M2 muscarinoceptor-associated ionophore at the cat adrenal medulla

Vol. 144, No. 2, 1987

April 29, 1987

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 965-972

M 2 MUSCARINOCEPTOR-ASSOClATED IONOPHORE AT THE CAT ADRENAL MEDULLA

Ricardo Borges, Juan J. Ballesta and Antonio G. Garci~*

Departamento de Neuroquimica,~ Universidad de Al icante, Al icante, Spain

~epartamento de Farmacolog~a, Universidad Aut6nomade Madrid, Arzobispo Morc i l lo , 4, 28029-Madrid, Spain

Received March II, 1987

SUMMARY: Atropine and pirenzepine displaced 3H-quinuclydinyl-benzylate binding and inh ib i ted methacholine-evoked catecholamine release with a s imi lar order of potencies, atropine being 200 fold ~ r e potent than pirenzepine. In contrast to high-K, methacholine-evoked ~Ca uptake or catecholamine release were not blocked by (+)PN200-110. Bay-K-8644 did not modify the secretory response to methacholine e i ther in the presence of Ca or Sr but potent iated K-evoked secretion. In depolarized glands, methacholine s t i l l evoked i t s usual secretory response. The resul ts suggest that muscarinic st imulat ion of cat adrenal chromaffin ce l ls stimulates Ca entry though an ionophore other than voltage-dependent Ca channels; such ionophore seems to be chemically operated through a M 2 muscarinoceptor. ~ 1987Ac~demice ..... ~c.

Muscarinoceptor st imulat ion causes d i f fe ren t biochemical and

physiological responses in chromaffin ce l ls from various animal species;

the subtype of muscarinoceptor mediating those responses is unknown. In the

cat (1,2) , ~ gerbi l (3), guinea-pig (4) and rat (5-7) a muscarinoceptor

mediates an increase of the rates of catecholamine release. In contrast,

muscarinic st imulat ion does not enhance catecholamine secretion from

f reshly isolated (8,9) or cultured bovine adrenal chromaffin ce l ls (10-14),

yet i t increases cGMP levels (10,15), phospholipid turnover (12,13) and

i n t r ace l l u l a r free Ca concentrations (16). How these changes re late to the

physiological control of the st imulus-secret ion coupling process fol lowing

st imulat ion of chromaffin ce l ls by endogenously released acety lchol ine, and

why in several species ( fe l i ne , rodents), but not in others (bovine),

muscarinoceptors t r igger catecholamine release, are fundamental questions

that might be explained by assuming a coupling of th is receptor to a

speci f ic membrane ionophore or channel in the fe l i ne , but not in the bovine

adrenal gland. In th is paper, we provide evidence suggesting that the cat

adrenal chromaffin cel l p re fe ren t ia l l y secretes adrenaline in response to

*To whom correspondence should be addressed.

965

0006-291X/87 $1.50 Copyright © 1987 by Academic Press, Inc.

All rights of reproduction in any form reserved.

Vol. 144, No. 2, 1 9 8 7 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

methacholine by act ivat ing Ca entry into the cell through an ionophoric

channel associated to or controlled by a M2-type muscarinic cholinoceptor;

such channel is chemically operated and seems to be unrelated to voltage

sensit ive Ca channels.

METHODS

3H-quinuclydin¥] benz~late binding

Aliquots (100 ~g protein/ml) of an 800xg supernatant homogenate from cat adrenomedullary or at r ia l tissues (20 volumenes of 50 mM Tris-HCl buffer, pH 7.4) were incubated at ~7gC for i h in 50 mM phosphate buffer pH 7.4 containing 0.02-I ~M (-) H-QNB (Amersham, specif ic ac t i v i t y 33 Ci/mmol). The procedure was based in that of Kayaalp and Neff (17). Non- specif ic binding was defined as the rad ioact iv i ty bound in the presence of I~M atropine.

Catecholamine release

Both cat adrenal glands were isolated and perfused at room temperature (25 + 2QC) with Krebs-bicarbonate solution bubled with 95% 0p-5% CO 9 at pH L4 . Solutions containing high K were prepared by adding KCl :and reducing isoosmotically NaCI. Catecholamine release were continuously monitored by on-line connection of the perfusion f lu id emanating from the glands to a Metrohm electrochemical detector; in some experiments, noradrenaline and adrenaline were separated by high performance l iquid chromatography (Series i0 Perkin-Elmer) (18).

