatively the activation of certain growth factor receptors

leads to a set of biochemical events which results in resetting

of the Na^/H^ exchanger which is actually inhibited by phorbol

esters. Stimulation of the exchanger may occur with tyrosine

kinase activity (Whitely et al. 1984; Vara and Rozengurt I985).

Furthermore other investigators have shown that Na^/H^ resetting

is triggered by a rise in cytoplasmic free calcium, possibly via

kinase activity which is stimulated by the Ca^^/calmodulin

complex (Owen and Villereal 1982). In fact the mechanisms

involved in Na^/H^ resetting can differ dramatically between

various types of fibroblasts (Muldoon et al. 1987) and can occur

via different pathways in the same cell (Huang et al. I987).

Whatever the mechanisms, the simplest and most attractive

hypothesis is that the increased sensitivity of the Na*/H*

exchanger to internal H^ ions is due to a phosphorylation at or

near the modifier site which raises the 'set point' of the

antiporter (Moolenaar I986). This theory is strengthened by the

finding that phorbol ester-induced Na*/H* activation is more

sensitive to ATP depletion than activation due to an acute acid

load (Grinstein et al. 1985). Also in the A431 cell line

activation of the Na^/H* exchanger decreased its affinity to

internal sodium which would favour Na*/H* exchange to Na /Na

exchange (Green and Muallem 1989).

23

If an elevation in pHi plays a crucial role in the cell response

to growth factor stimuli, this would suggest that certain key

enzymes are critically activated by the rise in pHi which is

observed, typically 0.1-0,3 pH units (Moolenaar 1986). In the sea

urchin egg, protein synthesis can be stimulated by elevating pHi

(Winkler et al. 1980) which can be demonstrated in cell free

systems (Winkler 1982). Also the activity of the DNA and RNA

polymerases increases as pH is increased (Gerson 1982).

Furthermore ribosomal protein S6 phosphorylation which may

control protein synthesis activation and re-activation of DNA

synthesis on stimulation of quiescent cells is a highly pH

sensitive processe (Chambard and Pouyssegur1986 ).

Therefore abberations in pHi control may result in abnormal

growth control. Transformed Chinese hamster embryo fibroblasts

maintain a higher resting pHi than the non-transformed cell line

due to a raised set-point of the Na^/H^ exchanger. In this

instance it is thought that the pHi rise is relatively benign

alone, but it may result in an exaggerated growth response to

appropriate stimuli (Ober and Pardee 1987). On the other hand

there are data which suggest that a persistent elevation in pHi

may stimulate cell growth per se (Perona 1988). Certainly in some

cases, oncogene-mediated cell proliferation is associated with

Na*/H* activation (Doppler et al. 1987).

Amiloride and its analogues have been used to try and demonstrate

that alkalinisation via Na*/H* exchange activation is a crucial

part of the growth process. Indeed, amiloride and its analogues

have been shown to inhibit DNA synthesis and a close relationship

24

between the potency of the analogue for Na /H exchange

inhibition and abolition of DNA synthesis was observed

(L’Allemain et al. 1984). This finding must be interpreted with

great caution since amiloride and its analogues can directly

inhibit DNA and protein synthesis and a myriad of other cell

functions (Besterman et al. 1984). In addition, if related to

pHi, amiloride and its analogues may not inhibit growth by

inhibiting the pHi rise but by inducing a pHi fall to potentially

cytotoxic levels (Grinstein et al. 1988).

The kidney renal brush border membrane Na*/H* exchanger has been

shown to be activated by various stimuli including metabolic

acidosis, a chronic reduction in renal mass and thyroid hormone.

The effect of these stimuli on Na*/H* exchange activity takes

between several hours to several days hence the term 'slow

activation type' (Grinstein and Rothstein 1986; Frelin et al.

1988). In the case of metabolic acidosis (Kinsella et al 1984)

excess thyroid hormone (Kinsella et al. 1986) and partial

nephrectomy (Vigne et al. 1985) the effects of these stimuli on

Na*/H* exchange are kinetically distinct from the growth factors

discussed previously. Na^/H^ exchange activity is increased by

the latter stimuli not by an increased sensitivity to internal

ions due to a raised set point of the modifier site, but due to

an increased rate of the Na*/H* transport system. This finding

suggests either an increased activity of the original

transporters possibly due to a reduction in a rate limiting step

(probably translocation and/or reduced binding of ions) or an

increase on the concentration of functional antiporter units in

the membrane (Kinsella et al. I986). The latter possibility is

25

attractive since the time taken for the stimuli to elicit a

response is consistent with the synthesis of more exchanger units

(Grinstein and Rothstein 1986). This finding is of interest since

all the above stimuli are known to induce renal hypertrophy (Nord

et al 1985).

Finally, some evidence against an elevation in pHi being a

mediator of proliferation should be presented. Mitogenic

stimulation of lymphocytes results in increased metabolic acid

production which outweighs Na*/H* activation and pHi actually

falls (Gelfand et al 1988). Expressions of the proto-oncogene C-

fcs which is an early response to mitogenic stimulation (Marx

1986) is unaffected by omission of external sodium, Na*/H*

exchange inhibition, and artificial cytoplasmic alkalinisation

(Grinstein et al. 1988). Most importantly with regard to pHi and

cell growth, most studies have been performed in bicarbonate free

media, thus negating the influence of CO^/HCO^ buffers on

intracellular buffering power and potential bicarbonate dependent

regulation. It is important to note that Cassel (I985) failed to

detect a pHi rise in bicarbonate containing media. Indeed is

resetting of the Na*/H* physiologically a mediator of a pHi rise,

or does this enable the cell to extrude acid more rapidly in the

event of increased metabolic acid production due to cell

stimulation ? (Boron 1984).

pHi and Vascular Tone

The possibility that intracellular pH is an important modulator

of vascular smooth muscle contractility and hence blood vessel

tone arose from the numerous reports that hypercapnia (elevations

26

in PCO^ hence intracellular acidosis (Jacobs 1920) is associated

with blood vessel dilation (e.g. Olsson 1981; Case et al. 1978;

Haddy and Scott 1968). In the human forearm induced reactive

hyperaemia is associated with an increase in blood pCO and a

reduced pH. In addition, infusions of sodium bicarbonate to

maintain pH had no effect on reactive hyperaemia whereas

hypocapnia due to voluntary hyperventilation reduced the response

(Kontos and Patterson 1964). In normal circumstances i.e. without

inducing reactive hyperaemia, hypercapnia induced by breathing

00^ had little effect on forearm resistance regardless of blood

pH. However, pre-treatment with phenoxybenzamine and propranolol

to inhibit the activity of the sympathetic nerves and circulating

catecholamines resulted in a decrease in forearm resistance

during the period of hypercapnia regardless of blood pH. These

workers proposed that the increased PCO2 causes vasodilation due

to a smooth muscle cell intracellular acidosis which is

counteracted by increased sympathetic activity (Kontos 1968).

Interestingly, studies on pial vessels suggest that vasodilation

is mediated via a decrease in extracellular pH (Kontos et al.

1977; Kushinsky et al. 1972); thus the mechanism of CO^ induced

vasodilation may differ in different vascular beds. In addition

it is important to release that changes in pHi in the blood

vessel wall may influence the release of vasoactive substance

from the endothelium and/or nerve terminals (Wray I988).

Data obtained form cultured vascular smooth muscle cells have

demonstrated that the vasoconstrictor agonist angiotensin II and

noradrenaline activate the Na*/H* exchanger which raises

cytoplasmic pH (Hatori et al. 1987; Berk et al. 1987; Owen I986).

27

These contractile agonists stimulate the polyphosphoinositide

system and therefore protein kinase C is thought to be activated

(Griendling et al. 1987) which is thought to be crucially

involved in the maintenance of vascular smooth muscle cell

contraction (Rasmussen 1987). In cultured vascular smooth

muscle cells sustained diacyglycerol formation on All stimulation

results from the hydrolysis of polyphosphoinositides, this

process being further stimulated by inducing cytosolic

alkalinisation and inhibited by acidification; thus it has been

suggested that cytosolic alkalinisation via activation of the

Na^/H^ exchanger may be critical for tonic contraction

(Griendling et al. I988). This concept has been strengthened by

the observation that activation of protein kinase C with phorbol

esters induces a tonic contraction (Danthuluri et al. 1987). So

far the data presented would suggest that an increase in vascular

smooth muscle cell pHi would enhance blood vessel tone and vice

versa as suggested by Mahnensmith and Aronson (I985).

Nevertheless, the rare investigations which have used intact

segments of vascular tissue have to yet substantiate this. Using

skinned muscle tissue, a depressive effect of acidosis has been

observed on the microfilaments of skeletal and cardiac muscle

(Fabiato and Fabiato 1978). Again with skinned vascular smooth

muscle only a relatively small (13%) reduction in force was

observed when pH was decreased from 6.9 - 6.5. In addition a

reduction in pH increased the sensitivity of the contractile

apparatus to calcium and reduced the rate of relaxation (Gardner

and Diecke I988). Addition of ammonium chloride to isolated

segments of rabbit aorta preconstricted with noradrenaline causes

a transient reduction in tone and a transient enhancement of tone

28

is observed when ammonium chloride is washed out suggesting that

intracellular alkalinisation has a vasodilator action and vice

versa for acidification (Furtado 1986). Similar changes in vessel

tone using the ammonium chloride pre-pulse techniques have also

been observed in rabbit ear artery (Ighorje and Spurway 1985),

the expected simultaneous changes in pHi and vascular tone were

confirmed using phosphorous nuclear magnetic resonance (Spurway

and Wray 1987). Danthuluri and Deth (1989) demonstrated that

addition of ammonium chloride to isolated segments of aorta

preconstricted with noradrenaline caused a transient reduction in

tone which was followed by an enhancement of tone to a level

greater than observed on the addition of noradrenaline alone.

Also addition of ammonium chloride to resting segments of aorta

had no effect initially but after a lag period a tension

development was observed, these effects being dose-dependent.

It appears that changes in intracellular pH do have marked

effects on contraction although the mechanism is not known. In

contrast to skeletal muscle (Mainwood and Renaud 1985) under

certain circumstances an acidosis can lead to enhancement of

vessel tone. If the findings of Danthuluri and Deth (1989) can be

ascribed to change in pHi rather than 'non-specific' effects of

ammonium chloride, this would suggest that the acute and chronic

changes in pHi on vascular tone may differ.

Resistance Vessel pHi and Hypertension

Changes in pHi as a result of alterations in cellular metabolism

leading to enhanced metabolic acid production or consumption,

environmental influences (e.g. changes in extracellular weak acid

29

or base concentration) or active pHi regulation may have profound

effect on cell function (see Busa and Nuccitelli (1984)). I have

limited myself to a brief discussion of two such cell functions,

the reason being that they may be of importance during the

development and maintenance of hypertension, implying a possible

link between intracellular pH and this disease state.

In the established phase of the disease essential hypertension is

characterised by an increase in total peripheral resistance at a

time when cardiac output is normal (Lund-Johansen I98O). The

blood vessels responsible for elevating peripheral resistance and

therefore raising arterial blood pressure are the pre-capillary

resistance arteries intimately involved in the local regulation

of blood flow (Folkow 1978). Indeed, Bright (I838) remarking on

the cardiac hypertrophy he observed in autopsy material from

'hypertensives', suggested that the blood "... so affects the

minute and capillary circulation, as to render greater action

necessary to force the blood through the distant sub-divisions of

the vascular system".

Johnson described wall thickening in the small arteries and

arterioles, a finding confirmed by Ewald (see Folkow, 1984).

Folkow pointed out that the artery wall thickening could

influence peripheral resistance profoundly (Folkow 1958). Folkow

(1982) has explained how an increased media thickness and a

modest reduction in lumen diameter in the resistance vessels,

results in a large increase in the media thickness/lumen diameter

ratio. The consequence of this is twofold: first, there will be

an increased resistance to blood flow which is proportional to

30

the fourth power of the vessel radius (Poiseuille's Law), at

maximal blood vessel dilation, secondly, the pressor response to

a given degree of myocyte activation will be amplified due to the

changes in vascular geometry (Lever I986; Mulvany 1987).

Folkow (1982) has proposed that in essential hypertension an

initial disturbance which raises blood pressure slightly,

stimulates vascular hypertrophy (hypertrophy meaning either an

increase in cell size or cell number in this instance). If the

initial disturbance persists, although perhaps intermittently,

vascular hypertrophy will amplify the pressor signal and thus a

positive feedback cycle is set in motion leading to further

hypertrophy and eventually established hypertension. Folkow

(1986) has proposed that in genetically hypertension-prone

animals and man this hypertrophic response is genetically

reinforced which would lead to a genetic predisposition into the

positive feedback cycle described.

Alternatively, there may be trophic stimuli (possibly excess

sympathetic activity or circulating factors with mitogenic

potential) which act on vascular smooth muscle resulting in a

structural change, increasing media thickness/lumen diameter

which raises blood pressure (Lever 1986). Whatever the mechanism,

it seems that the structural changes in the resistance vessel and

the blood pressure rise proceed hand in hand in both human

essential hypertension, and the spontaneously hypertensive rat

(Heagerty et al. 1988).

The mechanism by which pressure rise stimulates vascular

hypertrophy is not known. It is possible that an increased

31

pressure load stimulates Na^/H^ exchange and elevates pHi

Certainly, such increased load appears to stimulate

phosphoinositide hydrolysis (Ollerenshaw et al. 1988), which as

discussed, can indirectly lead to resetting of Na^/H^ exchange.

