SELECTIVE INHIBITION OF HEME OXYGENASE BY

iSIIETALLOP0RPHYRINS

b?,

SCOTT DAVID APPLETON

A thesis submitted to the Department of Pharmacology and

Toxicology in conformity with the requirements for

the degree of blaster of Science

Queen's University

Kingston, Ontario, Canada

September 1999

Copyright O Scott David Appletoo

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Abstract

Studies on the physiological role of heme oxygenase (HO) require an inhibitor

that will selectively inhibit HO activity without inhibiting any other function. particular1 y

without affecting the activity of either nitric oxide synthase (NOS) or soluble guanylyl

have previously been shown to inhibit HO activity, for their ability to inhibit HO without

inhibitins NOS or sGC activities. Measurement o i activity of HO in rat brain

microsomes and NOS in rat brain cytosol was conducted in samples incubatcd with

metalloporphyrins (0.15 to 50 M ) including zinc protoporphyrin I?<. ~ i n c

dcuteroporphyrin I?< ?,-I-bis ethylene glycol (ZnBG), chromium mrsoporphyrin IS

(CrMP). tin protoporphyin I?( and zinc N-methylprotoporphyrin IS. CrMP and ZnBG

were found to be the most selective inhibitors of HO activity; i.e. caused the greatest

inhibition of HO activity. 89 and 80%. respectively. without inhibition of NOS activity.

Based on these results, sGC activity in rat lung cytosol was determined in the presence of

CrMP or ZnBG (0.15 to 15 pM). ZnBG did not affect basal sGC activity. bur it did

potenriate S-nitroso-N-acetylpenicillamine (SNAP)-induced sGC activity. CrMP did not

affect either basal or SNAP-induced activity. It was concluded that of the five

metalloporphyrins studied. CrMP. at a concentration of 5 pM, was a selective inhibitor of

HO activity and was the most useful metalloporphynn For the conditions tested. Thus.

CrMP appears to be a valuable chemical probe in elucidating the physiological role of

HO.

Acknowledgements

There are several people that I would like to acknowledge for their contributions

towards this thesis. Although different in nature, all were greatly appreciated. To Dr.

Gerald S. Marks, I would like to thank him for his entertaining stories and helpful insight

Into the field of carbon monoxide research. To Dr. Kanji Nakatsu. for his attempts to

show me how to write and his careful ~uidance and concern for my future. I could not

have asked for better supervisors. I am also fortunate enough to have been blessed with

such wonderful parents and grandparents. who have selflessly sacrificed many things in

order for me to further my education. To Marc Chretien. for his work with the NOS

assays and the rest of the Nakatsu lab for providing me with some entertainment whilr 1

was writins this thesis. To Dr. Donald t-l. Maurice. for making the soluble yuany1yI

cyclase assay "child's play". To Dr. James F. Brien. for his guidance and his ability to

insight the "bloody obvious test". To Brian E. McLaughlin. for persevering through my

inane questions and his continual assistance throughout my project including teaching rnc

the HO assay and the value of proper controls. To the Adam's lab. for providing me with

a computer to work on and endless entertainment while writing my paper. Finally. I

would also like to thank the rest of the faculty and students in the Department of

Pharmacology for their friendship and support.

To papa, you will be "still kicking" in our hearts and our thoughts.

Table of Contents

Page

ABSTRACT ....................................................................................... I I

... ACKNOWLEDGEMENTS ..................................................................... 111

TABLE OF COPU'TENTS ........................................................................

LIST OF TABLES ................................................................................

.......................................................................... LIST OF FIGURES

................................................................ LIST OF ABBREVIATIONS

Chapter One GENERAL lNTRODUCTION ....................................................... A . Heme osygcnase ...................................................................

............................................ B . Suggested physiological roles of CO 1 . Suggested molecular mechanisms of action of CO ...................

.......................... 2 . Role of CO as a neuromodul;ttor in the CNS ........................... . 3 Role of CO as a modulator of vascular tone

...................................... 4 . Role of CO in other organ systems ..................................................................... C . Inhibition of HO

................................................. D . Statement of aims and objectives

Chapter Two SELECTIVE INHIBITION OF HEME OXYGENASE BY

............................................................ METALLOPORPHYRINS .......................................................................... A . Introduction

....................................... ................. B . Materials and Methods .. 26 ........................................................ 1 . Drugs and solutions 26

7 . Preparation of subcellular fractions of rat brain and lung ............ 27 3 . Measurement of HO enzymatic activity in microsomal fraction of rat

...................................................................... brain 29 4 . Measurement of NOS enzymatic activity in cytosolic fraction of rat

.......................................................................... brain 31 5 . Measurement of sGC enzymatic activity in cytosolic fraction of rat

.......................................................................... lung 32 ................................................................ . 6 Data Analysis 34

.............................................................. C . Results and Discussion 3 5

Chapter Three .............................................................. FUTURE DIRECTIONS 47

Page

........................................................................................ References 5 1

Vita ................................................................................................ 59

List of Tables

Page I . Comparison of rnetalloporphynn inhibition of HO.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... 40

List of Figures

Pagc Figure 1 .1 The heme degradation pathway .................................................... 4

Figure 1.2 The endogenous production of NO ................................................ 7

Figure 1.3 Activation of sGC by NO and CO ................................................. 8

Figure 1.1 Chemical structureofYC-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1

Figure 1 . 5 Chemical structure of metalloporphyrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Figure 2.1 Inhibition of HO and NOS activity by CrMP and SnPP ........................ 36

Figure 2.2 Inhibition of HO and NOS activity by ZnBG and ZnPP ........................ 37

Figure 1.3 Inhibition of HO and NOS activity by ZnMePP ................................ ?S

Figure 1.4 Effects of ZnBG and CrMP on basal sGC activity .............................. 1 2

. . . . . . . . . . . . . . . . . . . Figure 2.5 Effects of ZnBG and CrMP on SNAP-induced sGC activiry 43

viii

List of Abbreviations

ABT

BSA

de rlovo

EDTA

e.g.

c2t id.

ET- 1

GTP

'H

HO

HO- 1

HO-2

HO-3

hr

irl viiro

1 -aminobenzotriazole

bovine serum albumin

carbon- 1 3

charybdotoxin

carbon monoxide

chromium mesoporphyrin IN

of new

ethylenediaminetetraacetic acid

exernpli gratia (for example)

st alia (and others)

endothelin- 1

guanosine 5'-triphosphate

tritium

heme oxygenase

heme oxygmase- 1

heme oxygenase-2

heme oxygenase-3

hour@)

id est (that is)

in glass

inside the living body

calcium-activated K channels

L-NAME

LPS

LTP

rn

M

min

MS I concentration

NANC

NMDA

NO

NOS

ODQ

PBS

sGC

S bIC

SNAP

SnMP

SnPP

TEA

VIZ.

w/v

Zd3G

ZnPP

il""-nitro-L-arginine methyl ester

lipopoly saccharide

long-term potentiat ion

milli

molar

minute(<\

maximum selective inhibitory concentration

non-adrenrrgic. non-cho lincrgic

N-methyl-D-aspartate

nitric oxide

nitric oxide synthasr

1 H-[l.2.4]-oxadiazolo[- 4.3 -u]qui nosa

phosphate buffered saline

soluble guanylyl cyclase

smooth muscle cell

S-ni troso-iV-acety lpenicillamine

tin mesoporphynn iX

tin protoporphynn IX

tetraethylammonium

narnel y

weight per volume

zinc deuteroporphyrin IX 2,4-bis ethylene glycol

zinc protoporphyrin IX

zinc N-methylprotoporphyrin IX

degrees Celcius

plus or minus

micro

Chapter One

GENERAL INTRODUCTION

In 199 1 , Dr. Gerald S. Marks and colleagues introduced to the scientific

cornmunitv the notion that carbon monoxide (CO) may possess a phvsiological role.

These investigators drew comparisons between carbon monoxide and another

endogenous. gaseous molecule that had been recently shown to have a physiological

etfect. namely nitric oxide (NO) (Furchgott and Jothianandan. 199 1 ) Marks L'I d

( 199 1 ) highlighted the similarity between the structures of NO and CO, but noted that CO

has geater chemical stability than NO. The presence of an unpaired electron in YO

compared with the absence of unpaired electron in CO underlies the relative stability of

C'O and its greater half-life relative to NO Since NO has recently been demonstrated as

a primary messenger. it then follows that CO may also act as a primary messenger as

well. The following criteria (modified from Goodman and Gilman (9"' edition)) must be

satistied lor a substance to be considered as a primary messen9t.r particularly as thrv

apply to CO.

1 Exogenous application of the pirimary messenger must mimic its proposed

biological eflect. Liu el trl (1992) mimicked CO-induced relasation in rabbit

aortic strips precontracted with phenylephrine by bubbling CO into the tissue bath

containing the tissue.

2 . .A mechanism for the termination of action of the primary messenger must esist

Beins a gaseous molecule. the main mechanism for its termination of action

locally is ditksion away from the target area. CO is eliminated from the body

through exhalation as demonstrated by several investigators including Horvath rt

'11. ( 1998) who measured and used exhaled CO as an index of HO activity i l l w o .