45Ca uptake

After equi l ibrat ion with Krebs-bicarbonate solution, glands were ~rfused at i ml/min during 90 min with solutions containg 16 ~Ci/m] of ~Ca (Amersham, sp., act. 40 mCi/mg) and then washed for 5 min with

radioactive-free fresh solution. The stimulating and washing solutions contained 25 mM Na" (as NaHCOq), 236 mM sucrose and 250 }JM CaCI~ as well as the rest of the components of the Krebs solution. At the end o~the washing period, glands were frozen in l iquid nitrogen, thei r medullae careful ly dissected out, digested overnight in 1 ml of 2% sodium dodecylsulfate at 37QC and thei r rad ioact iv i ty contents counted in a Beckman 2800 model sc in t i l l a t i on counter.

RESULTS AND DISCUSSION

Both, radioligand binding studies and secretion suggest the M 2

nature of the cat adrenal medulla muscarinoceptor (Fig. 1).

3H-quinuclydinyl benzylate (3H-QNB) binding to cat adrenomedullary and at r ia l membranes was highly specif ic (more than 90%), saturable and the

Scatchard analysis revealed a single population of receptors with a n H

Hi l l coeff ic ient of 1.07 for the adrenal medulla. 3H-QNB bound was

displaced by atropine and pirenzepine with a similar order of potency,

atropine (a non-specific antagonist) being 200 fold more potent than

pirenzepine (a M 1 specif ic antagonist) (9). Various muscarinic agonists

given at 3-30~M for 30 s enhanced catecholamine release from perfused cat

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Vol. 144, No. 2, 1987 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

..~ A : Atrium

"A°reno, u3 100 o

rfl

I r~ 601 Atrnnin~\ ~ ~pirenzepine

"~ 20

10 9 8 7 6 5

- log [Drug] (M)

o IO0 E o 80

~ 60

m

L. 20 < u

••Plrenzepine 10 9 8 7 6

- tog [D rug ] (M)

Fig. 1. A. Displacem@nt by muscarinoceptor antagonists of 3H-Quinuclydinyl benzylate (~H-QNB) binding to cat adrenomedullary and atr ial membranes. Atropine or pirenzepine were preincubated for i0 min with the homogenate at the c4)ncentrations shown in the abscissa; a concentration of 0.5 nN of ~H-QNB was used. Displacement data are the means ~ s.e. of 3 experiments made in t r ip l icate.

B. Inhibition by muscarinoceptor antagonists of catecholamine release eveked by methacholineo Methacholine pulses (3~M for 30 s) were given at 30 min intervals. Once the secretory response stabilized, cumulative concentrations (abscissa) of atropine or pirenzepine were added and the methacholine pulses repeated in their presence 10 min later. Data are means + s.e. of 4 experiments.

adrenal glands with the fol lowing re la t ive order of potencies:methacholine>

pilocarpine>oxotromerine>McN-A-343>betachenchol>muscarine. Being the

most potent, methacholine was selected to perform the fol lowing

experiments. Atropine was lO0-fold more potent than pirenzepine in

inh ib i t i ng methacholine-evoked release, suggesting again that the

muscarinoceptor involved in th is response is of the M 2 subtype.

Methacholine- and K+-evoked catecholamine release with 30 s pulses

have in common (Fig. 2): ( I s t ) that secretion reaches a quick r ise to a

peak and a decline to basal levels; (2nd) that repet i t i ve ident ical st imul i

(applied at 15-min in terva ls) give s imi lar responses showing l i t t l e

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5 m i n , , , , , i

• • • m 0 Co 2 • • • i

• Methachol ine(3jaM 30s.)