Alternatively, abberations in pHi regulation such that vascular

smooth muscle cells are more alkaline may enhance the growth

response to appropriate stimuli and thus contribute to the

'genetic reinforcement' as proposed by Folkow. Furthermore

excessive neurohumoral stimulation may induce myocyte

proliferation via activation of Na^/H^ exchange: noradrenaline

and angiotensin II have been shown to induce proliferation of

myocytes held in cell culture. Also it has been suggested that

an elevation in pHi may enhance vascular tone and contribute

towards the increased peripheral resistance (Mahnensmith and

Aronson 1985).

Na*/H* Exchange and pHi in Circulating Blood Cells

Circulating blood cells are easily accessible and for this reason

are frequently used to study putative ion transport abnormalities

in hypertension (Bing et al. 1986). One such abnormality is

increased erythrocyte Na*/Li* countertransport which transports

lithium from 'loaded' erythrocytes against its electrochemical

gradient, this being energized by the influx of sodium down its

own electrochemical gradient (Canessa et al. 1980). Similar

findings have been reported by other investigators in both

essential hypertensives and sons whose parents were both

hypertensive (Woods et al. 1982), although occasionally there

have been conflicting data (Duhm et al. 1982). Lithium is not a

natural substrate for this transport system which normally

32

mediates Na^/Na^ exchange and therefore no net ion flux. The

pathological role of such an exchange system, if it occurred in

vascular tissue, remained obscure until Aronson (1982a) suggested

that erythrocyte Na^/Li* countertransport maybe the Na*/H*

exchanger or at least share structural components that are under

common genetic control.

The rate of cell swelling, due to sodium influx via the Na*/H*

exchanger after inducing an acid load with sodium proprionate

suggest that this antiporter activity is increased in lymphocytes

from the spontaneously hypertensive rat (SHR) compared with

normotensive rats (Feig et al. 1987) and in platelets from mild

hypertensives (Livne et al. 1987). Furthermore, amiloride

sensitive sodium influx is enhanced in leucocytes from essential

hypertensive patients (Ng et al. 1988). However reports of

intracellular pH in circulating cells have been inconsistent. A

decrease in erythrocyte pHi has been observed in human and

experimental hypertension (Resnick et al. 1987) although Na^/H^

exchange activity is not substantial in this cell type. Similarly

a decrease in pHi has been reported in lymphocytes from the SHR

(Batlle et al. 1990). On the other hand leucocyte pHi is

significantly more alkaline in hypertensive patients compared

with controls (Ng et al. 1989) whereas in platelets no difference

has been found (Weder et al. 1989).

Extrapolation of findings in circulating blood cells to vascular

smooth muscle cells, particularly those present in the resistance

arteries and thus the hypertensive process is speculative. With

regard to one aspect of ion transport, the sodium efflux rate

33

constant, a significant correlation between leucocytes and

resistance arteries has been demonstrated (Aalkjaer et al. 1986).

Aims of the Study

It is as a result of these possibilities being important in

causing and maintaining hypertension that I performed the

experiments described below with the purpose of trying to examine

vascular pHi in genetic and experimental forms of hypertension

and in human essential hypertension.

34CHAPTER TWO

MATERIALS AND METHODS

The Myograph

The myograph was developed in 1977 and allows both measurement of

vascular morphology (adventia, media and intima thickness) and

contractility under standard conditions (Mulvany and Halpern,

1977).

The instrument consists of a 15ml volume bath in which there are

two separate pairs of mounting heads (only one being used in the

experiments described below). One head is connected to a

micrometer screw and is thus mobile while the other head is

connected to a force tranducer via a connecting pin which passes

through the side of the bath. Each head has two screws which are

used to grip the wire upon which the artery is mounted. The bath

is heated to 37°C by heating blocks either side of the bath which

is warmed by circulating water. The bath has an inlet for passing

the buffer solution and two drainage rods attached to a suction

pump for bath emptying. The force transducer is fixed into the

wall of the bath and connected to a pre-amplifier which in turn

is connected to a chart recorder. (Grass Instruments Co.,

Quincey, Mass, USA). The artery is threaded over two 40 um

diameter stainless steel wires which pass down the lumen. The

wires are firmly held in place by the mounting head screws

(Figure 1).

35

Morphology Measurements

The myograph is designed with small perspex window situated

directly beneath the mounting heads. When the myograph is placed

on the stage of a light microscope (Model KHC Olympus) the

arterial wall can be visualised using water immersion microscopy.

A calibrated micrometer eyepiece (x8, Zeiss) is used to measure

the thickness of the adventitia, media and intima. A salt water

immersion objective lens was used (x25 Leitz-Wetzlar, (Germany).

All morphology measurements were made when the wires are

separated so that the vessel is held just under tension.

Measurements include the gap between the wires. The morphology

measurements were converted to 'normalised values' (see below)

assuming a constant media volume. Thus normalised media thickness

(m ) is the media thickness when the vessel is stretched to a

normalised diameter (1 ).

Normalisation Procedure

This procedure allows the setting of a lumen diameter of the

arteries at which active tension is maximal (Mulvany et al. 1977)

as opposed to many vascular preparations where all arteries are

simply placed under a given tension load. The procedure is based

on the observation that vascular smooth muscle displays a 'dome

shaped' active tension-length relationship. The force generating

ability of the contractile elements increases with stretch upto

an optimal point after which further stretch reduces active

tension and passive tension increases sharply. From the Laplace

formula T = Pr where T is tension, P is pressure and r is the

vessel radius, the effective pressure inside a section of vessel

can be calculated for any given internal circumference and wall

36

tension. With respect to the myograph, changes in tension and

internal circumference can be measured on the chart recorder and

micrometer screw respectively. Thus for each vessel a tension-

internal circumference (the smooth muscle stretch) relationship

can be plotted and the internal circumference at which the

vessels have an effective internal pressure of lOOmmHg (13.3 kPa)

determined. A curve fitting program is run on a programmable

calculator (Hewlett Packard HP41 CV) and the artery undergoes a

series of stretches by turning the micrometer screw. When the

vessel has been stretched to the point when the effective

internal pressure is equal or greater than 100 mmHg the procedure

is halted, the vessel passive tension reduced and the calculator

determines the effective internal diameter of the vessel when

relaxed and under an effective transmural pressure of lOOmmHg. In

addition the calculator determines the micrometer reading at

which the vessel will have an internal diameter (1 ) which is 90%

of the diameter when the vessel is under a transmural pressure of

lOOmmHg. Previous work (Mulvany et al.1983) has demonstrated that

at this internal diameter, active tension development in response

to contractile stimuli are mean maximal, this finding has been

confirmed in this laboratory (Bund, personal communication).

Expressions

i. ^100 the internal circumference when P is lOOmmHg.

ii. ^100 the 'effective diameter' corresponding to

since the vessel is stretched flat by the wires.

iii. lo is the normalised effective diameter such that lo = 0.9

^ 100*

The contractile responses can be expressed as follows:-

37

i. Active tension ( T) is expressed as mN/mm segment length

ii. Media stress ( S) is the tension change divided by the2normalised media thickness expressed as mN/mm . This

describes contractility per unit volume of smooth muscle

rather than actual magnitude of the contractile response.

iii. Effective active pressure ( P) is the pressure against

which the artery can contract expressed as the tension

change divided by the normalised lumen diameter according

to the Laplace equation p = T/r.

Indirect Systolic Blood Pressure Measurement

Rats were placed in an ether box until they showed no response to

a slight shake of the box. They were removed and placed on a

warmed pad. An inflatable cuff connected to an ordinary mercury

sphygmomanometer was placed around the tail and distal to this

was placed a light source and photomultiplier cell connected to a

plethysmograph. An oscilloscope displayed the plethysmograph

output. Pulse waves in the tail were visualised on the

oscilloscope screen. The cuff was inflated until the waves were

eliminated and then the cuff was slowly deflated until the waves

were just visible and the pressure recorded by the

sphygmomanometer was noted on the indirect systolic blood

pressure. For each rat the inflation-déflation procedure was

repeated 2-3 times to confirm the recorded measurement.

Dissections

Rats were killed by stunning and cervical dislocation. Mesenteric

resistance arteries were obtained from the mesenteric circuit

which was exposed and excised after longitudinal incision in the

38

abdomen. Dissection was performed in physiological saline

solution (composition described below) using trabecular scissors

and watchmaker forceps filed down further to permit finer

handling.

Solutions

Vessels were dissected and normally held in the myograph in2 5physiological saline solution, PSS * of the following

composition (mmol/1)

NaCl 119, KCl 4.7, CaCl^ 2.5, NaHCO 25, Mg S04 1.17, kH^PO^

1.18, EDTA 0.026 Glucose 5.5. K-PSS^'^ is on the same2 5composition as PSS but with an equimolar substitution of KCl

for NaCl. NAK is 10 umol/1 noradrenaline in K-PSS^*^

Animals

Male spontaneously hypertensive (SHR) and normotensive Wistar

Kyoto (WKY) rats were used for study at 5 weeks and 12 weeks of

age. The rats were obtained from the Leicester University

breeding colony, this strain of rats having been established for

14 years. Animals were given free access to standard chow and tap

water. Blood pressures were measured using the tail cuff

plethysmography method and rats used for study were not less than

one day and no more than three days after the blood pressure

measurement. They were killed by stunning and cervical

dislocation as previously described and one second order

mesenteric artery branch was dissected, cleaned free from

adherent fat and connective tissue and mounted in the myograph.

After gasing in 5% CO^ in 0^ and warming to 37'C for 30 minutes

arterial morphology measurements were made and the segment

39

normalised.

Standard Start Procedures

The standard start procedure in all rat experiments was two NAK

activations, one activation with 10 umol/1 noradrenaline alone,

one K-PSS activation and one further NAK activation. When human

resistance arteries were used the standard start procedure was

three K-PSS activations and a NAK activation. Activations were

for 2 minutes followed by washout in PSS, a full recovery was

permitted between contractions.

Intracellular pH Measurement

After the standard start procedure the myograph was placed on the

stage of a standard 16 microscope (Carl Zeiss Oberkochen Ltd,

W.Germany) converted into a fluorescent microscope. Attached to

the microscope was a XBO 75 W/2 high pressure xenon lamp

connected to a voltage stabilised power supply unit. Also

connected to the microscope was a photometer. This consisted of a

photo-tube attachment connected to a photomultiplier tube. The

photomultiplier tube contained control electronics which converts

the generated photocurrent (due to light emitted by the specimen

incident on the photomultiplier) into analogue voltage. The

photomultiplier tube is connected to a power supply which

possesses a digital voltmeter on which the signal is displayed

varying from 0.1 to l80 units and a signal amplifier. This was

interfaced with a BBC computer programmed to display the mean

signal during the period of data aquisition (2.5 secs) which was

permanently recorded on paper using a daisy wheel printer (DA

Computers, Leicester, UK). The fluorescence micoscope apparatus

40

was purchased from Carl Zeiss Oberkochen Ltd, W.Germany.

Above the microscope objective lens (Leitz Wetzlar x25) were two

sets of filters which could be alternately slid into the light

path from the xenon lamp light passed through the filter set when

a shutter was opened. This enabled a fluorescence intensity ratio

to be calculated.

Each filter set consists of three filters (Carl Zeis Oberkochen,

W.Germany).

1. An exciter filter, allowing the appropriate waveband to be

presented which was either 485 nm, bandwidth 10 nm or 455 nm

bandwidth 10 nm.

2. A chromatic beam splitter which is angled at 45° to the

oncoming light path which reflects light below 510 nm and

transmits light above this wavelength.

3. A barrier filter which transmits light in the range 520-560

nm.

Using these filter sets, fluorescence emission 520 - 560 nm could

be recorded when the artery, which has been loaded with the pH

sensitive fluorescent dye BCECF was excited at 485 nm and 455 nm

(Figure 2).

The organ bath was covered with a black plastic lid with a hole

which just enclosed the objective lens. The perspex porthole was

also covered and the microscope eye pieces replaced with black

plastic caps after focussing on the artery wall. Hence the signal

obtained was not influenced from external light sources

41

eliminating the need to work in a dark room.

When focussing through the vessel wall a faint striated pattern

could be seen. This indicated that the field of vision was

focussed on the smooth muscle layer.

Background Cell Autofluorescence

Before loading the arterial segment with fluorescent dye,

background cell autofluorescence was measured for 2.5 sec at 485

nm and 455 nm excitation wavelength. Five readings at each

wavelength were taken, each separated by a period of 1 minute.

The light source was attenuated by 80% using a neutral density

filter. From this manoeuvre the mean background fluorescence was

calculated at each excitation wavelength. Background fluorescence

was typically around 0.5 units at 485 nm excitation and slightly

less at 455 nm. This allowed the artery to be loaded (see below)

with fluorescent dye without the signal obtained going out of

range. The signal amplifier settings were never altered after

recording of cell autofluorescence.

Dye Loading

The acetoxymethyl ester of the pH sensitive fluorescent dye

2',7'-bis (carboxyethyl) 5,6-carboxyfluorescein (BCECF AM)(5

umol/1) was added to the myograph bath and left to load for 60

minutes at which time the excess was washed out. This compound

diffuses across the cell membrane and is hydrolysed by internal

esterases in the cytoplasm releasing the poorly permanent BCECF

(Rink et al. 1982; Grinstein et al. 1984). A fluorescence ratio

was determined every 60 sec, this was achieved by dividing the

42

emission with excitation at 485 nm minus the background at 485 nm

by the emission with excitation at 455 nm minus the background at

455 nm. As with the background measurements, excitation was for

2.5 sec at each wavelength.