3 . Drugs that decrease the endogenous production of the primary messenger should

also decrease the biological effect it, \ivo. Several studies have used this criterion

to demonstrate a physiological role For CO, for example Zakhaq L.I d. (1996)

demcnstrated !hat CO played a role as a vasodilator using competitive inhibitors

for HO. \iz. metalloporphyrins, despite issues concerning their selectivity

-I There must be an endogenous source for the primary messenger and. as hlarks C I

t r l pointed out. heme ovyyenase (HO) produces CO Heme biodegradation is

catalvzed by HO and produces CO as one of its products

HO is a widespread enzyme. which leads to funher speculation as to the putatix

physiological roles of HO and its products. Imrnunohistochrmical studies have

demonstrated that HO is found in the endothelial and the adjacent medial laver of certain

blood vessels (hlarks c.1 trl.. 1997) The localization of HO in both endothelium and

smooth muscle lavers of blood vessels has been shown by Johnson c~ r r l ( 1999) to

provide a functional role in times of endothelial dyshnction where any physiological

effect of NO on vascular tone is removed. This conclusion is based on studies in which

HO-derived CO determined the vascular tone in vessels where the endothelial NOS was

blocked or removed by denuding the vessel of rndothelium. Thus, simulating endothelial

dysfunction, either by removing the endothelium or blocking one of the major

vasodilating factors (EDRF) involved in the equilibrium between depressors and

constrictors, allowed HO-derived CO from the smooth muscle layer to become the major

mediator of vasodilation.

.A. Heme oxygenase

There are several mechanisms by which carbon monoxide (CO) is produced in the

body Minor sources of CO include auto-oxidation of phenols, tlavonoids. and

halomethanes. photo-oxidation of organic compounds; and peroxidation of membrane

!iplds (Iluines. 1084. \Inines. ! Q Q 7 ) The major w u c e r ~ f CO i s heme degradation bv

heme ovygenase (HO) to form CO as one of the products The HO system. in general. is

responsible for the catabolism of heme in the presence of NADPH and 0: ro form

biliverdin I?(. iron (Fe), and carbon monoxide (CO) (Figure I 1 ) The products of this

system are thought to be biologically active Bilirubin. subsequently formed from

biliverdin by biliverdin reductase. is an antioxidant. Fe has been shown to regulate the

expression of certain genes. including that of nitric oxide svnthase (NOS) and ferritin.

and it has also been ~mplicated along with an excess of intracellular heme ( a pro-oxidant)

in endothelial cell damage (hlaines. 1997. Juckett d.. 1998) Fcrritin binds and stores

Fe in a form less apt to contribute to oxidative injurv in endothelial cells and in

coordination with HO provides protection and constitutes an adaptive response to excess

heme (Juckett rr ul., 19%). And finally, it has been proposed that the endogmously

formed carbon monoxide (CO) pliiys a role in the reylation of cell hnction and

communication (Marks rt d.. 199 I , Verma rf d.. 1993).

The enzyme responsible for heme degradation is HO. which is a

ubiquitous enzyme for which there are two well-identified isoforms: heme oxygenase- 1

(HO- I ) and heme oxvgenase-2 (HO-2). HO- I is an oxidative stress-inducible protein.

ADPH BILIVER DIN-IX

HEME OXYGENASE

C (P450) REDUCTASE

Figure I . I : The heme degradation pathway. HO degrades heme in the presence of ovvaen d - and NADPH to form iron (Fe). biliverdin IX. and CO HO requires NADPH- cytochrome c(P450) reductase as a cotactor.

which has also been referred to as heat shock protein 32 (HSP 3 2 ) , localized in the

endoplasrnic reticulum. I t has a wide distribution in the vasculature, but it is highlv

expressed in liver and spleen. HO- 1 expression is upregulated upon exposure to heme as

well as heavy metals ( ~ d " . CO' + ), trivalent arsenicals. heat shock. ischemia. GSH-

depletion, radiation. hypoxia, hyperouia. endotoxin. intlammatory cytokines.

prost3ylandins (PG.4:). hormones. and other cellular transformations and disease states

(hlainrs. 1097) HO- 1 is responsible for the marked increase in CO svnthrsis during

pathological conditions and based on its wide variety of inducers. it has been proposed

that HO- 1 may a play role in maintaining cellular llorneostasis.

HO-2 is the constitutive isozyrne and is localized in rnicrosornes where it is

responsible for CO production under normal physiological conditions. It is not induced

bv the factors that increase HO-1 espression. but it has been shown that HO-2 is induced

by adrenal glucoconicoids it is widelv distributed in endothelium and neurons. its

greatest expression being in the brain as well as other pans of the nenaus system HO-2

has been shown to have similar distribution patterns with both nitric oxide synthase

(NOS) and soluble guanylyl cyclase (sGC).

Recently. a third isozyrne has been identified. namely heme onygenase-3 (HO-3 )

(McCoubrey rt d.. 1997). It exhibits approximately 90% homology in amino acid

sequence to HO-2. but is the product of a different gene. HO-3 is a hemoprotein unlike

the other two isozymes and has relatively lower heme catalytic abilities. This third

isozyme is located in the spleen. liver. thymus. prostate. heart. kidnev. brain and testis

and its physiological role requires hrther characterization.

B. Suggested Physiologic;rl Roles of Carbon Monoxide

Manv of the proposed phvsiological roles for CO are similar to ones which ha\,e

been previously elucidated for NO. it was not too long ago that endothelial-derived

relaxing factor (EDRF) was introduced to the scientific community as nitric oxide (YO)

This gaseous. chemically labile molecule is produced endogenously by nitric oxide

svnthase (NOS). which converts L-arginine to L-citrulline and NO (Moncada cJr (11 .

199 1 ) (Figure 1 7 ) . The bodv of work published since the discovery of NO as EDRF has

been enormous and continues to grow Elucidation of other phvsiological roles of LO

outside of its hnctional contribution to vascular tone include its role as an antitrophic

factor for vascular structure. a retrograde messenger in long-term potentiation (LTP). an

inhibitor of platelet aggregation, a causative factor in platelet disaggregation. and a

neurotransmitter in the central nervous system (CN S).

Many of the biological roles proposed for NO parallel those of another

endogenously formed gaseous molecule. rrz. C O Both gaseous molecules can mediate a

physiological effect, such as blood vessel relaxation. by activation of soluble guanylyi

cyclase (sGC) (Figure 1.3) (Vedernikov rt d, 1989; Moncada ri tr / . 199 1. Furchgott and

Jothianandan. 199 1 ; Hussain et (11, . 1997). However. CO has n considerably lower

potency than NO as a vasodilator (Furchgott and Jothianandan, 199 1 ) .

It has recently been shown that, in the presence of a compound known as YC-l ,

CO can attain a level of activation of sGC similar to that achieved by N O Based on

oenous these observations, several investigators have postulated the existence of an endo,

YC-l -like substance that would allow CO to attain a great potency irr vico. Furthermore.

NO SYNTHASE s

co 0

H3

Lcitrulline

Figure 1.2: The endogenous production of NO. NOS is a ubiquitous enzyme responsible toi the production of endogenous NO it converts the guanidino group of L-arginine to the urea group in L-citrulline. A s a result of this alteration in the structure of L-aryinine. NO is formed along with L-citrulline.

I GUANYLYL CYCLASE I I m

GTP cG

Figure 1 . 3 Activation of sGC by NO and CO Both NO and CO are able to stimulate sGC via interaction with the heme moiety. which is the predominant mechanism through which these gaseous molecules mediate their physiological effects.

the original state of knowledge concerning CO potency was based on tissue bath studies

where aiiquots of CO saturated solution are added. The amount of CO contained within

each aliquot was determined theoretically and the potency of CO was interpolated from

these theoretical calculc?tions We measured the CO concentration in Krebs' medium

following the addition of CO-saturated solution to tissue baths by a gas chromatograph-

headspace method i t was found that the cnncentrarinn nf CO in I I W I C bath medium at

maslmum tissue relaxation was approximately one-third of that calculated based on

theory Thus, the potency of CO as a vasorelaxant is higher than previouslv beliebed to

be the case

8.1 Suggested klolecular hlechimisms of CO

CO and NO bind to the heme moiety of sGC, which produces an increase in the

conversion of guanosine 5'-triphosphate (GTP) to cvclic guanosine 3 '.Y-monophosphate

(cG\IP) The heme regulatory subunit is essential for activation of sGC by CO as

demonstrated by the inabiiity of CO to activate sGC where the heme moiety was absent

(Brune ei t i / . . 1990). The increase in intracellular cGMP levels begins a cascade of

cellular events that produces many of the physiological erects of CO and KO.

Preventing CO-mediated increases in cGhZP by inhibiting sGC activity blocks many of

the physiological eRects of CO such as vasorelauation. The ferrous heme in sGC

possesses diRerent properties to that of the heme found in hemoglobin in that it has an

extremely low affinity for oxygen binding. The difference in the potency of NO and CO

was thought to be in their relative abilities to break the bond between the heme Fe and a

proximal histidine in sGC When CO binds to the ferrous heme of sGC, it is unable to

cleave the Fe-His bond, but instead assumes a slightly bent conformation NO. however.

can rupture this bond. which is thought to account for its more effective activation of

sGC.