• • • m 0 Co2", • • •

• Potassium (177 mM 3 0 s )

Fig. 2. Profiles of catecholamine secretory responses obtained upon perfusion of cat adrenal glands with high K concentrations or with methacholine. The graphs show the release of total catecholamines evoked by 30-s pulses of methacholine (top) or K (bottom). On Ca deprivation (horizontal bars), the release to methacholine or K was abolished; upon Ca restoration, the response recovered fu l l y . The horizontal top bar reflects the elapsed time and the vert ical bar the oxydation current obtained in nanoamperes at the electrochemical detector. The curves were drawn direct ly from the recording paper and are taken from a typical experiment out of 5.

desensitization; and (3rd) that, as previously shown (20), Ca deprivation

abolishes the secretory response. These similarities might lead to the

conclusion that both, methacholine and K enhance catecholamine release by

a similar mechanism, i .e . , activation of voltage-sensitve Ca channels.

However, when the secretory responses are carefully analysed, they

considerably dif fer, as the following experiments demonstrate.

Methacholine (3fM for 30 s) released 207 ~ 23 ng/pulse (n = 20) of

total catecholamines from which, 80% accounted for adrenaline; in contrast,

K (17.7 mM for 30 s) released 310 ~ 30 ng/pulse of catecholamines from

which 48% was noradrenaline and 52% adrenaline, suggesting that only

muscarinic stimulation discriminates between adrenergic and noradrenergic

chromaffin cells.

The second important d i f ference concerns the membrane po ten t ia l ;

although pi locarpine and acety lchol ine depolarize gerbi l (3) and rat (6)

chromaffin ce l l s and atropine blocks i t , i t is un l i ke l y that muscarinic

depolar izat ion is responsible for the secretory ef fects of methacholine.

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Two facts support th is assert ion: ( i s t ) methacholine-evoked secretion does

not inact ivate af ter prolonged st imulat ion, but the K response quickly

inact ivates (21,22); and (2nd) methacholine evokes a f u l l secretory

response even in depolarized glands. In 3 experiments, methacholine (30~M

for 30 s) was applied on top of various K concentrations (1.2, 5.9, 17.7,

35, 59 and 125 mM) once the K response was inact ivated. I t is known that

chromaffin ce l ls remain depolarized af ter long periods of K exposure, in

spi te of the decline of the rate of release to almost basal levels (23,24).

Methacholine evoked a s imi lar secretory response at a l l levels of

depolar izat ion. This f inding contrasts with one obtained using nicot ine as

secretagogue in an otherwise s imi lar protocol: at low K depolar izat ions,

n icot ine evoked a healthy secretory response but at higher depolarizations

nicot ine fa i led to enhance catecholamine release (A.R. Arta le jo and A.G.

Garc~a; unpublished resu l ts ) . So, the nicot ine-secretory effects seem to

require cel l depolar izat ion but methacholine, no.

The th i rd dif ference was established using the S(+) enantiomer of the

dihydropyridine PN200-110, a potent and select ive Ca antagonist in the cat

adrenal medulla (25). In 15 glands, 35 mM K pulses given at 30-min

in terva ls released 3.15+_0.23~g of catecholamines (N=94 pulses); cumulative

concentration-response curves gave an IC50 for (S)(+)PN200-110 to i n h i b i t

K-evoked secretion of 0.9 nM (N=8). At a concentration of I0 nM, that

e f fec t i ve ly blocks high K-stimulated release, the methacholine response was

unaffected (Fig. 3). Furthermore, there was no dif ference in the a b i l i t y to

i n h i b i t methacholine-stimuZated release between the more act ive (+) and the

less active (-) stereoisomers of (+)PN200-110; in contrast, K-evoked

release was inh ib i ted by the (-) enantiomer at concentrations lO0-fold

higher than those required with the (+) enantiomer (25). These resul ts show

a high degree of stereospecif ic i nh ib i t i on of K-evoked release by

(+)PN200-110, but no stereospecif ic i nh ib i t i on of methacholine-induced

release suggesting a d i f fe ren t patway for Ca entry during both types of

st imulat ion. We also performed experiments to test the effects of

muscarinic st imulat ion on 45Ca uptake into adrenomedullary ce l ls (Fig.

3). Methacholine doubled and K t r i p led the basal 45Ca uptake into

adrenomedullary chromaffin ce l l s ; while i0 nM (+)PN200-110 inh ib i ted

K-evoked uptake by 80%, the methacholine effects were not affected by the

dihydropyridine.