Calibration

Calibration of the fluorescence intensity ratio was achieved

using the nigericin high potassium buffer technique as first

described by Thomas (1979).^Nigericin is a potassium/hydrogen ion

exchange ionophone which sets up the following equilibrium

[K] /[K]. = [H] /[H].. Thus when [K] is approximately equal too 1 o 1 o[K]^, intracellular pH will be clamped to that of the external

solution (Grinstein et al 1984). It has been demonstrated that

the derived pHi value is not critically dependent on the

potassium concentration in the high potassium buffer (Chaillet

and Boron 1985). At the end of the experiments three solutions of

140 mmol/1 K-Hepes were set to different pH values in the range

6.4 - 7.6 by addition of sodium hydroxide and 10 umol/1 nigericin

added to each. The composition of the K-Hepes buffer was as

follows :(mmol/1) KCl 140, MgCl2 1, CaCl2 1.6, EDTA 0.026 glucose

10, Hepes 5. These solutions were warmed to 37°C and the exact pH

of each was measured using a Corning microcombination pH

electrode (Fisons, Loughborough, UK). The first solution was

applied to the myograph bath for 8 minutes and subsequent

solutions for 5 minutes each. Fluorescence ratios were recorded

every minute throughout the procedure. The last three readings

with each solution reached a plateau indicating that pH^ and pHi

had reached equilibrium and the mean value calculated. By pH

43clamping a resistance artery over a wide pH range a linear

relationship between the fluorescence intensity ratio and pH is

observed in the pH range used for calibration (Figure 3), a

finding in agreement with all other reports using BCECF. The

relationship between the actual fluorescence intensity ratio

values and pH are in agreement with previous reports (e.g.

Boyarsky et al. 1988a; Kobayashi et al. 1990). Thus, using this

technique a linear regression line could be calculated and the

other fluorescence intensity values evaluated to give true pH

readings. In all experiments the correlation coefficient was 0.95

or greater. Such an in situ calibration procedure avoids the

possibility of shifts in the spectral properties of the

intracellularly trapped fluorescence probe influencing the

results obtained; this is a potential pitfall when the

calibration curve is derived using BCECF free acid in solution.

The procedure described was performed on each vessel at the end

of the experimental protocol.

Fluorescence Changes

Throughout every experimental protocol dye loss occurred. This

was due to photobleaching rather than dye leakage since no loss

of fluorescence intensity was observed when resistance arteries

were loaded with BCECF and left for at least 2 hours. Using

resistance arteries pH clamped near neutrality a proportional

decline in fluorescence was observed with excitation at 485 nm

and 455 nm (Figure 4a) yet the fluorescence intensity ratio

remains constant (Figure 4b), although this results in an

inherently noiser record. A similar observation has recently been

reported (Putnam and Grubbs, 1990). There was upto 80% reduction.

44though never greater, in fluorescence before calibration in any

experimental protocol. Nevertheless, the signal was always

greater than ten times background fluorescence.

Intracellular pH and pHi changes

Resting intracellular pH was defined as the mean pH obtained from

five ratio determinations at the beginning of each experiment.

Before the pHi response to an experimental manoeuvre was

investigated, three ratio determinations were made to define a

base line. The pH change (' pHi) at a given time during any such

manoeuvre was defined as the pHi at that given time minus the

mean pHi of the three determinations which defined the baseline.

Chemicals Used

Noradrenaline, nigericin, N-2-hydroxyethyl-piperazine-N'-2-

ethanesulfonic acid (Hepes), 4,4-diisothiocyanatoslibebe 2, 2-

disulfonic acid (DIDS), and amiloride were purchased from Sigma

(Poole, Dorset, UK) and 2’, 7'-bis (carboxyethyl ) 5,6-

carboxyfluorescein (BCECF) and BCECF-AM were obtained from

Molecular Probes (Eugene, Oregon, USA). Ethylisopropylamoride

(EIPA) was obtained from Dr. EJ Cragoe and synthesized as

previously described (Kleyman and Cragoe, 1988). All other

chemicals were of standard laboratory grade.

Analysis of Data

Results are expressed as mean + the standard error of the mean

(SEM) for each set of experiments. Differences between means were

analysed using non-parametric testing (Mann-Whitney U). For

changes in pHi with time, results were compared between groups

45

using two-way analysis of variance (ANOVA). In all cases p 0.05

was considered significant.

46

MicrometerForce

Tra nsdu cer

2mm

Figure 1 Schematic representation of a segment of resistanceartery suspended on stainless steel wires between the mounting heads of a myograph. One mounting head is linked to a micrometer screw gauge and the other to a force transducer.

47

Oin LT\ —M3 m

o "3

o <- m mX -i CD u QJ U. ^ ^

□ " U CD CO D)

CD <-HO -K

DO) > " §"-3%W .LJ [U - (U -4 N M O —M M

g U-2 CD Oa; O O O J-J o X in u cn o.

T30) (U CO u

s iCO Mu pCO f - i

a «

crH "H

u X • H à + j CO Q, f-l I—< O CO - H

I— I cO o; 3 +-’x:+->

ao

cuT3( Ucj u CO o fn aE

CO!L ,bO

C + ->H X_ ( D CO ^

H CD !h CD 3 C /3 COCO •

CO ^

T )

> j

CD k 4-JECD

u o ^c / 3 "Cw CO

fSJ0 )k

bO

48

in£ 3.5 1

in00

O 3.0 -

a 2.5 - c o ■«-»

0 2.0 -cmin<D

1 1.5-4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5

pH

Figure 3 pH-dependent changes in fluoresence intensity ratio in a resistance artery using the nigericin high potassium buffer technique. Each point represents the mean + SEM (when visible) of three ratio determinations

49

II

14

13

12 485

1110

g87

65

455432

0100606040200

limaiminil

limelminil

Figure 4 The change in fluoresence intensity with excitation of 485nm and 455nm (a) and the fluoresence intensity ratio (b) recorded over 100 minutes.

50

CHAPTER THREE

RESISTANCE VESSEL pHi IN THE SPONTANEOUSLY HYPERTENSIVE RAT

COMPARED WITH THE NORMOTENSIVE WISTAR KYOTO CONTROL

Introduction

The genetically hypertension prone spontaneously hypertensive rat

(SHR) developed by Okamato and Aoki is a frequently used model

assumed to possess similar characteristics to human essential

hypertension. The main blood pressure elevation occurs between 4

and 12 weeks (Mulvany et al. 1980). During this time adaptive

refashioning of the vasculature, including the resistance vessels

will be taking place due to myocyte hyperplasia and/or

hypertrophy (Mulvany et al. 1978). Therefore, it was decided to

measure intracellular pH in mesenteric resistance arteries from

the SHR and compare the results with those obtained from vessels

from control normotensive Wistar Kyoto (WKY) rats at 5 weeks of

age when blood pressure is rising and at 12 weeks, when it is

established.

Methods

Male SH and WKY rats were obtained from the Leicester University

breeding colony. Animals were allowed free access to standard rat

chow and tap water. Blood pressure was measured using the tail

cuff plethysmography, as described above. Rats were used

individually for each experiment. On the day of study animals

were stunned and sacrificed by cervical dislocation and a 2mm

segment of second order branches of the superior mesenteric

artery were dissected for study.

51

Resting intracellular pH was measured in mesenteric resistance

arteries from the SHR at 5 and 12 weeks and compared with WKY

controls. The effects inducing contraction of pHi were investigated

using noradrenaline (10 umol/1) and K-PSS. The effects of the

addition and washout of ammonium chloride (10 mmol/1) were studied

in both strains at both time points. Further pertubations in

pHi were examined using sodium acetate (50 mmol/1) substituted

on an equimolar basis with sodium chloride. The effects of

the Na^/H* ionophore monensin (10 umol/1) on pHi were also

investigated. Both these experiments were only performed on

mesenteric arteries from 12 week WKY rats. Inhibition of Na /H

exchange with amiloride (1 mmol/1) and bicarbonate transport

with DIDS (200 umol/1) was observed over ten minutes in resistance

arteries from the SH and WKY rats from 5 weeks of age.

Results

Blood Pressure,Body Weight and Media/Lumen Ratio

The mean indirect systolic blood pressure was significantly higher

in SHR at 5 weeks compared with WKY rats. The experiments were

performed on one vessel from each animal and the media/lumen

ratio was significantly increased in SHR compared with WKY rats

indicating that structural changes were already beginning to

take place. At twelve weeks mean indirect systolic blood pressure

remained significantly higher in SHR and the SHR remained heavier.

Media/lumen ratio remained significantly raised in SHR; WKY vessels

52

failed to show significant structural changes when comparing this

strain at 5 and 12 weeks, whereas SHR showed a highly significant

increase at 12 weeks (Table 1).

Intracellular pH

At 5 weeks of age pH was significantly more alkaline in vessels

from SHR compared with those from WKY rats (7.36 _+ 0.04 versus

7.22 _+ 0.03 pH units, p = 0.0151, Figure 5). However, at 12 weeks

of age this difference was no longer evident (SHR 7.34 + 0.06

versus WKY rats 7.30 +_ 0.04 pH units, NS, Figure 6). This

appeared to be due to the pH in WKY vessels becoming more

alkaline as this strain of rat has aged, although it did not

reach significance when the values at 5 and 12 weeks were

compared.

Experiments in K-PSS

After exposure to K-PSS for 5 min at 5 weeks of age results in a

significant acidification in both rat strains (Figure 7). The

change in pH was greater in SHR than in WKY rats (&pHi -0.17

0.03 versus -0.07 ± 0.02 pH units, p = 0.0356). At 12 weeks of

age both rat strains again showed an acidification after 5 min

exposure to K-PSS. The response in both rat strains was now no

longer different (Figure 8).

Experiments with Noradrenaline

At five weeks of age activation of vessels with 10 umol/1

noradrenaline again led to a significant acidification of a

similar magnitude in both strains (Figure 9). However, at 12

weeks of age the two strains showed a highly significant

53

difference in response when exposed to noradrenaline (p < 0.001,

Figure 10). The arteries from WKY rats showed a small

alkalinisation after 5 min, whereas the SHR vessels continued to

show acidification (Figure 2.7). A comparison of the responses at

5 and 12 weeks in WKY rats revealed a significantly different

noradrenaline induced effect (p<0.01). A representative trace of

the effects of contraction on pHi in a 5 week WKY resistance

artery is shown (Figure 11).

Experiments with NH, C1

Addition of 10 mmol/1 NH^Cl caused an acute alkalinisation which

returned towards a baseline over the next 10 min (Figure 12). The

pH change did not differ between the two strains at 5 weeks (SHR

0.29 0.03 versus WKY rats 0.23 0.03 pH units, NS). Washout of

10 mmol/1 NH4C1 caused an acidification which undershot the

baseline (SHR -0.56 + 0.08 WKY -0.42 + 0.05 pH units NS). This

was followed by a return to baseline in 10-15 min. Neither strain

displayed any difference in the initial rate of recovery as

determined from the pHi recovery over 3 minutes after maximal

acidification (SHR 0.061 + 0.014 versus WKY rats 0.048 _+ 0.006 pH

units/min, NS). Addition of 10 mmol/1 NH^Cl at 12 weeks of age

again caused alkalinisation returning towards baseline over 10

min. The pH change was not different between the two strains (SHR

0.31 2 0.07 versus WKY rats 0.26 _+ 0.03 pH units, NS). There was

no significant difference within strains comparing results at 5

and 12 weeks. Wash-out of NH4C1 caused an acidification which

undershot the baseline (SHR -0.49 2 O.O3 , WKY -O.58 2 O.O6 pH

units NS). Again the initial rate of recovery did not differ

between strains (SHR O.O5I + O.O6 versus WKY rats O.O5I + 0.007

54

pH units/min, NS).

From the ammonium chloride-induced alkalinisation, the

intracellular buffering power (BT) was calculated using the

formula of Szatkowski and Thomas (1989) which is numerically

identical to the formula of Roos and Boron (1981). No significant

difference in the apparent buffering power was observed at 5

weeks (SHR 39 + 10 versus WKY 51 ^ 5 mmol/1 x pH) or 12 weeks

(SHR 35 ^ 9 versus WKY 42 _+ 7 mmol/1 x pH).

Contractility Studies

At 5 weeks activation with noradrenaline (10 umol/1) resulted in

the production of media stress which did not differ between the

two strains (SHR 153 + 16.4 versus WKY 157 + 13.4 mN/mm^,NS).

Similarly K-PSS did not induce a difference in media stress

production (SHR (126 _+ 9.8 versus WKY 131 13.2 mN/mm^, NS).

Wash out of ammonium chloride also produced slight contractions

in vessels from both strains of rat but mean media stress was not

significantly different (SHR 54 9.3 versus WKY 60 + 14.2

mN/mm^, NS).

At 12 weeks of age in the presence of noradrenaline mean media

stress produced in the two strains was not different (SHR I58 _+221.8 versus 210 + 12.1 mN/mm , NS). K-PSS induced a mean media

stress which was significantly lower in SHR (SHR 104 + 15.62versus 152 7.3 mN/mm , p<0.005). Wash out of ammonium chloride

failed to produce contractions in vessels from either strain at

this age.

55

Discussion

These experiments suggest that at five weeks of age the SHR

arteries have a more alkaline pH than vessels from control rats.