A compound known as YC-1, a benzyl indazole derivative, stimulates sGC at an

allosteric site independent of the heme moiety (Figure I -I) .As mentioned earlier. in the

presence of CO YC-I activates sGC to the wme specitic activitv as attained with T O

YC-I has no effect on the afinitv of CO for the heme of sGC. and also permits the

maximal activation of sGC bv CO it was postulated by Friebe cJr tr l ( 1 gC)b) that YC- I

acts through stabilization of the activated configuration of sGC. based on experimental

observations that YC-1 potentiation was independent of the mode of activation (NO. CO.

or protoporphvrin I?() One criticism of CO is that it is less potent than YO in activatmg

sGC resulting in a less substantial physiological erect However. the presence of an

endogenous Y C- l -like substance may allow more serious consideration to be given to the

biological roles of CO Despite its perceived shortcomings as an activator of sGC. CO

has been implicated in many biological roles

Wang rt trl utilized the rat tail artery as a model for studving the mechanisms

underlying CO-induced relaxation in the belief that this vessel possesses all three of the

cellular targets for CO mentioned below The first target is sGC where CO binds to the

heme moiety of the enzyme. Following sGC stimulation, the subsequent increase in

intracellular cGMP concentration produces several effects in smooth muscle cells

( SMCs). The elevated intracellu!ar cGMP levels inhibit inositol I .4,5-triphosphate (IP;)

formation, inhibit voltage-dependent calcium channels, and activate ~ a ' ' ATPase which

all result in a decrease in intracellular free calcium in SMCs (Lincoln rt a/ . . 1994). Wang

Figure 1.4: Chemical structure of YC- I This benzyl indazole derivative stimulates sGC at a site independent of the heme regulatory unit and is thought to act as an allosteric modulator of sGC acti~ity.

and colleagues determined that cGMP has a partial role in the signal transduction

pathway to induce vasorelasaion of rat tail artery since methylene blue. an inhibitor of

sGC. reduced but did not eliminate CO-induced vasorelaxation (Wang. 1998) CO-

induced elevation of cGMP was hrther supported by the observation that a membrane-

permeable inhibitor of cGW-dependent protein kinase (PKG), Rp-8-Br-cGklPS. also

reduced CO-induced mscrelauation c.f the rat tail artery In the rabbit aortic .;trip IRA<!

CO-induced usorelasation appears to be mediated exclusively by cGhlP since I H-

[ 1 .?.-I]-ouadiazolo[4.3-tr]quino.ralin- I -one (ODQ)(Hussain er tri . 1997). a more specific

inhibitor of sGC (Ganhwaite rr trl . 1995) completely inhibits CO induced relaxation in

this tissue

The plasma membrane of ShlCs have a high input resistance By activating m l y

a small number of K ' channels, hyperpolarization of the membrane could be achieved

This hyperpolarization results in inactivation of voltage-dependent calcium channels.

1.

inhibition of agonist-induced increase in [PI. and a decrease in Cam sensitiwty (Quast.

19%) This modulation of calcium-activated K - channels (Kc.,) by CO would account t'or

the decrease in muscle-relaxing effect of CO when precontractrd with KC1 relative to

relaxation seen with muscle precontracted with vasoactive hormones or neurotransmitters

(Utz and Ltllrich. I99 1 ).

Various vascular contraction studies by Wang (1998) showed that CO-induced

relaxation of rat tail artery was significantly reduced in the presence of

tetraethylammonium (TEA, 30 rnM) At high concentrations, TEA may interfere with

various types of K - channels, with the greatest effects on Kc, channels and delaved

outward rectit'ying K ' channels. Thus, studies using more specific K - channel blockers

would be required to hrther elucidate the roles of the different K* channel subtypes.

Charybdotoxin (ChTX) and apamin are used as selective inhibitors of high conductance

and low conductance Kc, channels, respectively. ChTX inhibited the CO-induced

vasorelasation in a similar manner to that of TEA. while aparnin had no effect Based on

these results. Wang suggests that high conductance Kc., channels are involved in the

mcchanicm for CO-induced vasnrelauari(-rn Finally. these workers aholishrd CO-induced

relaxation by concurrently blockin certain elements of the cGhtP pathway as wll as the

activation of high conductance Kc,, channels in vascular SMCs. From these studies it was

strongly suggested that both sGC and high conductance K ' channels are involved in

vasorelaxation mediated by CO.

Wane - t d ~ K e r hrther evidence to support the idea that CO-induced

vasorelaxation is mediated by the opening of high conductance Kc., channels in addition

to the activation of the cGhlP pathway in L ' S M Despite conflictins results from other

studies to determine the etlkcts of CO on Kc., channels. these investiyators den~onstrated

that CO. when applied cvtracellularly or intracellulariy, increased the open probability of

single high conductance Kc, channels in a concentration-dependent manner (Wang.

1998) This group postulated that Kc., channels may have modulatory sites that arc

sensitive to CO and that increasing CO concentrations would increase the probability that

these sites would be modified. Modification would entail an increased exposure of ca2 ' -

binding sites via conformational changes in the channel protein. it was also shown that

CO acts directly on Kc, channels independent of membrane-associated G proteins (Wang

r i nl.. 1997; White r i trl., 1993). Wang and Wu (1997) suggest that CO "increases the

open probability in a reversible fashion probably as a result of a relatively weak reaction

between CO and the imidazole group of histidine via the ':ormation of a hydrogen bond "

Due to the lack of permeability of the drugs used in this study. hnher experimentation is

required in order to determine the identit)i(ies) and location(s) of the residues involved in

the hnctioning of the channel.

Another mechanism bv which CO has been proposed to act is via the inhibition of

c!.tnchrome P450 (Coceani r ( ( I / , 198% Activation of this y t e m may produce

vasoconstricting substances, such as an arachidonic metabolite (Coceani r i d.. 198-1.

Harder r / trl. 1996) or endothelin-I (Coceani et d. 1996) I f the basal activitv of this

svstem contributes to the maintenance of vascular tone. it is hypothesized that mhibition

of cytochrorne P l 5 O by CO would result in a decrease in vascular tone or vasorelaxation

Coceani and co-workers demonstrated the rehat ion of lamb ductus arteriosus a i t h 1 -

aminobenzotriazole (ABT), a suicide substrate for monooxygenase. thus implicatins the

cytochrome P l S O based monooxygenase reaction mediating the production of endot helin-

1 (Coceani er d . . 1996; Coceani et d.1997). The role of the cytochrorne P1SO based

mechanism in the vascular et'Yect of CO has not been well characterized with the

exception of certain fetal vessels and, therefore. requires further investigation.

B.2 Role of CO as a neuromodulator in the CNS.

One role in which CO has been implicated is as a neuronal messenger. CO has

been shown to play a role in LTP, which is a proposed mechanism for learning and

memory formation, which occurs in the CAI region of the hippocampus. In fact. both

NO and CO are thought to act, either alone or in combination, as retrograde messengers

activating guanyiyl cyclase and, in turn, cGMP-dependent protein kinases. This is the

proposed mechanism by which CO and NO produce activity-dependent presvnaptic

enhancement durins LTP in the hippocampus (Hawkins et t r l . 1994) CO has been

shown to play a role in mechanical hyprralgesia along with mrtabotropic glutamate

receptors. similar to the roles which NO and the N-methyl-D-aspartate (NMDA) receptor

assume in thermal hyperalgesia (Aleller er t d , 1994) I t has also been demonstrated that

CO pr[rduce.; ctTect~ In the rat hvpothalamr~.; \\here i t may h n c t i o n ;I.; a nrr~rntransm~ttrr

in the modulation of neuroendocrine function. Thus, hemin, the substrate of HO. dose-

dependently inhibited the release of KCI-stimulated corticotropin-releasln_r hormone

(CRH) wlth no effects on basal CRH release (Pozzoli c3f d. 1994) CO has also been

indirectly shown to stimulate the release of gonadotropin releasing hormone (GnRH) In a

dose-dependent manner (Lamar rt trl . 1996).

When cobalt protoporphyrin 1X. an inducer of HO. was injected into the medial

nuclei of the rat hypothalamus. w i ~ h t loss occurred Based on these results. it has been

postulated that CO is involved in the signal transduction pathway in the medial

hypothalamus to suppress appetite (Marks. 1994)

B.3 Role of CO as a Modulator of Vascular Tone

CO mav play a physiological role in regulating vascular tone. CO-induced

vasorelaxation, as a result of a direct action on vascular smooth muscle, has been

demonstrated in a variety of isolated blood vessels from a number of species such as

porcine coronary meries (Graser rt c t l . . 1990), rabbit and rat thoracic aorta (Furchgott

and Jothianandan, 1991; Lin and McGrath, 1988), rat femoral and tail arteries

(Vedernikov rr d . . 1989; Wang rt nl., 1997), and the lamb ductus arteriosus (Coceani et

d.. 1984) CO dilates resistance vessels in the heart. lungs. and liver W a n ( 1999)

tested the effects of CO on the rat tail artery and considers it to be representative of

peripheral vascular tissue. This group demonstrated a concentration-dependent relaxation

bv CO in a rat tail artery precontracted with phenylephrine. This relaxation was not

mediated bv blockade of the a-adrenoreceptor as CO also relaxed U-16619

(prostaglandin L,, analog) i o n i r a c i ~ d tissuc, an agonisi that causes con:raction -:io

1 .

release of intraceilular Ca- The investigators reported an cndothelium-independent

sustained relaxation. which was reversed upon removal of CO.

The relaxation of blood vessels by CO is not uniform throughout the vasculature.

\'aqing degrees of vasorelusation are produced bv CO in ditferent ~clscular beds. In ;i

study by Brian cr d. ( 1994). the middle and basilar cerebral arteries in rabbits and dogs

were apparently not affected when CO was administered rtr w r o ( I to 300 phl) (Brian et

d. 1994). but were relaxed by NO However. in a later study LrfTler and co-wrkers

( 1999) using the cerebral microvasculature of newborn pigs demonstrated CO-induced

dilation of pial arterioles in the cerebral vascular bed. These investigators offer the

following reasons for the variation in response between the two preparations Brian d.