An experiment using Sr and Ca as permeant cations established a

c lear-cut f i t h dif ference between K and methacholine. Sr permeates

chromaffin cel l Ca channels better than Ca, causing a greater secretory

969


A

100 -

C 0 u

5 0

O.

o t..)

0 - Potassium

Fig. 3.

(5

Methacholine

B

100 " C 0 u

I/1 0

~ 5C

C

E _a 0 c" u

o 0 L3

(4) (4)

Potassium

i

l ~]Control

[~(+) PN 200-110(10"8M)

Methacholine

A. 45Ca uptake into adrenomedullary chromaffin .cells upon stimulation with methacholine o ~ . Basal uptake of 4bCa amounted to 245+ 30 cpm~g protein; net = Ca uptake evoked by methacholine 4u aQ~C K, with or without (+)PN200-110 (10 nM added 10 min before the

pulse), were calculated by subtracting the basal to the total tissue uptake. Data are means + s.e. of 4-5 experiments.

B. Effects of (S)(+)PN200-110 on catecholamine release evoked by 30 s pulses of methacholine (30 ~M) or K (35 mM). Pulses of methacholine or K were given ~o para l le l glands at 30 min intervals; once the responses were s tab i l ized, cumulative concentrations of (+)PN200-110 were introduced in the perfusion f l u id . Data are means of peak catecholamine release + s.e. of 4 paired experiments.

K ~-

(23.6 mM)

Methacholine (30 ;uM)

A 5min

B haaL .,,,,.,....,..,,, .,,.,,,.....,,,,,,

Ca 2 . S r 2 . Ca2 + S r 2 .

t i

Boy- K-8644 (0.1 ja M)

Fig. 4. Effects of Bay-K-8644 on the secretory responses to sustained st imulat ion with methacholine or high K. In gland B, methacholine (30~M) Was applied f i r s t in the presence of 2.5 mM Ca and then in 2.5 mM Sr; the~e stimulations were repeated in the presence of Bay-K-8644 (10- M). In gland A, a s imi lar protocol was performed but this time using i7.7 mM K as secretagogue. Results from a typical experiment out of 3. The ver t ica l bar corresponds to the oxidation current in nanoamperes.

970


response to high K and a much slower inactivation (21,22,24). Therefore, Sr

should produce different secretion patterns to K and methacholine i f the

cation permeates dif ferently through two possible Ca permeability pathways.

Fig 4a shows that 17.7 mM K produced a much larger response in the presence

of 2.5 mM Sr than in 2.5 mM Ca, agreeing with previous data from our

laboratory (21,22). Interesting enough, Bay-K-8644 (I0-7M), a

dihydropyridine that selectively enhances Ca channels-mediated secretion

(26-28) potentiated drastically the response to K only in the presence of

Ca; although the secretory response to Sr was faster, the drug did not

enhance secretion in the presence of this cation. Fig 4b shows the

responses to methacholine; in the presence of Sr, the i n i t i a l peak is lost

and no potentiation of secretion was seen with respect to Ca. Neither, the

secretory responses to methacholine in Ca or Sr were signi f icant ly affected

by Bay-K-8644.

All these results agree with the idea already suggested by Kirpekar et

al. (2) that on muscarinic stimulation of cat adrenal medulla, Ca enters

the chromaffin cell through an ionophore regulated by this cholinoceptor.

ACKNOWLEDGEMENTS

Supported by grants from C.A.I.C.Y.T. No 621/81, F. I .S.S.. Fundaci6n Ram6n Areces and U.S.- Spanish Joint Committee for Sc ient i f ic and Technological Cooperation No CCA8411029. We thank Dr. P. Hof, from Sandoz Ltd, Basel, Switzerland, for the kind g i f t of (+) and (-)PN 200-110, Prof. F. Hoffmeister from Bayer, A.G., Wuppertal, FRG, for the g i f t of Bay-K-8644, Mr. Miguel A. Diez for the drawings, Mrs. N. Tera for typing the manuscript, and Mr. Victoriano Mandado for expert technical assistance.

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M2 muscarinoceptor-associated ionophore at the cat adrenal medulla

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