At twelve weeks this difference was lost due mainly to a change

in basal pH in vessels from the WKY strain. In all studies, the

application of ammonium chloride induced alkalinisation followed

by acidification on washout. From the induced alkalinsation, the

intracellular buffering power was calculated. Although not

significant, the buffering power appeared to be lower in the SHR

at 5 weeks compared with the WKY. This may be a reflection of the

different resting pHi at this age, thus buffering power was not

estimated from the same interval on the pH scale. At 12 weeks of

age the estimated buffering power between strains was not

significantly different. The cell membranes of the myocytes in

the resistance artery are also highly permeable to undissociated

weak acid and intracellular pH promptly falls when sodium

acetate is applied to the myograph bath. There is a rapid

alkalinisation on removal of sodium acetate but contrary to the

findings of Korbmacher (1988) there was only a negligible

overshoot (Figure 13). It is also possible to artificially

elevate intracellular pH using the Na^/H^ ionophore monensin

(Figure 14). Neither of these pHi manipulations had any effect on

resting tension. At 5 weeks of age, K-PSS induced an

acidification in the arteries of both strains of rat with the

vessels of the SHR becomming significantly more acid. This

difference was lost at twelve weeks although both strains

displayed a prolonged acidification. Similarly, contraction

elicited by the agonist noradrenaline induced intracellular

acidification. At twelve weeks of age, arteries from the SHR

56

demonstrated the same response to noradrenaline but those from

the WKY showed a slight alkalinisation. Both K-PSS and

noradrenaline induced anticipated contractions in the resistance

vessels, when expressed in terms of media stress there was no

difference in magnitude of contraction produced except with K-PSS

of twelve weeks. At this time media stress was significantly

reduced in the SHR and may be due to inadequate removal of

nigericin from the myograph after some of the previous

experiments. It was noted that force production was sometimes

associated with changes in emission with excitation at 485 and

455 nm wavelengths. Although the contractions elicited are

isometric, small increases and decreases in emissions recorded

are probably attributable to variations in the amount of dye

visible in the microscope field induced by contraction.

Regardless of the direction of these small changes consistent

changes in the intensity ratio and hence pHi were observed. This

finding is in agreement with the observations of Aalkjaer and

Cragoe (1988) who have also measured pHi in resistance arteries

in a myograph.

At five weeks of age the intracellular pH was more alkaline in

the SHR resistance arteries; at this time the SHR were already

more hypertensive and the media/lumen ratio was also increased.

Nevertheless, at 5 and 12 weeks of age the media volume was not

significantly increased in the arteries from the SHR and thus a

clear growth response was not observed. Although the

morphological measurements are similar to previous reports in

mesenteric arteries (Mulvany et al. 1978; Mulvany et al. 1985) in

these publications significant media volume increases were

57observed. However, these findings were observed in the SHR at 20

weeks of age when further morphological change may have occurred,

resulting in a significant media volume increase. In addition, in

my experiments there may have been a selection bias resulting in

the dissection of arteries from the SHR which were not from

precisely the same anatomical site as the WKY (see below).

Therefore, the alkaline pHi in the resistance arteries at 5 weeks

may be critical for the development of vascular hypertrophy. The

higher pHi could be a genetically predetermined abnormality which

predisposes the myocytes of the SHR to proliferate in an

exaggerated fashion to appropriate growth stimuli.

Alternatively the pH difference may be a consequence of the

increased pressure load placed upon the blood vessel wall.

Certainly it appears that an increased pressure load on the aorta

proximal to a stenosis between the renal arteries stimulates

phosphoinositide hydrolysis (Ollerenshaw et al. 1988). This may

result in an increased production of diacylglycerol and protein

kinase C activation which in turn can influence Na^/H* exchange.

Experiments were performed where Na*/H* exchange and bicarbonate

dependent pH regulation were inhibited in 5 week rats. As can be

seen in Figure 15 DIDS caused a small fall in resting pHi which

did not differ between strains. Amiloride also caused a small

fall in resting pHi which was significantly greater in the SHR

(Figure 16). This finding suggests both Na*/H* exchange and a net

bicarbonate influx system are active in the resting state and

that Na*/H* exchange activity is increased in resistance arteries

in the SHR. Despite this finding no difference was observed in

58the initial rate of recovery from an acid load. When the pHi

values were log transformed a linear relationship with time was

observed over six minutes. Analysis of the regression lines did

not demonstrate any difference between the SHR and WKY animals.

Contrary to findings in cultured VSMC, where stimulation with

noradrenaline and angiotensin II result in cell alkalinisation

(Owen et al. 1987; Berk et al. 1987) activation with

noradrenaline induced acid changes in pHi in most experiments. In

cell culture systems bicarbonate free Hepes buffered media have

been used. In the WKY resistance arteries at 12 weeks there was a

small non-significant alkalinisation on stimulation with

noradrenaline indicating that in this strain the pH response to

this agonist changes with time. It is unclear why the pHi changes

induced with K-PSS and noradrenaline differ between strains and

change with times. Nevertheless these experiments argue against

the role of Na*/H* exchange induced cell alkalinsation in tonic

VSMC contraction. Furthermore, since the contractile response in

terms of media stress was not increased in the SHR, differences

in resting pHi and pHi change during contraction are unlikely to

contribute to the increased vascular resistance in this strain

due to a potentiating effect on myocyte contractility.

59

5 weeks

SHR WKY

Blood pressure (mmHg) 119 + 6.3 99 + 3.2 P=0.018

Weight (g) 94 + 4.1 58 + 3.9 p=0.006

mQ, (um) 11.22 + 0.56 9.78 + 0.51 NS

1q (um) 197 + 7.47 208 + 9.89 NS

Media volume (um /um) 7396 + 468 6748 + 556 NS

m /I (%) ratio o o 6.1 + 0.27 4.8 + 0.25 p=0.004

12 weeks

SHR WKY

Blood pressure (mmHg) 158 + 2.6 114 _+ 2.8

Weight (g)

m o (urn)

1 o (urn)

Media volume

m. /l J % )ratio

311 + 3.9 280 + 7.9

15.62 + 1.3 11.94 + 0.62

198 + 12.2 242 + 9.4

10591 + 1231 9642 + 6l2

8.1 + 0.7 5.0 + 0.4

p 0.001

p=0.003

NS

p=0.021

NS

p=0.004

TABLE 1

Mean + SEM of the indirect systolic blood pressure, body weight and morphological parameters on the SHR and WKY at 5 and 12 weeks of age.NS = not significant

60

7.7 -I

7.6 -

7.5 -

7.4 -pHi

7.3 -

7.2 -

7.0SHR WKY

Figure 5 Resting intracellular pH of mesenteric resistance arteries from SH and WKY rats at 5 weeks of age. Horizontal bar, mean value; vertical bar, _+ SEM * p<0.05.

61

7.7 -I

NS7.6 -

7.5 -

7.4 -pHi

7.3 -

7.2 -

7.0SHR WKY

Figure 6 Resting intracellular pH of mesenteric resistance arteries from SH and WKY rats at 12 weks of age. Horizontal bar, mean value; vertical bar, _+ SEM; NS, Not significant.

62

ApHi

0.1

0.0

- 0.2

-0 .31.00.0 2.0 3.0 4.0 5.0

time(mins)

Figure 7 Change in pHi induced over 5 minutes by 125 mmol/1K-PSS in resistance arteries from SH (O) and WKY (#) rats at 5 weeks of age. Each point represents the mean _+ SEM at minute intervals.** p<0.01 (ANOVA SH versus WKY rats). N=ll

63

0.1

0.0

ApHi -0.1NS

- 0.2

-0 .31.00.0 2.0 4.03.0 5.0

tlme(mlns)

Figure 8 Change in pHi induced over 5 minutes by 125 mmol/1 K-PSS in resistance arteries from SH (O) and WKY (4 rats at 12 weeks. Each point represents the mean _+ SEM at minute intervals. Not significant (ANOVA SH versus WKY rats). N=10

64

0.0

ApHi -0.1 - NS

— 0.2 -

-0 .30.0 1.0 2.0 3.0 4.0 5.0

time(mins)

Figure 9 Change in pHi induced over 5 minutes by noradrenaline(10 umol/1) in resistance arteries from SH (O) and WKY (#) rats at 5 weeks. Each point represents the mean _+ SEM at minute intervals. Not significant (ANOVA SH versus WKY rats). N=ll

65

0.0

ApHi —0.1 -

— 0.2 -

-0 .31.00.0 2.0 3.0 4.0 5.0

time(mins)

Figure 10 Change in pHi induced over 5 minutes by noradrenaline (10 umol/1) in resistance arteries from SH (O) and WKY (•) rats at 12 weeks. Each point represents the mean _+ SEM at minute intervals.** p<0.01 (ANOVA SH versus WKY rats). N=10

66

7.5 -I

7.4 -washoutwashout noradrenalineK-PSS

7.3 -

pHi7.1 -

7.0 -

6.9 -

6.820 25155 100

timelmins)

Figure 11 Intracellular pH in a mesenteric artery from a 5 week WKY rat showing the effects of K-PSS and noradrenaline induced contractions.

67

pHi

7.8 n

7.6 -

7.4 -

7.2 -

7.0 -

6.8 -

6.6 -

6.4

N H 4CI washout

—I-------- T"10 15

timelmins)

— T- 20

I25

Figure 12 Representative trace of the changes in pHi induced by addition and washout of NH^C1(10 mmol/1). The mesenteric resistance artery was taken from WKY rat aged 5 weeks.

68

7.8 n washoutsodium acetate

7.6 -

7.4 -

pHi7.2 -

7.0 -

6.80 2 4 6 8 10 12 14 16 18 20

time(mins)

Figure 13 Representative trace (N=5) of the changes in pHiinduced by the addition and washout of sodium acetate (50 mmol/1). The mesenteric resistance artery was taken from a WKY rat aged 12 weeks.

69

pHi

7.6 -I

7.4 - monensin

7.2 -

7.0 -

6.8 -

6.60 2 4 6 8 10

time(mins)

Figure 14 Representative trace (N=5) of the change in pHi observed after the addition of the Na /H exchange ionophore monensin (10 umol/1). The mesenteric resistance artery was taken from a WKY rat aged 12 weeks.

70

0.0

NSApHi -0.1 -

— 0.2 -

-0 .30 6 8 102 4

time(mins)

Figure 15 Mean _+ SEM change in resting pHi in thepresence of DIDS (200 umol/1) over 10 minutes in resistance arteries from SH (O) and WKY (#) rats at 5 weeks. NS not significant (ANOVA SH versus WKY rats) N =8

71

ApHi

0.0

— 0.2 -

-0.30 2 4 6 8 10

★ ★

time(mins)

Figure 16 Mean + SEM change in resting pHi in thepresence of amiloride (lmmol/1) over 10 minutes in resistance arteries from SH (O) and WKY (#) rats. ** p<0.001 (ANOVA SH versus WKY rats). N =8

72

CHAPTER FOUR

RESISTANCE VESSEL pHi DURING THE DEVELOPMENT OF EXPERIMENTAL

HYPERTENSION

Introduction

In the previous chapter it has been demonstrated that the resting

pHi in the cells of resistance arteries of SHR was significantly

higher when compared with that found in vessels from normotensive

Wistar-Kyoto control animals at five weeks. Such a finding might

be present as a consequence of the vascular growth process or

alternatively might be a necessary event for the morphological

changes to occur. In addition it is possible that the increased

resting pHi is genetically predetermined in SHR; as such, if it

is related to growth, this would support the hypothesis that

subjects or small mammals with a genetic predisposition to

developing hypertension have cells with the ability to

demonstrate an enhanced growth response when faced with a trophic

stimulus (Folkow, 1982). In order to investigate further whether

this increased pHi is a genetically determined abnormality or one

that occurs as growth is stimulated it becomes necessary to

impose hypertension on the circulation of an animal not bred to

develop high blood pressure. Accordingly, it was decided to

measure pHi in mesenteric resistance arteries after the induction

of coarctation of the aorta over a period of one month whilst

recording morphological changes in the arteries using the

myograph.

73

Methods

Aortic Coarctation

Female Wistar rats (10-12 weeks of age, l80-210g body weight)

maintained upon normal rat chow were used throughout the study

eight animals were studied in each group of 3 and 9 days and 10

rats in each group were studied at 28 days. Coarctation of the

aorta was induced under ether anaesthesia using the modified

technique of Selge and Stone (1946). A silk ligature was tied

around the aorta between the origins of the two renal arteries,

with a wire of 0.4 mm diameter included inside the ligature and

withdrawn thereby allowing a stenosis of constant diameter to be

induced. Unlike the original technique, the left ureter was not

ligated. Sham operations were performed by manipulating the aorta

between the renal arteries but no ligature was applied. All

experiments were performed at 3, 9 and 28 days after the surgical

procedure.

Measurement of blood pressure by direct arterial cannulation

Cannulation of the carotid artery was carried out on rats under

ether anaesthesia. Catheterisation was performed through an

incision on the anterior surface of the neck. The control

catheter (p50 Portex, Kent, UK) had internal and external

diameters of 0.58 mm and 0.96 mm respectively and was inserted

and then secured in position with three braided silk ties (3/0,

Ethicon, Edinburgh, UK). This catheter was exteriorised between

the scapulae passing to the right side of the trachea between the

muscle layers. The catheter was protected externally by a

stainless steel spring 40 cm in length. Four hours after surgery

74

blood pressure recordings were made.

Arterial catheters were connected to a Statham P23 strain gauge

transducer (Staff Instruments, Henley-on-Thames, UK) via a metal

tubing adapter (Clay Adams, NJ,USA) and a plastic three way tap

(Vygon, Cirencester, UK). A continuous recording of blood

pressure was made using a chart recorder (Grass Instruments Co.,

Quincey, MA, USA) to which the transducer was linked. A

continuous recording was thus obtained and mean arterial blood

pressure could be calculated as diastolic plus 1/3 pulse

pressure. After blood pressure had been recorded for at least 45

minutes the animal was sacrificed.