( 1994) examined the responses of major cerebral arteries whereas Lettler and colleagues

( 1999) utilize the arterioles that are among the smallest precapillary vessels on the brain

surface. It is important to recognize that blood vessel tone is controlled in the arterioles

and not in the large arteries. Second. the latter study was conducted on intact brain l tr

w o . whereas the former used a tissue bath apparatus with precontracted anery rings

Lefler r i a/. (1999) suggest that CO dilation is less functional under the ill ~ r - o

conditions employed by Brian et ( I / . and that underlying neurons in the i ~ r ~iw

preparation are possiblv contributing to the CO-induced vasodilation. Moreover. t h e

studv , . by Leffler rt d . involves newborn animals where the HO-CO system is

developmentally regulated. Finally. it is suggested that the species difference may

account for the divergent findings of the two studies since Brian rt t t l . studied dog and

rabbit cerebral arteries and LeMer ul used pigs.

There are several mechanistic evplanations for the ditferent etlkcts in the

vasculature bv NO and C O Firstly, it has been proposed that the cellular targets of CO

and NO may account for the differences. NO acts through sGC activation resulting in

elevation of cGMP content and in pan to hyperpolarization resulting from direct

interaction of NO with c a 2 ' -dependent K* channels (Bolotina rr t r i . . 1994) CO

stimulates sGC resulting in elevation of cGhlP content and mav have actions through the

cytochrome P450/endothelin- l system (Coceani er d., 1996) andior Kc , channels (Wang

c.t d . . 1997) depending on the vascular bed being esamined The differences in CO and

NO actions on vascular tone may also b e accounted for by their relative abilities to

activate sGC. On the basis of studies in the ductus arteriosus with methvlene blue. an

inhibitor of sGC of poor selectivity, CO was reported to produce an insignificant increase

in cGMP accumulation. It would be of interest to repeat this study with ODQ. a more

selective inhibitor of sGC (Garthwaite rt d.. 1995). On the other hand, sodium

nitroprusside (SNP), a NO donor, significantly stimulated sGC (Coceani rt d, 1996).

NO is more potent than CO in activating sGC, which also accounts for the varying

abilities of these two gaseous molecules to produce vasorelaxation.

CO has also been shown to play another role in modulating vascu

as a vasorelauant. In a recent review published by Johnson el a!. ( 1999),

liar tone. besides

CO is identified

as having a dual action. Endogenous CO production contributes to the functional

equilibrium between vnsoconstrictor and vasodilator factors. that determines a certain

level of blood pressure and its role in this equilibrium has been regarded as that of a

vasodilator. According to Johnson rt d. ( 1999), investigators have failed to differentiate

between the endothelium-dependent and -independent roles of endogenously formed CO

This gmup (Kozma rr ( I / , lq(JRl~) demonstrated CO-induced vasodilation as an

endothelium-independent event in isolated superfused rat gracillis first-order arterioles

denuded of endothelium. but when the endothelium was lef intact. a product of HO.

presumably CO (as these results were mimicked bv CO). elicited a vasoconstrictive

response Johnson LV '11 ( 1999) suggest that endothelium-dependent vasoconstriction is

either due to CO-induced release of a vasoconstrictive agent from the endothelium or to

CO-induced inhibition of NOS or to both. The latter possibility is supported by rvidmcr

tiom earlier studies where CO binds to NOS and inhibits NO (EDRF) production (White

and Marletta, 1992). Thus. removal of a vasodilating factor, such as EDRF. from the

equilibrium that contributes to blood pressure results in a vasoconstrictive effect.

Kozma r f id. ( 1997. 1998tr. 199Sh) have attempted to clarie the role of CO in

the vascuiature These investigators inhibited NOS in endothelium intact vessels by .V"-

nitro-L-aryinine methyl ester (L-NAME) pretreatment. When heme was added to the

tissue bath. CO was produced by the actions of HO resulting in vasodilation. The

vasodilation induced by heme addition was inhibited by HO inhibitors. These workers

suggest that heme-derived CO in the smooth muscle may mediate its vasodilatory effects

in a local manner and its vasodilatory effects may be buffered simultaneously by the

inhibition of the vasodilatory influences of endothelium-derived NO. Johnson el ol.

( 1999) express the need to acknowledge and clarify the potential interactions between the

CO and NO systems. Without the knowledge of the functional hemodynamic

significance of this interaction. accurate interpretations of results from future experiments

would be dificult.

CO has been proposed to assume an antitrophic role in the vasculaturr Under

! I \ ~ I Y ! C condition.;. it ha.: been ihown that CO conrrols the proliferation of vascular

SblCs (blorita d, 1997). I t was previously reported that CO. produced from HO- 1 in

hypoxic vascular SMCs. regulates the production of the growth factors. mdothelin-1

(ET-I ). and platelet-derived growth factor-p (PDGF-P) and decreases the proliferative

response of vascular S M C s to ET-1 (blorita and Kourembanas. 1095) CO has also been

shown to suppress E2F-1 expression, a prototype member of s family of transcription

kctors that participate in the control of cell cvcle progression. Taken together. these

reports susgest that CO. under certain pathophysiologic conditions. is responsible for not

only the immediate effects on vascular tone. but also producing more long-term

adaptations in vascular structure.

B.4 Role of CO in other organ systems.

It has been shown that CO is involved in intestinal neurotransmission using mice

with genomic deletions for HO-2 and nNOS resulting in the absence of these enzymes in

the rnyenteric ganglia. It was shown in these knockout mice that non-adrenergic. non-

cholinergic (NANC)-mediated intestinal relaxation and c 0 elevations induced by

electrical field stimulation were markedly reduced in both HO-7 and nNOS knockout

mice (Zakhary rt ul., 1997). These results indicate that NO and CO act as

neurotransmitters in the myenteric plexus and mediate their etrects via the cGhlP

pathway. Further studies with HO-2 knockout mice involving the bulbospongiosus

muscle, which mediates ejaculation and ejaculatory behavior, have demonstrated a

diminished retlex activity of this muscle (Burnett et id.. 1998) CO was concluded to be

the most plausible product of HO-2. that would account for the results obtained.

C. Inhibition o f HO

HO activity is inhibited by compounds that act as pseudosubstrates These

compounds are resistant to HO degradation and compete with heme (iron protoporphvrin

[X. FePP) for the hydrophobic heme pocket (Maines. 1997) These metalloporphyrins

ditfer from heme with respect to the metal cation associated with the porphyrin ring

and/or the substituents bound to the porphyrin ring (Figure 1.5) These variations from

heme affect their potency as HO inhibitors The hydrophobic pocket for which the

metalloporphyrins and heme compete. exhibits specificity for the porphyrin side chains

but not for the chrlated metal (hlaines. 1997). By definition of n competitive inhibitor.

the relative concentrations of heme and metalloporphyrins will determine the degree to

which HO is inhibited.

It was recently shown that a KO donor, in a dose-dependent manner. also

inhibited HO activity. It was suggested that the NO nitrosylates intracellular free heme

and prevents its degradation by heme oxygenase (Juckett r i d, 1998). This contributes

hrther to the close relationship that exists between the HO and NOS systems.

Metalloporphyrins are heme analogs that vary in the metal cation coordinately

bound to the njtrogen groups of the porphyrin ring as well as substituents of

Figure 1.5: Chemical structure of metalloporphyrins. Heme is a metalloporphyrin where the substituents RI and R2 are vinyl groups. For other metalloporphyrins. a different metal cation is coordinately bound to the porphyrin ring. For ZnPP: substitute Zn for Fe and RI= R2= CH=CH2; for SnPP: substitute Sn for Fe and RI= Rz= CH=CH2. for CrhlP substitute Cr for Fe and RI= R2= CHI-CH3; for ZnBG: substitute Zn for Fe and RI= R2= CHOH-CH20H; ZnMePP has the same chemical structure as ZnFP with the exception of a methyl group bound to one of the nitrogens in the porphyrin ring.

the porphyrin ring. These compounds have been used as pharmacological tools in the

past. but it has also been pointed out that they are produced endogenously under certain

physiological settings. Zinc protoporphyrin is formed when zinc replaces iron for

insertion into protoporphyrin, in a reaction catalyzed by ferrochelatase. i t has been

suggested that in iron deficiency states, zinc protoporphyrin will limit the amount of

l i m e catabolism

Endogenous zinc protoporphyrin production has also been hypothesized to play a

role. at least in pan. in the decrease in attention span and small decrements in IQ scores

seen in patients experiencing subclinical lead toxicity (Marks er i d . 19%) Production of

zinc protoporphyrin by ferrochelatase is increased in iron deficiency states as well as in

lead poisoning. The increased endogenous zinc protoporphyrin concentration has been

proposed to inhibit the formation of carbon monoxide by heme oxyrenase and thus

modulates LTP and memory. The inhibition of heme oxygenase in the hippocampus was

proposed to alter the neurophysiological phenomenon known as LTP. which might

account for the cognitive deficits observed in lead poisoned patients.

inhibitors of enzymatic activity are useful tools in establishing a physiological

role for specific enzymes. Thus. L-NAME has played an important role in elucidating the

biological roles of NOS (Moncada el ~ z l . . 1991). Metalloporphyrins have been shown to

inhibit HO. and their potency is affected by the metal cation associated with the

porphyrin ring as well by different ring substituents (Vreman rt dl . . 1993). Inhibition of

HO activity has been demonstrated for each of the metalloporphyrins used in this study.

specifically zinc protoporphyrin IX (ZnPP). tin protoporphyrin IX (SnPP), chromium

mesoporphyrin IX (CrbP) (Vreman rt 01.. 1993; Cook rt at., 1995; Marks rr d., 1997).

zinc deuteroporphyrin 1X 2.4-bis ethylene glycol (ZnBG) (Chernick r t d l . 1989; Vallier

e t d , 1991; Vreman et al., 1992) and zinc N-methylprotoporphyrin IX (ZnklePP) (De

\ latteis rt c r / . 1985) hletalloporphvrins have been used to test the hypothesis that CO

has a physiological role SnPP and ZnPP have been used to investigate a possible role for

CO as a vasodilator (Zakhary er id.. 1996) ZnPP (1-20 ph.1) also has been used to

demcnstr3te 3n apparent role for CO in LTP (Zhuo cf d.. P 3 ) and the inhibition of

depolarizat~on-induced glutamate release ( I O ~ L M ) by Shinornura ~ . r d. ( 19W)

Mrtalloporphyrins are also used in a clinical setting as HO inhibitors.