Resting pHi, in second order mesenteric arteries, was measured 3,

9 and 28 days after the induction of coarctation of the aorta.

The effect upon resting pHi of blockade of Na*/H* exchange was

examined using ethylisopropylamiloride (EIPA) (60 umol/1) with

fluorescence measurements being made every minute for 10 minutes.

Resting pHi was also investigated in the presence of DIDS (200

umol/1), an anion transport inhibitor which blocks bicarbonate

dependent pH regulation. These experiments were performed at 9

and 28 days. The effects of contraction on pHi was investigated

using noradrenaline (10 umol/1), a concentration which is known

to bring about a maximum response in this tissue and was studied

over a 5 minute period.

75Results

Carotid Blood Pressure and Body Weight

Carotid mean arterial pressure was significantly higher in

experimental animals at 3 days compared with control rats

(p<0.01, Figure 17), and remained significantly higher at 9 and

28 days (p <0.01 at both time points, Figure 17). At 3 and 9 days

the animals with aortic coarctation were significantly lighter

than sham controls; body weights were comparable at 28 days

(Table 2).

Morphology

Mean media thickness, lumen diameter, media thickness/lumen

diameter ratio and media volume values were similar in arteries

from rats with coarctation compared with sham operated control

animals 72 hours after surgery (Table 3, Figure 18). Similarly at

9 days although all parameters had increased indicating vascular

growth was occurring in rats with coarctation none attained

statistical significance (Table 3, Figure 18). At 28 days after

surgery mean lumen diameter was not different between the two

groups of animals (Table 3); however media thickness, mean media

thickness/lumen ratio and media volume had all increased

significantly in arteries from rats with coarctation (Table 3,

Figure 18).

Intracellular pH

The resting artery pHi was not different between rats with

coarctation compared with sham operated control animals at 72

hours (Figure 19). At 9 days pHi had risen sharply in both groups

of animals but was not statistically significantly different and

76

also did not change at 28 days (Figure 17).

Experiments with EIPA and DIDS

At 9 days the addition of EIPA resulted in a fall in pHi in

arteries from rats with coarctation and from sham-operated

animals, which was identical over a 10 minute period (Figure

20). The addition of DIDS also produced a fall in pHi, but when

compared by analysis of variance this was significantly less in

vessels with coarctation (Figure 21).

At 28 days the application of DIDS produced a fall in pHi which

was no longer different between the groups of animals (Figure

22). However EIPA now produced a significantly greater fall in

pHi in vessels from rats with coarctation (Figure 23).

Experiments with Noradrenaline

Noradrenaline (10 umol/1) produced media stress values which were

significantly greater between rats with coarctation compared with

sham operated animals at 3 days (p<0.05, Table 4). At 9 and 28

days there was no significant difference in media stress between

the two groups of rats (Table 4). Activation with noradrenaline

resulted in a significant acidification over 5 minutes which did

not differ in the resistance arteries from rats with coarctation

compared with sham operated animals at any time point (Table 5).

Discussion

These results demonstrate that the production of coarctation of

the aorta brought about hypertension proximal to the stenosis and

morphological changes of mesenteric resistance arteries: the

77

significant increase in medial volume observed at 28 days would

indicate that vascular growth has taken place. The exact nature

of the growth response is uncertain. Previous work using the same

model of experimental hypertension demonstrated that hypertrophy

was brought about in the proximal aorta (Ollerenshaw et al. 1988)

but the response may not be uniform throughout the circulation.

Indeed in a Goldblatt 1-kidney model of experimental

hypertension, it has been demonstrated that the histological

change in the mesenteric resistance vasculature was that of

hypertrophy (Korsgaard and Mulvany, 1988). However, the

contractile response as represented by the noradrenaline-induced

media stress was reduced, whereas in the current experiment no

such reduction was found; therefore the two models may not be

exactly comparable. Intracellular pH was not significantly

different in the arteries from rats with coarctation and animals

with sham-operations at 72 hours. At 9 days when there was

evidence that media volume was increasing, pHi rose in rats with

coarctation but this rise was also seen in arteries from animals

with sham operations. Between 9 and 28 days no further pHi change

was seen in either group of rat. At no time point was pHi

significantly different between the two groups of rat. Because

pHi was lower in both the sham and coarctation group at 3 days

compared with 9 and 28 days it seems probable that the surgical

procedure with possibly prolonged exposure to the ether

anaesthesia has a depressive effect on resting pHi 72 hrs after

surgery. In addition the pHi change on activation with

noradrenaline was slightly attenuated at this time point compared

with 9 and 28 days.

During the course of the experiment, the effects on resting pHi

78

of applying DIDS and EIPA were examined. At 9 days in the

presence of DIDS there was a significantly reduced fall in pHi in

arteries from rats with coarctation compared with that seen in

rats which had undergone sham procedures. At 28 days this

difference was no longer seen. DIDS inhibits bicarbonate

dependent acid extrusion, possibly via a Na^ dependent HCO^ /Cl

exchanger and/or inwardly directed Na^-HCO^ co-transport and

Cl /HCO^ exchange which extrudes alkali. It is not possible to

determine whether one or more mechanisms have been altered at 9

days in arteries of rats with coarctation, but the results

suggest that bicarbonate dependent pH regulation is changed at

this time. The addition of EIPA produced a fall in resting pHi in

resistance arteries which was similar in both groups of rat at 9

days. However at 28 days the fall in resting pHi was greater in

the arteries from rats with coarctation in the presence of EIPA,

thus it appears that although resting pHi was not increased in

arteries from rats with coarctation pHi is maintained due to a

greater Na*/H* exchange activity. Furthermore, the greater

dependence on Na*/H* exchange to maintain pHi would indicate that

there is an increase in intracellular acid loading, either as a

result of an increased membrane permeability to protons

permitting a greater influx, increased proton production within

the cell or a reduced cell buffering power. Which of these

mechanisms is responsible is uncertain from the results of these

studies: however there is evidence that increased membrane

permeability to protons does occur in the renal brush border of

the kidney undergoing hypertrophy (Kinsella et al. 1986).

Nevertheless, it is more attractive to postulate that there are

major changes in cellular metabolism in the growing vasculature

79

of the hypertensive rat, which may lead to changes in the

production of metabolic acid equivalents. At 9 days, net DIDS

sensitive acid extrusion may be reduced and perhaps metabolic

acid production is reduced at this time due to an increase in

proton consuming reaction, although the Na*/H* exchanger appears

unchanged at this time; at 28 days excess proton generation may

be occurring. Thus changes in pHi do not appear to be of primary

importance in the development of vascular growth. The finding

that resting state pHi does not differ in the resistance vessels

from the hypertensive rats compared with sham controls but the

changes in pHi induced by DIDS and EIPA do differ argues that

precise pHi regulation is of fundamental importance in cellular

function.

Throughout this study noradrenaline induced contractions induced

significant acidification. Unlike the finding in SHR compared

with the WKY, no difference in the agonist induced pHi change

was observed in the resistance arteries of the hypertensive

animals compared with the controls.

Sham Coarctation

Before surgery 194 + 4 196 + 4 NS

3 days 216 + 3 1 8 5 + 5 P = 0.001

9 days 230 + 5 2 0 9 + 7 p = 0.02

28 days 246 + 5 255 + 8 NS

TABLE 2

80

Mean + SEM of the body weight, in grams, of the rats before aortic coarctation or sham operations and 3,9 and 28 days post surgery.

81

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82

Sham Coarctation

3 days 357 + 40 514 +34 p = 0.01

9 days 440 + 42 459 + 39 NS

28 days 470 + 31 457 + 40 NS

TABLE 42Mean _+ SEM of the media stress (mN/mm ) on exposure to 10 umol/1

noradrenaline in rats with aortic coarctation and sham controld at 3,9 and 28 days post surgery. NS = not significant.

Sham Coarctation

3 days -0.17 + 0.02 -0.15 + 0.04 NS

9 days -0.24 + 0.02 -0.26+0.04 NS

28 days -0.21 + 0.02 -0.24+0.02 NS

TABLE 5

Change in intracellular pH induced by noradrenaline

(10 umol/1) for 5 minutes in mesenteric resistance

arteries from rats with aortic coarctation and sham

controls. NS = Not significance.

83

84

180 -I

o>XEE<D35 1 4 0 -q>k _Û.T>o 120 -L _<oo

1000 10 20 30

days

Figure 17 Mean _+ SEM direct carotid artery pressure(mmHg) at 3» 9 and 28 days post surgery from rats with coarctation of the aorta (O) and rats with sham (' operations. *P<0.001. N=5,9 and 7 for rats with coarctation of the aorta at 3,9 and 28 days respectively. N = 4,6 and 5 for rats with sham operations at 3,9 and 28 days respectively.

85

COo

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E3.COE 3 o E _2 o >2 io m E

9 -

8 -

7 -

6 -

NS

NS

"T“10

—T— 20

I30

days

Figure 18 Mean _+ SEM media volume at 3 » 9 and 28 dayspost surgery from mesenteric resistance arteries from rats with coarctation of the aorta (O) and rats with sham operation (#). *P<0.01. N=8 at 3 and 9 days. N=10 at 28 days.

pHi

7.7 -I

7.6 -NS NS7.5 -

7.4 - NS7.3 -

7.2 -

7.00 10 20 30

time(days)

Figure 19 Mean _+ SEM intracellular pH at 3, 9 and 28 dayspost surgery from mesenteric resistance arteries from rats with coarctation of the aorta (O) and rats with sham operations (#). NS = Not Significant.N=8 at 2 and 9 days. N=10 at 28 days.

87

0.0

A pHi ”0.1 -NS

— 0.2 -

-0.30 2 4 6 8 10

time(mins)

Figure 20 Mean _+ SEM change in resistance pHi in thepresence of EIPA (60 umol/1) over 10 minutes in resistance arteries from rats with coarctation of the aorta (O) and sham controls (#) 9 days post surgery. N=8.

0.0

ApHi “0.1 -

— 0.2 -

-0.31086420

time(rriins)

Figure 21 Mean _+ SEM change in resting pHi in the presenceof DIDS (200 umol/1) over 10 minutes in resistance arteries from rats with coarctation of the aorta (O) and sham controls (#) 9 days post surgery. **P<0.001. N=8.

89

0.0

ApHi -0.1 -NS

- 0.2 -

-0.30 2 4 6 8 10

tlme(mins)

Figure 22 Mean _+ SEM change in resting pHi in the presenceof DIDS (200 umol/1) over 10 minutes in resistance arteries from rats with coarctation of the aorta (O) and sham controls (#) 28 days post surgery.NS = Not significant. N=10.

90

0.0

— 0.1 -

ApHi

— 0.3 -

— 0.40 2 4 6 8 10

tlme(mlns)

Figure 23 Mean _+ SEM change in resting pHi in the presence of EIPA (60 umol/1) over 10 minutes in resistance arteries from rats with coarctation of the aorta (O) and sham controls (•) 28 days post surgery. **P<0.001. N=10.

91

CHAPTER FIVE

RESISTANCE VESSEL pHi IN HUMAN ESSENTIAL HYPERTENSION

Introduction

As already discussed above, there are data from circulating blood

cells that indicate that Na^/H^ exchange activity is increased in

essential hypertension. If this abnormality occurs in VSMC the

result may be cell alkalinisation. Alternatively, Na^/H* exchange

may be increased in hypertension to offset an increased metabolic

acid production. This concept is supported by the observation

that erythrocyte pHi is lower in hypertensive patients (Resnick

et al. 1987). The possibility that this may arise from a

disturbance in calcium metabolism has been proposed based on data

obtained from lymphocytes from the SHR (Batlle et al. 1990). To

date there is no information concerning intracellular pHi and

Na^/H* exchange activity in VSMC in human essential hypertension.

Therefore, intracellular pH was measured in subcutaneous

resistance arteries from untreated essential hypertensives and

matched controls.

Methods

Subjects

Fourteen patients with essential hypertension were recruited form

the outpatient clinic of the Department of Medicine, Leicester

Royal Infirmary, Leicester, UK. No subject had ever received any

antihypertensive medication and all patients were thoroughly

screened for secondary causes of hypertension. All patients had

blood pressures greater than 140/95 mmHg when measured on at

92

least three occasions using a Hawksley random zero

sphygmomanometer. The results obtained from this group of

patients were compared with those from a group of normotensive

subjects matched for age, sex and body weight who were recruited

by the use of an advertisement placed in a local newspaper. All

participants were informed of the nature of the experiment and

gave their consent in accordance with the regulations of the

local ethical committee.

Preparation

Artery segments, about 2mm long, were dissected from biopsies

(about 0.5 X 0.5 x 1.5 cm) of skin and subcutaneous tissue taken

under local anaesthesia (3-5 ml 2% lignocaine hydrochloride) from

the gluteal region.

Results

Resistance arteries were studied from 14 patients (9 male) with

essential hypertension. These were matched with 14 normotensive

control subjects (9 male). There were no significant differences

in mean age, height or body weight although the control subjects

were slightly older (Table 6). The hypertensive patients had

significantly higher blood pressures in both lying and standing

positions (Table 6).

Morphology

Fourteen arteries were studied from each group. Mean media

thickness was increased in vessels from hypertensive patients

although this did not attain statistical significance when

compared with control arteries (14.9 ± 0.72 vs 12.8 _+ 0.75 um, p

93= 0.059 NS). Similarly media volume was not significantly

different between vessels from hypertensive patients compared

with control subjects (9656 _+ 734 vs 8887 ± 759 um^/um length

NS). Mean lumen diameter was not different between the two groups

of vessels (190 _+ 9.0 vs 206 _+ 10.3 um, NS). Mean media/lumen

ratio was significantly raised in arteries from hypertensive

patients (7.92 + 0.4 vs 6.4 _+ 0.47%, P = 0.022). The 16.4%

increase in media thickness was less than previously recorded

(Aalkjaer et al. 1987) although the current patients had lower

blood pressures compared to the previously studied group the

other parameters are in agreement with previous work.

Resting Intracellular pH

The resting intracellular pH in resistance arteries from

hypertensive patients was not different from that observed from

vessels from control subjects (7.24 _+ 0.06 v 7.25 ^ 0.04 units, n

= 14). There was no correlation between age and pHi in arteries

from patient or control groups or when the data were pooled.

Experiments in K-PSS

Exposure to K-PSS for 5 min resulted in a significant

acidification in arteries from both hypertensive patients and

normotensive control subjects (Figure 24). The change in pHi was

not significantly different between the two groups of artery.

Experiments with Noradrenaline

Activation of vessels with 5 umol/1 noradrenaline again led to a

significant acidification (Figure 25) which was significantly

94

attenuated in vessels from hypertensive patients (p<0.01, Figure

25).

Contractility Studies

Activation with noradrenaline (5 umol/1) resulted in the

production of media stress which did not differ in vessels from2patients compared with controls (117 11.4 vs 109 11.0 mN/mm ,

NS). Similarly K-PSS did not induce a difference in media stress2production (hypertensive 91 + 10.3 vs control 94 + 12.3 mN/mm ,

NS).

Experiments with EIPA

Ethylisopropylamiloride (60 umol/1) applied for 10 minutes caused

a fall in pHi in both groups of artery (Figure 4.3). The change

in pHi was not different between vessels from patients compared

with control subjects -0.11 _+ 0.02 vs 0.13 2 0.03 NS, Figure

26).

Experiments with DIPS

Exposure to the anion exchange inhibitor DIDS also resulted in

acidification in both groups of vessel (Figure 4.4). Again the

mean fall in pHi was not different in arteries from patients

compared with control subjects - 0.097 0.05 vs -0.091 ± 0.03

units NS; Figure 27 .

Discussion

In this study intracellular pH has been measured in human

resistance arteries in essential hypertension and no difference

was found compared with normotensive controls.

95

Blockade of the Na^/H* exchanger with EIPA resulted in an

acidification which was of the same magnitude in both groups,

suggesting that Na*/H* exchange is involved in maintaining

resting cell pHi and that the reliance of the cells on this

system is identical in arteries from both the hypertensive and

control groups. The anion exchange inhibitor, DIDS, also produced

a fall in pHi. The acidification observed indicates that in the

resting state, bicarbonate dependent acid extrusion is the

dominant mechanism as observed in rat mesenteric arteries.

Because the fall in pHi was identical in hypertensive patients

and control subjects the data do not suggest that there is any

disturbance in bicarbonate-dependent pHi regulation in resistance

arteries in established essential hypertension.

Stimulation of the vessels with K-PSS and noradrenaline produced

a sustained acidification over a 5 minute period similar to the

observed in rat mesenteric vessels. The acidification observed on

activation with K-PSS was not different between the hypertensive

and control groups. On the other hand, the acidification observed

when the vessels were stimulated with 5 umol/1 noradrenaline

(which elicits a maximum contractile response to this agonist in

human vessels) is slightly yet significantly reduced in vessels

from the hypertensives.

Contrary to the results found in leucocytes and erythrocytes we

have found no difference in resting intracellular pH in arteries

from hypertensive patients compared with the control subjects.

Moreover the acidification observed on blockade of the Na /H

exchanger was identical in both groups which is at variance with

96

the possibility of increased Na*/H* activity secondary to

increased metabolic acid production. There was a reduced

acidification in vessels from the hypertensives in the presence

of noradrenaline; whether this finding is a fundamental

difference or a consequence of the blood pressure rise is

uncertain from these data. However following activation with

noradrenaline media stress was the same in both groups; therefore

this finding does not appear to bear any direct relation to

vascular contraction.

It could be argued that differences in arteries from hypertensive

patients compared with controls, such as increased pHi and Na*/H*

exchange activity are lost when studied under standard conditions

in the myograph. The finding in the genetic and experimental

models of hypertension in the previous chapters do not support

this possibility.

If disturbances in pHi are related to an increased propensity for

cellular growth in the hypertensive vasculature it is possible

that in the established phase of the disease the abnormality is

no longer apparent at a time when all arterial remodelling has

taken place. In this regard, in the SHR, resting artery pHi was

not significantly increased at twelve weeks. If this is the case,

these experiments call into question the usefulness of studying

pH regulation or any other putative cellular disorder in

circulating blood cells in established hypertension.

97

Male

Age(yrs)

Height(m)

Weight(kg

BP(mmHg)

Systolic

Essential Hypertensive

Patients

14

9

49 + 2.4

1.70 + 0.03

77 + 2.7

Supine Standing

169+5*** 170+4***

Control Subjects

14

9

54+3.1 NS

1.73 + 0.03 NS 78 + 5.0 NS

Supine Standing

134+4 127+5

Diastolic 102+2*** 110+2*** 74 + 3 81+3

TABLE 6Demographic data from the untreated group of essential

hypertensive patients and matched normotensive control

subjects. ***p<0.001 NS not significant.

98

0.0

ApHi —0.1 -

NS— 0.2 -

-0.30.0 1.0 2.0 3.0 4.0 5.0

time(mins)

Figure 24 Change in pHi induced over 5 minutes by 125 mmol/1K-PSS in subcutaneous arteries from hypertensives (O) and control (#). Results show mean and SEM at minute intervals. Not significant (ANOVA hypertensives versus controls). N=l4.

99

0.0

ApHi -0.1 -

— 0.2 -

-0.30.0 1.0 2.0 3.0 4.0 5.0

time(mins)

Figure 25 Mean + SEM change in pHi induced over 5minutes by noradrenaline (5 umol/1) in subcutaneous arteries from hypertensives (O) and controls (#). Results show the mean and SEM at minute intervals.* p<0.01 (ANOVA hypertensive patients versus controls) N=14.

100

0.0

ApHi -0.1 -NS

— 0.2 -

-0.30 2 4 6 8 10

tlme(mlns)

Figure 26 Mean _+ SEM change in pHi in the presence ofEIPA (60 umol/1) over 10 minutes in subcutaneous arteries from hypertensives (O) pnd controls (#). NS = not significant (ANOVA hypertensive patients versus controls). N=14.

101

0.0

ApHi -0.1 - NS

— 0.2 -

-0.30 2 4 6 8 10

time(mins)

Figure 27 Mean _+ SEM change in pHi in the presence ofDIDS (200 umol/1) over 10 minutes in subcutaneous arteries from hypertensives (O) and controls (#). NS = Not significant (ANOVA hypertensive patients versus controls). N=4.

102CHAPTER SIX

RESISTANCE VESSEL pHi FROM OFFSPRING OF ESSENTIAL HYPERTENSIVE PATIENTS

Introduction

In established essential hypertension any cellular abnormality

which initiates the disease may be absent or masked by

disturbances which are a consequence of hypertension (Lever

1986). First degree offspring of essential hypertensive patients

are at increased genetic risk of developing hypertension and

usually have higher blood pressures than matched controls

(Pickering 1982). For this reason such offspring are likely to

display abnormalities which may be of primary importance in

raising blood pressure. In this regard, a significant increase in

intracellular pH was observed in resistance arteries from young

SHR when blood pressure was rising but not when the hypertension

was established. For this reason intracellular pH was measured in

subcutaneous resistance arteries from normotensive first degree

offspring of essential hypertensive patients and compared with

matched control subjects.

Methods

Subjects

Ten normotensive volunteers were studied, all of whom had at

least one parent known to be receiving treatment for essential

hypertension. The results from this group were compared with

those obtained from ten controls with no such family history of

hypertension.

103

Preparation

Artery segments were dissected from biopsies of skin and

subcutaneous tissue as described in the previous chapter. The

study was approved by the local Ethical Committee.

Results

Resistance arteries were studied from 10 first degree offspring

(3 male) of essential hypertensives. These were compared with

vessels from 10 control subjects (5 male). There were no

significant differences in mean age, height or body weight,

although the relatives were slightly smaller and heavier (Table

7). There were no differences in the systolic blood pressures in

either the lying or standing position between the two groups.

However, diastolic blood pressure was significantly increased in

the offspring in both the lying and standing positions (p<0.05)

(Table 7).

Morphology

Ten arteries were studied from each group and none of the

morphological parameters were significantly different in the

offspring. Media thickness (13.13 2 0.98 vs 11.14 _+ 0.77 um NS),

media volume (10577 2 1136 vs 8174 _+ 965 um^/um length, NS),

lumen diameter (238 _+ 10.9 vs 225 116.0 um NS), media/lumen

ratio (5.51 + 0.34 vs 5.24 + 0.35% NS).

Resting Intracellular pH

The resting intracellular pH in resistance arteries from

offspring of essential hypertensives was not different from that

104

observed from control subjects (7.26 _+ 0.04 vs 7.28 +_ 0.07 NS).

Experiments in K-PSS and Noradrenaline

Exposure to K-PSS for 5 minutes resulted in a significant

acidification which did not differ in arteries from the offspring

compared with the controls (Figure 28). Similarly, activation of

the vessels with noradrenaline resulted in an acidification which

did not differ between the two groups (Figure 29).

Contractility Studies

Activation with noradrenaline (5 umol/1) resulted in the

production of media stress which did not differ in vessels from

the offspring compared with controls (155 ± 17.5 vs 174 _+ 22.52mN/mm NS). Again K-PSS did not induce a difference in media

2stress production (141 _+ 18.7 vs I58 + 18.0 mN/mm ).

Experiments with EIPA and DIDS

Application of EIPA (60 umol/1) for ten minutes caused a fall in

pHi in both groups of artery (Figure 30). The change in pHi was

not different between vessels from offspring of essential

hypertensive patients and controls. Exposure to DIDS also

resulted in an acidification over ten minutes which did not

differ between the two groups of artery (Figure 31).

Discussion

In this study intracellular pH has been measured in resistance

arteries from the offspring of essential hypertensive patients.

No difference was observed compared with the control groups. In

agreement with previous findings (Aalkjaer et al. 1987) no

105morphological differences were observed betwen the two groups.

Although normotensive, the offspring displayed significantly

higher lying and standing diastolic blood pressures.

Blockade of the Na*/H* exchanger with EIPA and bicarbonate

transport with DIDS resulted in an acidification in both cases.

These acid changes were not different in the offspring compared

with the controls. Therefore, the data do not provide any

evidence that abberations in pH homeostasis in particular

increased Ng^/H* exchange are of relevance in the generation of

hypertension. In addition, no difference was observed in the acid

pHi change when contraction was induced using either K-PSS or

noradrenaline. In resistance arteries from hypertensive patients

the acid pHi change was significantly reduced compared with

arteries from normotensive controls. This difference was not

observed in this relative study which suggests that the finding

in the arteries from hypertensive patients is a consequence of

hypertension rather than a fundamental abnormality.

These findings are in contrast to the observations in the

mesenteric arteries from the SHR. In this genetically

hypertensive prone animal, intracellular pH was elevated at 5

weeks when the blood pressure was rising. Furthermore,

differences in the pHi change during contraction were observed

compared with arteries from the WKY. These experiments suggest

that the differences observed between the SHR and WKY control are

either unrelated to hypertension or not relevant to the

pathogenesis of human essential hypertension.

106It is possible that the group sizes were too small to detect

differences between arteries from the offspring compared with

controls since not all relatives of hypertensive patients will

develop hypertension in later life. Nevertheless, it was decided

to finish this relative study after examining the results for

several reasons. First, the results are convincingly negative,

therefore a type 2 error would be unlikely. Second, the diastolic

blood pressures are significantly increased in the offspring

compared with controls indicative of a genetic trait

Nevertheless it would not be overstating the data obtained from

this and the preceding chapter to conclude that in isolated

resistance arteries from humans there is no evidence that

abnormalities in resting state intracellular pH or pHi

homeostasis are of importance in the development and maintenance

of essential hypertension.

107

RELATIVES CONTROLS

Male

Age(yrs)

Height(m)

Weight(kg)

BP(mmHg)

Systolic

Diastolic

10

3

27 + 3.7

1.67 1 0.02

69 + 2.4

Supine Standing

115+3 NS 115+4 NS

68+3< 80+3'

10

5

28 + 3.4 NS

1.72 + 0.03 NS

63+5.3 NS

Supine Standing

121+4 114+6

55+4 69+3

TABLE 7

Demographic data from the first degree offspring of essential

hypertensives and control subjects with no family history of

essential hypertension.

*p<0.05 NS not significant

108

ApHi

0.0

— 0.2 -

NS

-0.30.0 1.0 2.0 3.0 4.0 5.0

time(mins)

Figure 28 Mean _+ SEM change in pHi induced over 5 minutesby 125 mmol/1 K-PSS in subcutaneous arteries from first degree offspring of essential hypertensive patients. (O) and controls (#). NS = not significant (ANOVA relatives versus controls) N=10.

109

0.1 -1

0.0

ApHi -0.1 - NS

— 0.2 -

-0.30.0 1.0 2.0 3.0 4.0 5.0

time(mins)

Figure 29 Mean ^ SEM change in pHi induced over 5 minutesby noradrenaline (5 umol/1) in subcutaneous arteries from first degree offspring of essential hypertensive patients (O) and controls (•). NS = not significant (ANOVA relatives versus controls) N=10.