Hyperbilirubinemia remains the most frequent clinical problem pediatricians must deal

ivith during the newborn period. and under certain circumstances may cause severe brain

damage even in healthy term newborns. Certain metalloporphyrins have been exploited

therapeutically to decrease hyperbilirubinemia in the neonate (Qato and hlaines. P W .

L'alaes r.1 d.. 1994. Jlaninez ' I / . 1990) Tin rnrsop~rphyrin IS ( SnbIP) was recently

used to control severe hyperbilirubinemia in hll-term newborns SnhlP 1s a potent

inhibitor of HO activity and was administered by Martinez and co-workers to healthy

newborns to decrease hyperbilirubinemia. This pharmacological means of preventing the

development of severe hyperbilirubinemia eliminates the need for phototherapy and

reduces the length of time the infants are under clinical care. This method offers some

logical advantages over the current methods for controlling hyperbilirubinemia. such as

phototherapy and eschange transhsion, that are used only after the problem has become

severe.

Several investigators have shown that metalloporphyrins are not specific

inhibitors of HO, but also inhibit NOS and sGC (Luo and Vincent, 1994; kletfert tcr d..

l994; Grundemar and Ny. 1997) Based on these findings. the conclusions reached by

investigators using rnetalloporphyrins to establish a physiological role for CO in

biological systems, in which NOS and sGC are also active, have been criticized

In contrast, Zakhary el d. ( 1996) have reported that SnPP was 10 times more

potent in inhibiting HO-2 than NOS or sGC and based on this finding have used SnPP as

R selective agent to study CO-induced vasodilation

D. Statement of aims and objectives.

In the past. it has been demonstrated that HO can modulate selective cytochrornr

PJSO isozyme activity by degrading the heme moiety of selected PJjOs. NOS is a

molecule closely similar to P-150 and its heme moiety mav also be subject to dewadation - by HO Furthermore, it was recently observed that when HO was incubated together with

cycloosygenase, a hemoprotein. cyclooxygenase activity was decreased. Co-incubation

with SnhlP prevented the reaction The results were interpreted as showing that HO. or a

product derived from heme degradation played a role in modulatins the activity of

cvclooxygenase. Thus. the activities of NOS and sGC may be modulated by degradation

of their heme moieties by HO

In order to study the interactions between HO and sGC or NOS. a selective

metalloporphyrin inhibitor of HO is required. The hypothesis which we wish to

investigate is the following: "A metalloporphyrin when used at an appropriate

concentration will hnction as a selective inhibitor of HO " To test this hypothesis ive

compared a range of concentrations of five metalloporphyrins as inhibitors of both 30s

and HO. Two of the most selective inhibitors of HO were selected and the effects of

these two metalloporphyrins on basal and SNAP-induced sGC activity were assessed

Chapter Two

SELECTIVE INHIBITION OF HEME OXYGENASE BY METALLOPORPHYRINS

.A. lntrodrlction

Ln the present study. the objecti~c was to test five rnetalloporphvrins that haw

been shown previously to inhibit HO activity. for their ability to selectivelv inhibit HO

relative to UOS and sGC activities For each rneta1loporphl;rin. a concentration lvns

determined that inhibited HO activity, without inhibiting h O S activitv The two most

selective inhibitors. CrMP and ZnBG. were hrther studied to determine whether they

atyected basal or 3'-nitroso-IV-penicillamine (SNAP)-induced sGC activitv it was found

that CrMP. at a concentration of 5 phl, was a selective inhibitor of HO activity and

appeared to be the most usetLl HO inhibitor based on the studies conducted

B. Materials iind Methods

B. 1 Drugs and solutions.

Erhvlenediaminetetraacetic acid (EDTA) disodium salt. hemin. ethanoiamine.

bovine serum albumin (BS A), leupeptin. Amberlite IRP-69, L-N AbE. heparin. cGhlP.

NADPH and SNAP were obtained from Sigma Chemical Co (St Louis, hlO) Tris-HCI

and 3-isobutyl- l -methylxanthine were purchased from ICN Biomedicals Inc. (Costa

Mesa, CA). CrMP, ZnSG, ZnPP, SnPP, and ZnMePP were purchased from Porphyrin

Products inc. (Logan, UT). rUI other chemicals were at least reagent grade and were

obtained from BDH Inc. (Toronto, ON). Stock solutions of methemalbumin ( 1.5 mi1

hemin and 0.1 5 mM BSA) and of each of the five metalloporphyrins ( 1.0 mM) were

prepared as previously described (Vreman rt d., 1993). Briefly, hemin or

metalloporphyrin was dissolved in 0.5 ml of 10Y0 (wiv) ethanolamine. BSA dissolved in

1 ml of deionized water was added to the hemin solution only The volume was made up

to 7 ml and slowly adjusted to pH 7 4 with 1 h 1 HCl and vigorous stirring. The final

?:c!urne f i r each stmk solut io~ was adjusted to ir! ml with deionized water The

mrtalloporphy-in vehicle was prepared as above without the addition of any

metalloporphyrin. The methemalbumin and metalloporphyrin stock solutions were

prepared with the laboratory lights turned off and were stored at -20°C for up to 1 month.

B.2 Preparation of ~ ~ b ~ e l l ~ l i ~ r fractions of rat brain and lung.

Adult male Sprague-Dawley rats (300-3SOg) were obtained tiom Charles River

Canada inc (Montreal. Q C ) Rats were given ~ l t l lihitrrm access to Ralston Purina

Laboratory Chow (no. 500 I , Rrn's Feed and Supplies. Ltd.. Oekville. O N and water

All animals were cared for in accordance with the principles and guidelines of the

Canadian Council on Animal Care, and the experimental protocol was approved by the

Queen's C1niversity Animal Care Committee For measuring HO and NOS activity. each

rat was killed by decapitation. and its brain was excised and weighed For measuring

sGC activity, the rat was injected i . p. with heparin (3m& body weight) and then 45 min

later anaesthetized with sodium pentobarbital (93 mykg body weight). The lungs were

perfused with 40 ml of ice-cold phosphate buffered saline (PBS) by inserting a syringe

into the pulmonary artery through the right ventricle. The left atrium was cut to allow

outflow of perfusate. During perfusion, the lungs were continuously inflated and detlated

with an air-filled syringe inserted into the trachea. Perhsion was continued until the

lungs were cleared of all blood and appeared white in colour The lllngs were excised

and rinsed with I0 rnl ice-cold PBS.

:Llicro.somd i+acriori of Rar Hruirr jbr hletr.s~rri~rg HO dcti~ity . .A homo yenat e

( 1 ~ O , O . wiv) of brains pooled from four rats was prepared in ice-cold. HO homogenizing

buffer (20 mhl KH:PO.:, 1 3 mM KC! and n ! 0 mhl EDTA; adjusted to pH 7 4 at -W

with I M KOH) using a Potter-Elvehjern homogenizing system with a ~ef lon ' pestle The

n~icrosomal fraction of the rat brain hornogenate was obtained by centrifugation at 10.000

u g for 20 min at J'C. followed bv centrifugation of the supernatant at 100.000 K ,c for 60

nlin at 4°C. The 100.00ir u g pellet (microsomes) was resuspended in 100 mhl KH2P04

butfer (adjusted to pH 7 4 with I bl KOH) using a Potter-Elvehjern homogenizins svstem.

The rat brain microsomal fraction was divided into equal aliquots, placed into

rnicrocentrihge tubes. and stored at -80°C for up to two months Protein concentration

of the microsomal fraction was determined by the Biuret method (Gornall rr trl. 1949).

which was modified as described previouslv (Marks el trl, 1997).

( > w . s o l i ~ * brtrcrroti c ~ f k r t Brtrirr $)re ilt~.ccsriri~g IVOS d cririty. Brains from four

rats were pooled and homosenized in ice-cold, NOS homogenizing buffer (50 rnM

HEPES. I mhl EDTA and 10 pyrni leupeptin; adjusted to pH 7 4 at 4°C with I 0 41

NaOH) with a Potter-Elvehjem homogenizing system. in a ratio of 1 y tissue: I . I mi

homogenizing buffer. The cytosoiic fraction was obtained by centrihgation of the

homogenate at 10,000 u g and then 100,000 x g, as described above for preparation of the

microsomal fraction of rat brain. The 100,000 x ,g supernatant (cytosol) was divided into

equal aliquots, placed into microcentrifbge tubes, and stored at -80°C for up to two

months Protein concentration of the cytosolic fraction was determined by the Biuret

method as described above

(:~voso/ic Frtzctror~ oflitit l.iuig-sji)r.Llc.ti.sitrnig.s(;C'.4~~tir~it~~. Lungs from one rat

were homogenized in ice-cold sGC homogenizing buffer (50rnM Tris HCl (pH 7 1).