110

0.0

ApHi -0.1 -

NS

-0.31086420

tlme(mlns)

Figure 30 Mean _+ SEM change in pHi in the presence of EIPA (60 umol/1) over 10 minutes in subcutaneous resistance arteries from first degree offspring of essential hypertensive patients (O) and controls (#] NS = not significant (ANOVA relatives versus controls) N=10.

Ill

0.0

NSApHi -0.1 -

— 0.2 -

-0.31086420

time(mins)

Figure 31 Mean _+ SEM change in pHi in the presence of DIDS (200 umol/1) over 10 minutes in subcutaneous resistance arteries from first degree offspring of essential hypertensive patients (O) and controls (#). NS = not significant (ANOVA relatives versus controls) N=10.

112CHAPTER SEVEN

GENERAL DISCUSSIONIn these studies I have carried out experiments enabling

resistance vessel pHi and isometric contraction to be recorded

simultaneously. Other reports of the simultaneous measurement of

these two parameters are scarce at present, notable exceptions

being Spurway and Wray (1987), Greider et al. (1988) and Aalkjaer

and Cragoe (1988) although the latter study is the only one to

use resistance arteries. This study was designed to measure

resistance vessel pHi from hypertensive rats and from human

essential hypertensives. Intracellular pH recorded in these these

experiments will primarily reflect vascular smooth muscle cell

(VSMC) pHi, the predominant cell type in the blood vessel wall.

Although the dissected resistance vessel contains substantial

amounts of adventia, this is mainly non-cellular material and

predominantly collagen (Spurway and Wray, 1987).

The mechanisms involved in the control of VSMC pHi are not

clearly understood although in the absence of bicarbonate, Na^/H

exchange will be the major pHi regulator (Berk et al. 1987;

Weissberg et al. 1987). In the presence of a CO^/HCO^ buffer

system bicarbonate transport is also involved. DIDS caused a fall

in resting pHi in all my studies demonstrating the existence of

net bicarbonate influx. DIDS has no effect on resistance vessel

pHi or femoral artery cultured myocytes in the absence of

bicarbonate (Aalkjaer and Cragoe 1988; Kahn et al. 1990). The

ionic mechanisms of DIDS sensitive acid extrusion were not

investigated but likely candidates are Na^-dependent HCO^/Cl

113exchange and/or inwardly directed Na -HCO^ cotransport.

In cultured VSMC of aortic origin, DIDS has no inhibitory action

on the recovery from an imposed acid load (Vigne et al. 1988;

Kortmacher et al 1988), although in the AlO cell line omission of

bicarbonate or addition of DIDS lowered pHi. In addition, in the

presence of EIPA pHi recovery from an acid load was observed

(Figure 32) in agreement with a previous report (Aalkjaer and

Cragoe, 1988). Recent results from cultured femoral artery

myocytes demonstrated that Na*/H* exchange was the major acid

extrusion system at a low pHi, whereas at physiological pHi,

DIDS-sensitive acid extrusion predominated (Kahn et al. 1990). In

all of the studies I performed amiloride or EIPA caused resting

pHi to fall suggesting that Na*/H* is involved in the maintenance

of steady state pHi. However in another study no evidence of

Na^/H^ exchange activity was observed in resting mesenteric

artery segments from adult (13-20 weeks) WKY rats (Aalkjaer and

Cragoe 1988).

A Cl /HCO^ exchanger has been detected in all smooth muscle

cells investigated so far (Aickin 1989). In mesenteric arteries,

removal of external chloride (substituted with iodide and

gluconate) caused a pHi rise of 0.2 in the absence of external

sodium and the pHi rise could be inhibited with DIDS. This

finding suggests a Cl /HCO^ exchange working in reverse mode. No

pHi rise has been observed in mesenteric vessels following

removal of external chloride in the nominal absence of

bicarbonate (Aalkjaer, personal communication). In mesangial

cells (Boyarsky et al. 1988) and in guinea pig ureter (Aickin

114

1989) this system enhances pHi recovery from an imposed alkaline

load. However, evidence that Cl /HCO^ exchange is an alkaline

pHi regulator in vascular smooth muscle is lacking. Korbmacher

(1988) found that inhibition of Cl /HCO^ with DIDS had little

effect on pHi back regulation after a sodium acetate pre-pulse. A

similar finding has been observed in rat resistance vessels

(Aalkjaer, personal communication).

In addition, the metabolic status of the cell appears to affect

pHi. In cultured aortic VSMC pretreatment with 2-deoxyglucose

inhibited pHi recovery from an acid load perhaps indicating that

a phosphorylation of the antiport is essential in maintaining its

internal pH sensitivity (Weissberg et al. 1987). On the other

hand, 2-deoxyglucose caused a considerable acid load without any

evidence of an inhibitory action on acid extrusion systems in the

ureter (Aickin 1989).

Since intracellular buffering is the first line of defence

against acid or base disturbances a large buffering power is

advantageous to pHi homeostasis. From the ammonium chloride

induced cell alkalinisation an apparent total buffering power of

around 40 mmol/1 x pH was observed in mesenteric arteries from

the SHR and WKY. This value is in good agreement with a previous

estimate in resistance artery (Aalkjaer and Cragoe, I988) and in

the range of 10-100 mmol/lxpH as generally reported (Szatkowski

and Thomas, 1989). Nevertheless data obtained from ventricular

muscle show that the pHi transients induced by the addition and

removal of ammonium chloride are much smaller in intact tissue

compared with the isolated myocyte (Bountra et al. 1990) which

115may be due to diffusion delays in the complex extracellular space

of multicellular tissue. This could equally apply to intact

resistance arteries which would result in a considerable over­

estimation of the intracellular buffering power. Acid/base

transport across the cell membrane will also affect the

calculated apparent buffering power profoundly (see Roos and

Boron, 1981). It is interesting to note that Aickin (1988) has

failed to demonstrate a significant increase in buffering power

in the guinea pig ureter in the presence of a CO^/HCO^ buffer

compared to a Hepes buffered medium. A similar finding has been

observed in resistance arteries (Aalkjaer and Cragoe, 1988).

Since the cell membranes of both preparations are clearly

permeable to CO^ this finding is difficult to interpret. Even in

the absence of carbonic anhydrase a functional increase in

buffering should be observed if CO^ is given time to equilibrate

(see Aickin, 1988).

In cultured myocytes the observations that vasoconstrictor agents

induced cell alkalinization (Owens et al. 1987; Hatori et al.

1987; Berk et al. 1987) and that inhibition of Na^/H^ exchange

causes vasodilation (Haddy et al. 1985) has led to the hypothesis

that Na*/H* exchange mediates cell alkalinisation which may play

a role in sustained smooth muscle contraction (Berk et al. 1987).

Nevertheless my studies using intact arteries have not confirmed

this. In all experiements (except those on 12 week WKY animals),

noradrenaline-induced contraction was associated with a

significant acidification. Why are these findings contrary to

those observed in cultured myocytes is not certain but there are

several possible explanations for this discrepancy. First,

116concerning the action of growth factors on pHi,(see below) the

majority, but not all, such studies, (Vigne et al. 1988), have

been performed in the nominal absence of bicarbonate. However,

Aalkjaer and Mulvany (1988) observed a sustained acidification on

stimulation with noradrenaline and angiotensin II in bicarbonate

-free buffer. Second, vascular smooth muscle cells in culture

medium become less contractile and more proliferative, a process

termed 'phenotype modulation' (Chamley-Campbell et al. 1981).

Thus it is questionable whether vasoconstrictor agonists elicit a

contractile response in cultured myocytes. In intact perfused hog

coronary artery measurements of pHi using nuclear magnetic

resonance (NMR) recorded a small but significant acidification

during stimulation with noradrenaline which increased the

perfusion pressure, (Grieder et al.1988). Furthermore, the

vasodilator action of amiloride and its analogues may not be due

solely to inhibition of Na*/H* exchange. We have demonstrated

that the amiloride analogue EIPA fully relaxes a potassium

induced contraction in the absence of external sodium where Na^/H

exchange will be inactive (Izzard and Heagerty, 1990).

The observation that acidosis occurs during stimulation with

noradrenaline is also in contrast to the findings in platelets

where thrombin stimulation induces an acid load due to activation

of proton producing reactions but stimulation of Na*/H* exchange

more than compensates for this and an alkalinisation is observed

(Zavoico et al. 1987). Contraction in resistance vessels may

result in an increase in anaerobic glycolysis in order to

maintain adequate levels of ATP leading to an increase in lactic

acid production. Contrary to what may be expected, production of

117lactic acid per se would not acid load the myocytes; the

conversion of glucose to lactic acid neither produces nor

consumes protons, rather it is the subsequent hydrolysis of the

synthesised ATP which is the proton-producing reaction (Gevers,

1977; Busa and Nuccitelli, 1984). On the other hand aerobic

metabolism is neither a proton-producing nor consuming process in

steady state conditions where the ATP/ADP ratio is constant. In

non-steady state conditions this is not the case, a declining

ATP/ADP ratio as a result of a faster hydrolysis of ATP than

synthesis will impose a threat of acidosis on pHi homeostasis,

(Busa and Nuccitelli, 1984).

Changes in the levels of cytosolic free calcium during

contraction may have a direct effect on pHi due to competition

with protons for common buffer sites as reported in cardiac

purkinje fibres (Bers and Ellis, 1982; Vaughan-Jones et al.

1983). Activation of the plasma membrane Ca^^ATPase pump when

cytosolic free Ca^* levels rise will occur via association of

these ions with calmodulin. It is believed that this pump retains

electroneutrality via the exchange of one Ca^* ion for two

external protons. This Ca^*/2H* exchange has been implicated in

the transient acidification observed when cultured VSMC are

stimulated with All (Hatori et al. 1987; Berk et al. 1987a).

Depolarisation in 125 mmol/1 K-PSS changes the membrane potential

to - 6.9 mV in resistance arteries (Mulvany et al. 1982) and

thus the inwardly directed proton electrochemical gradient is

abolished. This means that the acidification observed must be due

to changes in proton metabolism possibly as a consequence of the

118elevation in cytosolic [Ca^*]i. The K-PSS used in these

experiments only contained 25 mmol/1 Na/,and this reduction in

external sodium may also inhibit active acid extrusion.

Since the noradrenaline induced contraction is associated with a

fall in pHi, it is not possible to discern whether resetting of

the Na^/H^ exchanger has occurred, as observed in cultured VSMC,

due to phosphorylation of the antiporter modifer site possibly

via protein kinase C activation. Interestingly, although

noradrenaline does stimulate the hydrolysis of PIP2, in

resistance arteries, this agonist does not cause an increase in

total diacylglycerol possibly due to its rapid phosphorylation to

phosphatidic acid (Ohanian et al. 1990).

In the SHR compared with the WKY, the changes in intracellular pH

induced by high potassium buffer and noradrenaline differed at

5 weeks and 12 weeks of age respectively. Also a small but

significant reduction in pHi change was observed in the presence

of noradrenaline in vessels from essential hypertensives.

Nevertheless in all these instances the contractile response when

expressed as media stress did not differ. (The contractile

response in terms of media stress only differed in two instances.

A reduction in media stress to K-PSS stimulation was observed in

the SHR at 12 weeks possibly due to inadequate washout of

nigericin, later it was found that thorough rinsing of the organ

bath with 50% ethanol overcame this problem. An enhanced media

stress was observed in the mesenteric arteries from rats with

coarctation hypertension at 3 days). Thus the differences in the

intracellular pH changes mentioned above cannot be implicated in

119

the hypertensive processes via effects on vascular tone possibly

because these pHi change differences were not of a sufficient

magnitude to exert a significant effect on the contractile

response or other factors may compensate for an 'abnormal' pHi

change during contraction in vessels from genetically

hypertensive prone rats and in human essential hypertension.

Studies on isolated resistance vessels from hypertensive rats and

man have failed to demonstrate any increase in sensitivity to

vasoconstrictor agonists during the development and established

phase of hypertension (Mulvany, 1987; Heagerty et al. 1988). This

suggests that the vascular hyper-reactivity, observed in

hypertension, in the absence of an altered neuro-humoral

environment, is a consequence of an altered vascular structure

rather than an intrinsic abnormality of the VSMC. Therefore it is

unlikely that abnormalities in resting pHi or pHi change during

contraction play a part in the development or maintenance of

hypertension by increasing myocyte responsiveness to contractile

stimuli (Aalkjaer, 1990).

It is widely held that VSMC tone is sensitive to perturbations in

pHi and that an intracellular alkalosis enhances force

development (Mahnensmith and Aaronson, 1985; Berk et al. 1987)

yet evidence for the latter is very weak. Certainly there is

evidence that an acidosis may stimulate the sarcoplasmic

reticulum calcium pump (Grover and Samson, 1986) and inhibit

calcium influx (Irisawa and Sato, 1986), effects which one would

expect to reduce cell calcium and thus reduce vascular tone.

Furthermore evidence from cultured VSMC suggests that cytoplasmic

alkalinisation mobilises calcium from internal stores (Siskind et

120

al. 1989). However, in these studies no change in resting tension

on addition of ammonium chloride was observed, indeed in

resistance arteries from five week SHR and WKY controls, cell

acidification on washout of ammonium chloride caused a modest

transient contraction. Likewise, Aalkjaer and Cragoe (I988)

observed contraction in resistance vessels when pHi regulation

was inhibited with EIPA and DIDS after an ammonium chloride

prepulse. In addition, an acute alkalinisation resulted in a

transient dilation of mesenteric vessels pre-constricted with

noradrenaline and acute acidosis caused a transient enhancement,

these changes being brought about by ammonium chloride and carbon

dioxide prepulse methods (Aalkjaer and Mulvany, 1988a). This

enhancement of tone observed during an acidosis may be due to

competition between protons and calcium ions for common buffer

sites as occurs in heart purkinje fibres (Vaughan-Jones et al.