5mM MsCI:. 1 mM EDTA and 5mM benzamidine HCI) to produce a 20% (w/v)

homogenat r The cvtnsolic fraction \\as ohtamed hv di tfrrential wnlr ih~gat ion o f t he

hornogenate at 10,000 s g and then 100.000 x g, as described above for the preparation of

the microsomal fraction of rat brain. The 100,000 x g supernatant (cytosol) was divided

into equal aliquots. placed into microcentrifhge tubes, and stored at 4°C for less than 24

hours Protein concentrarion was determined using bicinchoninic acid (Smith ct d..

1985). which was obtained as pan of a protein assay kit from Pierce Chemical Co

(Rockfbrd. IL)

B.3 Measurement of HO enzymatic activity in microsomal fraction of rat brain.

HO activitv in the microsomal fraction of rat brain homoyenate was determined

by measuring the rate of CO formation during the NXDPH-dependent oxidation of heme

(Vreman and Stevenson, 1988) and modified as described by Cook et d. ( 19%) For

each ZnPP. CrMP, ZnBG, SnPP and ZnMePP sample, a reaction mixture consisting of

100 mhl KH2P04. 0.2 mg of microsomal protein and methemalbumin (final

concentration of 25 p M hemin and 2.5 ph.1 BSA), was pipetted into six 3.5-ml amber

glass vials (Chromatographic Specialties Inc., Brockville ON). The following

concentrations of metalloporphyrin were added to individual vials: 0 . I 5 . 0.5. I . 5 . 5 . 1

and 50 pM. Each vial was sealed with a silicon-~eflon' septum and a screw cap

(Chromatographic Specialties Inc.). and then was preincubated for 5 min in the dark at

3 7 O C . in a shaking water bath NADPH (0.5 mhl) then was added to each vial. the

headspace zas was displaced with CO-free air and the incubation was continued for

another 15 rnin. The reaction was stopped by placing the vial on pulverized dry ice t -

78°C). where it remained for 30 rnin until the headspace gas was analyzed. For each

metalloporphyrin reaction set. CO production was corrected for the CO produced in a

blank reaction vial that contained a rnetalloporphvrin concentration of 50 LN. but no

NADPH. To determine total HO activity in the microsornal fraction of rat brain. a

reaction vial was prepared that did not contain metalloporphyrin or vehicle (There was

no inhibition of HO activity 111 a reaction set containing rnetalloporphyrin vehicle

equivalent to a range of 0 15 to 50 pbl metalloporphyrin.)

CO in the headspace gas was quantitated by gas-solid chromatography using a

I3X molecular sieve as the stationary phase and a sprctrophotometric detector (RG.4-3:

Trace Analytical Inc.. Pvlenlo Park. CX) set at 254 nm to quantitate the Hg vapour formed

from the reaction of CO with HgO The headspace gas was injected onto the gas

chromatographic column via the carrier-gas stream of CO-free air (35 mlimin). ~bhich

was directed througl~ the headspace for 50 sec Analysis of the headspace continued over

the n e a I10 sec The amount of CO in the hradspace gas was determined by

interpolating the peak area of the chromatographic signal on the linear CO standard curve

( 10 - 170 pmol CO), which had a correlation coeficient of 0.999 (iV=9 determinations).

The rate of formation of CO in the microsomal fraction of rat brain hornogenate was

expressed as nmol CO formed/mg protein/ hr. NADPH-dependent formation of CO was

calculated by subtracting the value for CO produced in samples not containing NADPH

(blank) from the value for CO formed in samples containing NADPH.

B.4 Measurement of NOS enzymatic activity in cytosolic fraction of rat brain.

NOS activitv in the cytosolic fraction of rat brain hornogenate was determined

using a modification of an established procedure (Bredt and Snyder. 1990) that was

optimized tbr the hippocampus of the guinea pig (Brien et trl . , 1995. Kimura tv d..

1996). A 100-111 volume of reaction buffer (50 mhl HEPES. 1 mhl EDTA. 1 25 mR1

CaClz and 2 mM NADPH; adjusted to pH 7.4 at 37°C with 1.0 M NaOH) and a -3i-ld

volume of cytosol containing 0.525 mg protein were added to six test tubes. The

following metalloporphyrin concentrations. 0 15. 0.5. 1 5 , 5. 15 and 50 phi. were added

to individual test tubes in a volume of 100 p1 The samples were preincubated for 5 mi11

at 37°C. after which a S p I aliquot of an aqueous solution containing 35.000 dpm L-

["c] argir~ine (hew England Nuclear -- Xlandei. Guelph. OW) and I SO p?rl nun-

radiolabelled L-arginine was added. The samples were incubated for 15 min. and the

reaction was stopped by the addition of ice-cold 'stop' bufier (20 mM HEPES and 2 mhl

EDT.4: adjusted to pH 5 . 5 at 4°C with 1.0 M NaOH) Experimental blanks were

prepared by adding the 'stop' buffer to a sample containing reaction buffer, cytosolic

protein and a metalloporphyrin concentration of 50 pM. before the addition of L-['"c]

aryinine and incubation at 37°C for 1 5 min To determine total NOS activity. samples

were prepared that did not contain metalloporphyrin or vehicle. (There was no inhibition

of NOS activity in samples containing metalloporphyrin vehicle equivalent to a

concentration range of 0.15 to 50 pM metalloporphyrin.)

.Af er incubation. each sample was subjected to ion-exchange chromatography

using an Amberlire IRP-69 (sodium ion form) column (4-cm high. 0 4-crn diameter. and

0.75-ml volume) with a silanized glass wool plug .A 1 0-ml volume of deionized Lkatrr

was added to the column to elute L-[l"~]citrulline. The eluate was collected and mixed

with 10 ml of scintillation cocktail .A 35 -p I aliquot of L-[ ' ' '~lar~inine solution was

mixed with ! r? rnl scintillaticn cccktail to dewmine the tctal r3dicacti~ity 2dded :c a x ! ?

sample. Radioactivity was quantitated by liquid scintillation spectrometry. and the

radioactivity of the individual samples was corrected for experimental blank NOS

activity was espressed as nmol L-["c] citrulline formedimy proteini h r In a prelimman

experiment. incubation of rat brain cytosol with 1 m M L-NAME. an inhibitor of UOS.

1 .

inhibited Ca- -dependent formation of L-["c] citrulline in rat brain cvtosol by 97 b r

0 49.0 ( r ' V 4 )

B.5 Measurement of sGC enzymatic activity in cytosolic fraction of rat lung.

sGC activitv was measured in the cytosolic fraction of rat lung homoyenate using

a modification of the procedure described by Liu rr td . ( 1993). Rat lung cytosolic

fraction (25 p1) and 50 p1 of a reaction mixture ( 100 mM Tris-HCI (pH 7 4). 6 mhi

StgCIZ. 2 nihl hsobutyl- 1 -methyluanthine. I 0 mbt cGhlP. 1 mhl L-cyteine and 1 mg

BSNml) were added to each of five test tubes. The following metalloporphvrin

concentrations, 0.15, 0.5, 1.5, 5 and 15 pM, were added to individual test tubes in a final

volume of 10 pl. For the basal sGC activity experiments, the reaction was started by the

addition of 25 yl of an aqueous solution containing 500,000 dpm of ['HIGTP (New

England Nuclear-Mandel) and 200 pM non-radiolabelled GTP. The samples were

incubated at 30°C for 30 min and the reaction was stopped by adding 100 p1 of ice-cold

10% (w/v) trichloroacetic acid and putting the tube on ice. For the stimulated sGC

activitv experiments. the reaction was started by adding 100 phl SNAP. followed by 25

pI of ['HIGTP solution for a final volume of 120 p1. The reaction was carried out as

described for the basal sGC activity experiments. Experimental blanks were prepared by

a d d i r ~ ~ I-a1 lung cylusuiic ii-a1

metalloporphyrin concentrat

determine total sGC activity

rim that i ud been 'ooiittd for 5 min. ~,ttiiction nisrur t : and a

on of I S piLI ro a vial before starting the reaction. To

samples were prepared that did not contain

metelloporphvrin .After incubation was completed. approximately 1.000 dpm of

[ ' " C ] C G ~ I P (New England Nuclear-blandel) was added to each sample to provide an

index of column etficiency The precipitated proteins were removed from the acidified

sample by centrifugation at 11.000 s g for 3 min The supernatant was applied to a

column (4-cm high and 0 4-cm diameter) containing 0 5 5 of neutral alumina that was

equilibrated with 5*'0 (wiv) trichloroacet~c acid Each column was washed with I ml of

5% (wiv) trichloroacetic acid. followed by 3 ml of water and fmallv by 0.75 rnl of 200

m M sodium acetate (pH 6.0) [ 'H]CG~IP and ['' 'C~CGILLP were eluted with I ml of 200

mM sodium acetate (pH 6.0). The eluate was collected, mixed with 8.0 mi of

scintillation cocktail and quantitated by liquid scintillation spectrometry. Radioactivitv

measured for each sample was corrected for crossover of '"c radioactivity into the 'H

channel during liquid scintillation spectromeiry and for the recovery of ['"c]cG~.IP from

the column, which ranged from 60-70% The amount of cGMP formed during the

incubation was calculated from the specific radioactivity of the ['HIGTP (20 9 Ci/rnrnol)

and corrected for experimental blank sGC activity was expressed as prnol cGMP

formed/mg p r o t d h r ,

B.6 Data analysis.

The activity of HO, NOS and sGC following incubation with each concentration

of metalloporphyrin was expressed as percent of total activity. measured in the absence of

metnlloporphyrin. The data are presented as group means k SD of four tissue

preparations from ditierent afiimals. unless otherwise stated. Parametric statistical

analysis of the data was conducted by repeated-measures, one-way analvsis of variance

For a statistically sigificant I.' statistic (pc-0.05). a post hoc Newman-Keuls test was

conducted to determine which experimental groups were statistically different (p- O 0 5 )

C. Results and Discussion

The metalloporphyrins used in this study have been shown previously to inhibit

HO activity (De Matteis r i tr/. , 1985; Chernick rt d., 1989; Vreman ct LI/. . 1992. 1993 )

The use of metalloporphyrins in investigating a physiological role for HO has been

criticized because some metalloporphyrins have been shown also to inhibit %OS and sGC

activity (Luo and Vincmt. 1994: Meffert rr d.. 1994. Grundemar and %\iv. I W7) In

contrast. Zakhary L.1 (I/. ( 1996). in a study demonstrating HO-derived CO as a vasodilator.

reported a dose of SnPP that appeared to selectively inhibit HO relative to NOS Thus.

the potential exists for finding a metalloporphyrin and/or a metalloporphyrin

concentration that would inhibit HO activitv without inhibiting NOS or sGC activity i n

the present study. five metalloporphyrins were investigated to detrrnminr whether there is

a metalloporphyrin andlor a metalloporphyrin concentration that can be used as a

selective inhibitor of HO relative to NOS and sGC.

HO activity in rat brain rnicrosornes and NOS activity in ra

3.3 0.3 nmol CO formed!mg protrinihr (n=6) and 5 0 2 I . I nmo

t brain cytosol were

formedlmg proteidhr (n=4), respectively. .All five metalloporphyrins, viz. CrMP. ZnBG.

SnPP. ZnPP. and ZnMePP. inhibited both HO and NOS activity (Figures 2.1-2 3 )

However. there was a concentration for each metalloporphyrin. at and below which only

HO activity was inhibited. The metalloporphyrin vehicle did not affect HO or NOS

activity (data not shown).

CrMP

SnPP

I

0.1 I i'o 1 A0

Metalloporphyrin Concentration (pM)

Figure 2.1 : hrhihiliorr of HO tord NOS trcfivity hy C'nLP md 91PP. Concentration- response curves for HO (r ) and NOS (. ) activity in rat brain microsomes and cytosol. respectively, were obtained in the presence of CrMP and Sr.PP. The data are presented as group means k SD (N=4). The letter 'a' denotes the maximum selective inhibitory ( M I ) concentration at or below which there is selective inhibition of HO activity, with no effect on NOS activity; NOS activity for lower concentrations of metalloporphyrins were not ditierent from the MSI concentration. Group means with different letters are statistically different from each other, p<O.O5.

ZnBG

ZnPP

I

0.1 1 i o 160

Metalloporphyrin Concentration (pM)

Figure 3.1: I d ~ i h i t i o ~ t oj. H O turd ;VOS ~rclivi& hj. ZIIBG mid Z~rl'l'. Concentration- response curves for HO ( r ) and NOS ( . ) activity in rat brain microsomes and cytosol. respectively, were obtained in the presence of ZnBG and Z n P P The data are presented as group means + SD ( N = l ) . The letter 'a' denotes the maximum selective inhibitory (MSI) concentration at or below which there is selective inhibition of HO activitv. with no effect on NOS activity; NOS activity for lower concentrations of metalloporphyrins were not different from the MSI concentration. Group means with different letters are statistically different from each other, p<O.OS.

Metalloporphyrin Concentration (pM)

Figure 3. -3. l ~ i l ~ r h r r i o ~ i of HO toid W S i l ~ ~ r ~ r f y hrv ZtuLId'i'. Concentration-response curves for HO ( - ) and NOS ( m ) activity in rat brain microsomes and cytosol. respectively. were obtained in the presence of ZnhlePP The data are presented as group means i SD (X=l) The letter ' a ' denotes the maximum selective inhibitory (MI) concentration at or below which there is selective inhibition of HO activity with no effect on NOS activity; YOS activity for lower concentrations of metalloporphyrins \yere not different from the MSI concentration. Group means with different letters are statistically different from each other, p<0.05

In comparing the metalloporphyrins as selective inhibitors of HO. the first step

was to determine a maximum selective inhibitory (MSI) concentration for each

metalloporphyrin. at or below which there is inhibition of HO. with no effect on NOS or

sGC activitv (Table I ) The next step was to compare the percent inhibition of HO

~ctivity st h e M I concentration t'or each rnetallnpnrphvrin - \ l ~ > , the percent inhihition

of HO activity at concentrations N o i d and 10-fold lower than thc MSI concentration was

compared (Table I ) . This latter comparison was made because in an experiment. a

concentration lower than the MSI concentration, at which the metalloporphyrin becomes

non-selective. would normally be used. Based on these criteria. the inhibition of HO

activity was lowest and/or declined most rapidly with metalloporphyrin concentration fur

ZnMePP. ZnPP and SnPP (Table I ) Thus. it was concluded that CrMP and ZnBG at

concentrations at and below 5 pbl were selective inhibitors of HO activitv relative to

UOS activity in rat brain.

It is interesting to compare the above data (Figures 1.1-2 3 ) with that of bletfen cJr

d. ( 1994). These workers concluded that CrMP and ZnPP, but not tin mesoporphyrin or

ZnBG, inhibited NOS in the rat hippocampus Based on these findings. it was

emphasized that some metalloporphyrins are non-selective and would therefore not be

useful in biological studies involving CO. The concentration of metalloporphyrin used in

these studies ranged from 10 to 100 pM. while in the present study metalloporphyrin

concentrations from 0.15 to 50 pM were used. The results of Metfen r / d. ( 1994).

showing that CrMP and ZnPP inhibited NOS at concentrations of 10 pPvl and higher are

in agreement with the results of the present study. In contrast, it was found in this study

Xtetalloporphyrin " &IS1 inhibition of HO Activity (' O )

concent ration At blS1 .At 1/3 MSI .-it l l 10 \IS1 (PW concentration concentration concentration

ZnblePP 0.5 5 6 1 4 2 29 f 27.1 16 -C 20 9 "

Table 1 Comparison of metalloporphyrin inhibition of HO (N 4). " Maximum selective inhibitory (MSI) concentration, concentration at or below which there is selective inhibition of HO activity with no effect on NOS activity 11 Vote; the data for this concentration is not shown on the ZnhdePP graph in Figure 2. -3

that ZnBG inhibits NOS at concentrations higher than 5 pM. Tin mesoporphyrin was not

tested in the present s tudy The message that emerges in comparing the data is that to

achieve selectivity of metalloporphyrin-induced inhibition of HO versus NOS. the

concentration used is critically important and must be kept at or below 5 pM.

To characterize further the selectivit). of CrhlP and ZnBG as inhibitors of HO. the

c t k i t of the iuo rncitalloporphyrins on basal and SKIP-induccd ;GC actiiit; ;\as

determined for metalloporphyrin concentrations similar to those used for the study of HO

and NOS activity The basal and SNAP-induced sGC activities in the rat lung cytosol

were 12 1 i 56 (n=6) and 2059 2 82 1 (n=-3) pmol cGMP forrnedhg proteidhr.

respectively. Thus, the addition of 100 pM SNAP produced approximately a 1 Mold

increase in sGC activitv from basal level Neither CrMP nor ZnBG had any etfect on

basal sGC activity (Figure 2 4) at the concentrations tested Ho~tevrr . ZnBG elevated

SNAP- induced sGC activity (Figure ' S ) , which is consistent with reports from

investigators who found that certain rnetalloporphyrins. such as cobalt protoporphyrin.

enhance NO-induced sGC activity (Dierks r / trl.. 1997) CrMP had no rtfect on SNAP-

induced sGC activity for the concentration range tested, a range that included the %IS1

concentration of 5 pM, at and below which C r b P selectively inhibits HO activitv. with

no efTect on NOS activity Thus. CrhlP was found to be the most selective and useful

inhibitor of HO activity compared with NOS and sGC activities in rat brain and lung.

To our knowledge. no particular structural feature of metalloporphyrins has been

identified that allows the prediction of the efficacy of the compounds to inhibit HO or

NOS. For sGC, a mechanism to explain. at least in part, the interactions of

metalloporphyrins with this enzyme has been proposed by Serfass and Burstyn ( 1998).

Figure 2.4: Lflects qf ZttBC; mtd C'rhlP ujl Ekl.snl sCX' nczivitj. Concentration-response curves for basal (N=4) sGC activity were obtained for ZnBG ( - ) and CrMP ( m ) . The data are presented as group means k SD. Group means with different letters are statistically different (p<0.05) from other group means within the same concentration-response curve.

1401

120- w I-

> I-- 100- w

3 80-

601

* 40- s 20-

0

T T

-

I 1 1

0.1 I I 0 100

Metalloporphyrin Concentration (pM)

Metalloporphyrin Concentration (pM)

Figure 2.5 : Ii&fec~.s of ZnBG mid CrhlP or1 SIVA P-btdtrcrd sGC1 trctivil).. Concentration-response curves for SNAP-induced (N=3) sGC activity were obtained for ZnBG ( 7 ) and CrMP (. ). The data are presented as group means 2 SD. Group means with "a" are not statistically different (pC0.05) From each other. Group means labeled with "b" are statistically different @<0.05) to all other group means. Group means labeled with "c" are not different to means labeled with either -'a" or "b" within the sanle concentration-response curve.