1983). In the purkinje fibre however, although an acidosis

elevates cytoplasmic calcium levels, a depressive effect on

contraction still occurs possibly due to an inhibitive effect on

the contractile apparatus. Perhaps an enhancement of tone is

still observed in the resistance vessels because the contractile

machinery is not inhibited by a low pHi as observed by Gardner

and Diecke (1988). To date however, cytosolic free calcium levels

and contractions have not been reported in intact arteries during

perturbations in pHi to my knowledge, thus further studies are

required to understand the mechanisms whereby an acute acidosis

enhances vascular tone, which may not necessarily be mediated by

changes in cytosolic free calcium.

The relationship between intracellular pH and cell growth has

121

been much discussed (see Grinstein et al. 1989). More recent data

support the finding of Cassai et al (1985) that in the presence

of a CO^/HCO^ buffer system, which is clearly more

physiologically relevant than a bicarbonate free system, growth

factors do not elicit a pHi increase. In fibroblasts, the Na*-

dependent HCO^ /Cl exchanger sets pHi to a level which is beyond

the operating range of Na*/H* exchange even in the presence of

growth factor (Moolenaar et al. 1988). Mutant fibroblasts devoid

of Na^/H^ exchange activity can grow in the presence of

bicarbonate (L'Allemain et al. 1985). The pHi response to growth

factors in mesangial cells is a small acidification, although

alkalinisation occurs in the absence of bicarbonate. This occurs

because Cl /HCO^ activation outweighs that of the Na^/H^

exchanger and Na^-dependent HCO^/Cl exchange (Ganz et al. 1989).

From these studies it has been concluded that growth factors

increase pHi regulation in anticipation of an increase in

metabolic acid production on cell activation (Moolenaar et al.

1988; Ganz et al. 1989; Thomas, 1989).

Although the above demonstrate that cell alkalinisation is not a

necessary physiological response to growth factor stimulation, it

does not prove that cell pHi is essentially invariable and not

modulated by the Na*/H* exchanger. Stimulation of cell

differentiation with retinoic acid results in a pHi increase due

to raising of the set point of Na*/H* exchange in a leukaemic

cell line, whilst bicarbonate transport does not alter (Ladoux et

al. 1988). Spread cells have a higher pHi than round cells due to

increased Na*/H* exchange activity. Bicarbonate influx and efflux

systems are also increased but to the same extent at resting pHi

122

levels (Schwartz et al. 1990). In addition platelet stimulation

with thrombin elicits a pHi rise, bicarbonate dependent pH

regulators appear to be absent in this cell type (Sage et al.

1990).

Intracellular pH was more alkaline in resistance vessels from the

SHR at 5 weeks due most likely to increased Na*/H* exchange

compared with WKY animals. However, the studies from rats with

aortic coarctation suggest that a pHi increase is not an absolute

requirement for vascular growth. This finding along with the

observation that cultured smooth muscle cells from the SHR are

also more alkaline compared with WKY as a result of increased

Na*/H* exchange (Berk et al. 1989), suggest that this is a

genetic difference. Since enzymes critically involved in cell

growth have an alkaline pH optimum, the myocytes from the SHR may

respond more avidly to appropriate growth stimuli. Cultured cells

from the SHR display an enhanced growth rate compared with the

WKY (Berk et al. 1989). Thus the elevation in pHi in the SHR may

facilitate resistance vessel hypertrophy leading to an increase

in peripheral vascular resistance. On the other hand the pH

differences may be a genetic difference between the SHR and WKY

unrelated to blood pressure (Ives, 1989). Indeed, significant

differences in amiloride sensitive sodium influx (Kuriyama et al.

1988) and sensitivity to exogenous calcium (Mulvany and

Korsgaard, 1983) have been observed in cultured VSMC and

resistance vessels respectively between the SHR and WKY; however

in both reports no difference was observed between the SHR and

the normotensive Wistar rat. Clearly, further work is required to

clarify this issue.

123

In coarctation hypertension and most importantly essential

hypertension, there was no significant increase in resistance

artery pHi. In the coarctation model, the application of DIDS and

EIPA caused significantly different changes in pHi compared with

controls at 9 and 28 days respectively after hypertension was

induced. This finding implies that the activity of acid/base

transport systems may alter to maintain pHi near normal possibly

negating the effects of changes in cellular metabolism on

intracellular acid production. Thus Na*/H* exchange activity is

increased in this model of hypertension but at a time when

significant changes in vascular structure had already occurred

arguing against a causal role for this antiporter.

On the other hand, in resistance arteries from hypertensive

patients both EIPA and DIDS caused changes in pHi which did not

differ from the normotensive controls suggesting that pHi

homeostasis is normal. In addition, this negative finding was

also observed in first degree offspring of essential hypertensive

patients which argues against the possibility that disturbances

in pHi homeostasis play a role in initiating the hypertensive

process. It is important to realise that these studies do not

address the question of whether the kinetics of the Na*/H*

exchanger are altered in resistance vessels in essential

hypertension; but if it were, it clearly does not lead to an

increased intracellular pH, nor is Na*/H* exchange overactive in

resting resistance arteries. Furthermore, treatment of cultured

VSMC with glucocorticoids increases the Vmax of the exchanger

although pHi is not increased, yet glucocorticoids have an

inhibitory rather than stimulatory effect on the growth of VSMCs

124

held in culture (Berk et al. 1988).

Enhanced erythrocyte Na^/Li^ countertransport has been proposed

to be a marker for a global abnormality in Na^/H^ exchange.

However, although erythrocytes do possess an amiloride sensitive

Na^/H^ exchange system (Escobales and Canessa, 1982) data have

been presented which indicate that erythrocyte Na*/H* exchange

and Na^/Li^ countertransport are not indicated by the same

transport system (Kahn, 1987). Indeed the increased Na^/Li*

countertransport activity observed in patients with essential

hypertension has been shown to be amiloride insensitive (Carr et

al. 1988). Nevertheless a global physicochemical disturbance in

the cell membranes of patients with essential hypertension might

increase the activity of both these systems (Kahn, I987).

The data presented here demonstrate that increased Na*/H*

exchange and cell alkalinisation is not observed in resistance

vessels of patients with essential hypertension or relatives,

thus the pathological significance of the finding in erythrocytes

from patients with essential hypertension, if any, has yet to be

determined.

Finally, with regard to vascular structure, increases in media

thickness and media/lumen ratio are not conclusive evidence of

myocyte growth or proliferation, a media volume increase is the

relevant parameter in this regard. In the SHR, media volume was

not significantly increased compared with the WKY, in this study.

Nevertheless, I am unsure whether this is a true negative finding

for the following reasons. First, as already discussed, older

125rats may be required to detect a significant media volume

increase. Second, although all resistance arteries studied were

second order branches, no effort was made to dissect them from

the same anatomical site in each rat strain which could influence

the morphological findings due to investigator bias, i.e.

vessels from each strain will be dissected which appear 'the

right size' for mounting on the myograph. The importance of

dissecting vessels from the same site in each strain has been

pointed out previously (Mulvany et al. 1978). Thirdly, previous

studies using the same techniques have demonstrated media volume

increases in mesenteric arteries (Mulvany et al. 1978) due to

cell hyperplasia (Mulvany et al., 1985), media volume is also

significantly increased in the femoral artery of the SHR (Bund

and Heagerty, 1989) and in renal arteries when examined after a

perfused fixation technique (Smeda et al. 1988). In the aortic

coarctation study, greater care was taken to select vessels from

the same site and a clear media volume increase is observed.

Nevertheless the precise nature of the morphological changes with

regards to media volume changes may differ in these two models of

hypertension. For the reasons presented above, the non­

significant increase in media volume observed in this study in

subcutaneous resistance vessels in essential hypertension and in

a previous study (Aalkjaer et al. 1987) cannot be taken as

evidence for or against vascular growth in the resistance

vasculature. As in the heart, hypertrophy undoubtedly occurs in

the larger arteries (Barrett, 1963; Furuyama, 1962). In

intestinal autopsy material in chronic hypertension, an increased

media/lumen ratio was observed yet media cross sectional area was

not increased (Short, 1966). More information is needed to assess

126

the contribution of vascular growth to the increased peripheral

resistance in human essential hypertension.

127

7.6

7.4

7.2

pHi 7.0

6.8

6.6

6.4G 5 10 15 20 25

time(mins)

Figure 32 Representative trace of pHi from a resistanceartery pre-pulsed with ammonium chloride. Washout solution contained 60 umol/1 EIPA.

128

CONCLUSION

Intracellular pH is a fundamental parameter which can potentially

influence a myriad of cell processes. On the basis of

observations in circulating blood cells, aberrations in pHi

control due to overactive Na*/H* exchange, for example, have been

implicated in the genesis and maintenance of hypertension via

effects on resistance vessel tone and growth. It is possible that

factors which induce vascular hypertrophy - already defined,

perhaps pressure load itself, induce cytoplasmic alkalinisation.

These hypotheses have been examined using the pH sensitive

fluorescent dye BCECF to measure intracellular pH and contraction

simultaneously in arterial segments. Using the myograph, arteries

with an internal diameter under 300 um have been studied which

are of a size that contribute significantly to the increased

peripheral resistance in established hypertension. Also it is

important to stress that all experiments were performed in the

presence of bicarbonate in physiological concentrations (Thomas,

1989). Overall my conclusion must be that the relationship

between pHi and an intimate role in the genesis of hypertension

must be weak.

Vascular contractions elicited with the agonist noradrenaline

binding to its receptor or depolarisation in K-PSS, generally

induced an acidosis and certainly no significant alkalinisation

above resting pHi levels. This finding negates the hypothesis

that cell alkalinisation is a mediator of the tonic contractile

response. However this result does not mean that Na^/H^ exchange

is not necessary for agonist-induced sustained contraction. The

finding that the potent Na^/H^ exchange inhibitor EIPA has

129

vasodilator activity independent of Na*/H* exchange blockade

makes assessment of this antiport in resistance vessel

contraction very difficult. In the literature authors have

referred to VSMC alkalinisation causing enhanced blood vessel

tone. These studies have not addressed this methodically, but it

is worth noting that alkalinisation due to the addition of

ammonium chloride had no effect on resting tension. Furthermore,

recent evidence suggests that VSMC alkalinisation results in

vasodilation; all such studies are prone to error if the method

used to alter pHi has an effect on blood vessel tone independent

of the pHi change.

At five weeks of age, resistance arteries from the SHR were

significantly more alkaline than age matched WKY arteries which

could be relevant to the process generating hypertension. In

experimental hypertension no such pHi increase was observed

suggesting that the pressure load per se does not mediate this

rise in pHi. Nevertheless, it is possible that an increase in

blood pressure causes a different response in resistance vessels

of young rats. In this regard histological analysis of mesenteric

arteries suggest that Goldblatt-l-kidney 1-clip hypertension

induces myocyte hypertrophy as opposed to hyperplasia in the same

arteries in the SHR (Korsgaard and Mulvany, 1988). However, the

finding that intracellular pH is increased in cultured VSMC from

the SHR compared with the WKY (Berk et al. 1989) is indicative of

a genetic difference rather than a consequence of an altered

environment.

Curiously VSMC from stroke prone SHR have reportedly increased

130

Na*/H* exchange activity but resting pHi is decreased compared

with the WKY (Kobsayashi et al. 1990). But both these cultured

cell studies were unfortunately performed in the absence of

bicarbonate.

Therefore there is evidence that pHi is abnormal in VSMC in the

SHR compared with the WKY, although a causal role for this

abnormality in this model of hypertension is an attractive but as

yet untested hypothesis.

In these studies pHi has been measured for the first time in

human resistance arteries from essential hypertensive patients,

first degree offspring of essential hypertensive patients and

matched controls. The findings were negative. Intracellular pH in

the resistance vessels of hypertensive patients and relatives was

unchanged from normotensive controls. Blockade of Na^/H^ exchange

and bicarbonate dependent regulators caused changes in pHi which

were the same in each group. The only difference observed was a

slight reduction in the acid change in pHi in the arteries from

the hypertensive group on stimulation with noradrenaline. This

was not observed in resistance arteries from the relatives and

therefore may be secondary to changes in cellular metabolism

during noradrenaline contraction as a result of hypertension.

This is possible because sensitivity to exogenous calcium in the

presence of noradrenaline is reduced in resistance arteries from

hypertensives (Aalkjaer et al. 1987) but not in the resistance

arteries from relatives (Aalkaer et al. 1987a).

It is widely believed that changes in the vasculature due to VSMC

131hypertrophy account for the structural changes which are so

important in maintaining high blood pressure (see Owens, 1989).

Only recently has the sparse data concerning resistance vessel

structure in human essential hypertension been re-examined and it

must be concluded that in arteries of a calibre to contribute

significantly to the total peripheral resistance there is little

evidence of a growth response (Aalkjaer, 1990)

It is important to realise that it is the increased media/lumen

ratio that affords the structural advantage which permits

resistance vessels to regulate local blood flow in the presence

of a raised arterial pressure. Another consequence of this

structural change is vascular hyper-reactivity to pressor

stimuli. In the absence of medial hypertrophy, an increased

media/lumen ratio would arise due to a re-orientation of the pre­

existing myocytes around a smaller lumen, a morphological change

somewhat similar to a tonic contraction. Although lumen diameter

was not significantly reduced, a 'non-significant' change can

have a profound effect on the media/lumen ratio (Folkow, 1986).

Perhaps a clearer understanding of the structural changes in the

resistance vasculature should be the aim of future hypertension

research.

132

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