These investigators postulate that a key requirement for sGC activation by

metalloporphyrins is the absence of a bond between a proximal protein-histidine and the

metal in the porphyrin. This proposed mechanism is based on the observation that

activation of sGC by NO has been attributed to binding of NO to heme iron with

concomitant breaking of a bond between a proximal protein-histidine and iron. In their

itudt. the n ~ ~ n i n ~ a l acti\ .ati(~n (3f vCrC hc. ZnPP i.; ilttrihtltect t o the likelihood that the h o n d

between a proximal histidine and Zn atom is intact On the other hand. the marked

activation of sGC by SnPP is attributed to the absence of a bond between a proximal

histidine and the Sn atom Activation of sGC by SnPP. as demonstrated by Serfass and

Burstvn (19%). provides funher rationale for selecting CrhlP from among the

metalloporphyrins tested, as the most selective metalloporphyrin to elucidate the

physiological role of HO

Luo and Vincent (199.1) and Grundemar and Uv ( 1997) have concluded that

ZnPP. SnPP and ZnBG cannot be used to establish a messenger role for CO This

comment is based on the fact that these metalloporphyrins inhibit sGC in addition to HO

Ho~ever . in their study, the concentration of these metalloporphyrins ranged from 10 to

100 pM for sGC inhibition. This conclusion requires reconsideration in light of our data

which demonstrates that concentrations of metalloporphyrins less than 10 pM can inhibit

HO activity without inhibiting hOS and sGC activities. Thus, carehl exploration of

concentration-response relationships with a variety of metalloporphyrins potentially can

lead to the identification of an appropriate selective inhibitor for the biological model

being used. This conclusion is reinforced by the results of Zakhary er tri. (1997). who

used HO-2 knockout mice and SnPP to demonstrate that CO plays a role in NANC

relaxation evoked by electrical field stimulation of mouse ileal segments in wild-tvpe

mice. SnPP partially inhibited NXNC relaxation. However. in mice where the gene for

HO-2 had been deleted, SnPP did not affect NANC transmission.

There is considerable interest in the use of metalloporphyrins to inhibit HO in the

treatment of juvenile jaundice (Valaes et 01.. 19%; Qato and Maines. 1985) CrbIP and

ZnBG appear to ire promising candidates hecause of their high potency to inhibit HO 3.;

demonstrated in the present and other studies (Vreman ~~r t r l . . 1908). good oral absorption

(Vallier el c t l . 1991; Vallier ~ j r d . 1993). resistance to metabolism by HO. and inability

to upregulate HO- 1 in cell culture (Zhang, Contag, Stevenson and Contag, personal

sornrnunicatian). >loreover. CrhlP has the additional advantages of nut distributing

across the blood-brain barrier and being photochemically inactive. .Although ZnBG is a

photosensitizer. the potential low doses required for therapeutic use, due to its high

potency as a HO inhibitor, may restrict its photoreactivity For the above reasons.

therapeutic and toxicological studies of CrklP and ZnBG are warranted.

In summary, of the five metalloporphyrins tested. CrMP and ZnBG inhibited HO

to the greatest extent at and below the concentration for which there was no measurable

inhibition of NOS activity Furthermore. CrhlP was found to have no erect on basal or

SNAP-induced sGC activity, unlike ZnBG, which enhanced SNAP-induced sGC activity

Thus, in this study, CrMP, at or below a concentration of 5 pM, was found to be a

selective inhibitor of HO relative to NOS and sGC, in rat brain and lung The hypothesis.

which we set out to test, was the following: "A metalloporphyrin when used at an

appropriate concentration will function as a selective inhibitor of HOW. Clearly the

hypothesis has been shown to be correct. In other studies using different biological

models, it will be necessary to determine the concentration of CrMP or other

rnetalloporphyrins that will selectively inhibit HO activity without inhibiting NOS and

sGC activities.

Chapter Three

FUTURE DlRECTIONS

.A common feature of biological regulatory systems is that they themselves are

subject to modification by multiple input signals These highly integrated h n c t ~ o n s are

controlled bv multiple variables in the case of respiration. for example. blood CO: is the

major consideration but pH also plays a role. In the autonomic nervous system. it has

long been established that the release of the major transmitters is subject to autoinhibiton'

feedback mediated bv presvnaptic acetylcholine and norepinephrine receptors but many

other substances act pres)maptically as well. As CO and NO act on similar regulatory

systems. a naturally arising question is whether there are interactions between NOMOS

and C O H O systems.

On theoretical considerations alone one could build a case for HO altering NOS

nctivitv because heme. the HO substrate. is an essential prosthetic group of \OS

Evidence contributing tc, the reciprocal regulaton: interactions be twen CO and NO has

been reported where endogenously derived NO may mediate the induction of HO-I in

vascular SMCs (Durante and Schafer, 1998) Thus. three structurally dissimilar NO

donors induced HO-I sene expression in vascular SbICs. Moreover. the same

investigators demonstrated that blockade of endogenous NO production also blocked the

induction of HO-1 gene expression in vascular SMCs (Durante rt a / . , 1997). Other

studies in the vascular endothelium led to similar results (Motterlini rr o/.. 1996. Yee rr

d., l996).

Further evidence in the field of immunology has been reported for the interaction

between HO and NOS. Macrophages use agents such as NO to defend the host organism

against invading microorganisms NO also evens effects against normal cells located

proximally to activated macrophages resulting in tissue damage and intlammation.

Turcanu rr d. ( 1998~1. 199Sh) have reported HO- 1 modulation of NOS in macrophages in

situation.; nt' widative .;tre.;.;% and suggest !!!at HO may XI as a 5hc.n-term anti-

inflammatory asent and limit tissue damage under circumstances in which YO

hyperproduction leads to toxic effects. These workers suggested that HO inhibits NOS

by degrading its heme cofactor and that it might also reduce c / e trow) NOS synthesis by

decreasing the cellular heme levels (Turcanu rt t r l . . l998h) This postulate is based on the

following Bone marrow-derived macrophages were treated with lipopolysaccharide

(LPS) in order to induce NOS and NOS activitv was assessed bv NO production. When

HO- I was induced in bone-derived niacrophagrs for 24 hrs prior to LPS treatment. UOS

activity, as assessed by NO production, was decreased. The anti-intlarnmatory effects of

HO- I have also been characterized in situations such as experimental arthritis and acute

pleurisy (Willis rt d. 1996). HO was also shown to assume a protecti\.e role in

xenograft endothelium during accommodation (Bach rt tzl. . 1997). Further evidence

contributing to the interaction between the HOKO and NOSMO systems has been

reported in the vascular endothelium it was shown by White and Marietta (1992) that

CO inhibits NOS activity by binding to the heme moiety of the enzyme.

Further evidence implicating a potential interaction between the HO and NOS

systems and a possible role for HO modulation of NOS lies in the marked similarities in

the location of HO and NOS. Both isozymes of HO (HO-I and HO-2) along with NOS

and sGC were shown to be co-localized in select neurons of the brain using

imrnunohistochemical analysis (Yincent and Maines, 1994) I t was found that many

neurons expressing HO-2 correspond to those known to express high levels of sGC ( a

hemoprotein) as well In other studies in blood vessels. HO-2 and NOS displayed the

same localization in endothelium and adventitial neurons: in the intestine. HO-2 and

neuronal W S were both Iocdized to the rnyenteric plexus Due to the cc-localiza!!c\n (3'

HO with these hemoprotein enzymes. HO is ranted access to other sources of heme

~vithin these cells and thus. provides further rationale for the hypothesis of HO

modulating NOS and sGC activity

In our present studies. CrMP (5ph.l) has been shown to inhibit HO wthout

afiectiny NOS or sGC This will facilitate testiny the hypothesis that HO may regulate

the activity of NOS and sGC by degrading the heme moieties of these enzymes. similar to

the manner in which HO has been shown to degrade the heme moiety of certain

cvtochrome P150 isozyrnes We propose ro accomplish this by co-incubating sGC or

NOS with HO and then measuring the change in enzymatic activity followiny HO

exposure in addition to the broken cell preparations described above, we would measure

the effects of 140 on NOS and sGC activity in intact tissues .A selective HO inhibitor is

required to demonstrate whether any eflects seen in NOS or sGC activity are in b c t due

to HO or some other aspect of the reactions.

One of the criticisms of CO as a signaling molecule is that it has a lower potency

than NO. However, it has been recently shown that CO, in the presence of YC- I . can

stimulate sGC to levels similar to that observed with NO. Thus, the search for an

endogenous YC-1 analog has become increasingly important to CO research in order to

redefine the physiological relevance of CO. Certain structural characteristics have been

sugested by Sharma et ~ 1 1 ( 1999) to be responsible for the stirnulatory actions of YC- 1

These workers attribute the sGC-activating abilities of YC-I to the fact that it has two

nitrogen groups, which. according to Sharma rt of. are responsible for coordinating with

ihe heme iron by donating a non-bonding pair of electrons This structural feature along

wi th its hydrophobic nature are prcposed to acccun! f ~ r .;GC ~timulatinn hy YC-I Both

biliverdin and bilirubin possess four nitrogen groups and are hydrophobic The

poseibilitv exists that either of these compounds may serve as an endogenous YC-1

analog and we propose to compare the effects of biliverdin and bilirubin with the rtfects

of YC- I on sGC in the presence and absence of CO

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