Journal of Physiology (1996), 491.3, pp.669-675

Acetylcholine inhibits Ca2+ current by acting exclusively at asite proximal to adenylyl cyclase in frog cardiac myocytes

Jonas Jurevicius and Rodolphe Fischmeister *

Laboratoire de Cardiologie Cellulaire et Moleculaire, INSERM U-446,Universite de Paris-Sud, Faculte de Pharmacie, F-92296 Chatenay-Malabry, France

I The effects of acetylcholine (ACh) on the L-type Ca2P current (ICa) stimulated byisoprenaline (Iso) or forskolin (Fsk) were examined in frog ventricular myocytes using thewhole-cell patch-clamp technique and a double capillary for extracellular microperfusion.

2. The exposure of one half of the cell to 1 /SM Iso produced a half-maximal increase in ICasince a subsequent application of Iso to the other half induced an additional effect of nearlythe same amplitude. Similarly, addition of 1 /,M ACh to only one half of a cell exposed toIso on both halves reduced the effect of Iso by only -50 %.

3. When 10 /M Iso or 30 uM Fsk were applied to a Ca2+-free solution on one half of the cell,ICa was increased in the remote part of the cell where adenylyl cyclase activity was notstimulated. However, addition of ACh (3-10 /M) to the remote part had no effect on ICa'while addition of ACh to the part of the cell exposed to Iso or Fsk strongly antagonized thestimulatory effects of these drugs.

4. Our data demonstrate that ACh regulates Ica by acting at a site proximal to adenylylcyclase in frog ventricular cells. We conclude that the muscarinic regulation of Ica does notinvolve any additional cAMP-independent mechanisms occurring downstream from cAMPgeneration.

The cardiac negative inotropic effect of acetylcholine (ACh)in the heart results from the modulation of severalregulatory pathways and ion channels (Hartzell, 1988;Lindemann & Watanabe, 1991). One of the best studied ofthese is the inhibition of the L-type Ca2+ current (Ica) Withthe exception of myocytes in the sino-atrial node (Petit-Jacques, Bois, Bescond & Lenfant, 1993), ACh has no effecton basal Ica but it strongly antagonizes the stimulatoryeffect of isoprenaline (Iso; Hescheler, Kameyama &Trautwein, 1986; Fischmeister & Hartzell, 1986), orforskolin (Fsk; Fischmeister & Shrier, 1989). Both Iso andFsk stimulate Ica through activation of adenylyl cyclaseand subsequent cAMP-dependent phosphorylation (Hartzell,1988). Although ACh clearly inhibits adenylyl cyclaseactivity in cardiac myocytes (Hartzell, 1988; Lindemann &Watanabe, 1991), several recent studies have shown thatACh may inhibit Ica via a number of additionalmechanisms, such as activation of protein phosphatase(Herzig, Meier, Pfeiffer & Neumann, 1995) or nitric oxide(NO) synthase (Han, Shimoni & Giles, 1994, 1995;Balligand et al. 1995; Wang & Lipsius, 1995).

It was our aim in this study to evaluate the contribution ofthese mechanisms to the muscarinic regulation of Ica infrog ventricular cardiac myocytes. To delineate thesemechanisms, we examined the effect of ACh underconditions in which ACh inhibition of adenylyl cyclase didnot take place. This was made possible by the use of arecently developed double-barrelled microperfusiontechnique. This technique allows the exposure of only partof a cell to a drug and independent recording of the 'local'and 'distant' effects of the drug on I.. (Jurevicius &Fischmeister, 1996). As shown in our recent study, the localeffect of Iso or Fsk is due to adenylyl cyclase activation inthe part of the cell exposed to these drugs, while thedistant effect is due to passive diffusion of cAMP from theexposed part to the remote part of the cell (Jurevicius &Fischmeister, 1996). We have now used this method toexpose either one or the other part of the cell to ACh, inorder to separate the effect of ACh not related to adenylylcyclase inhibition from the overall effect of the drug.

Preliminary results have appeared in abstract form(Fischmeister & Jurevicius, 1995).

* To whom correspondence should be addressed.

This manuscript was accepted as a Short Paper for rapid publication.

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J Jurevicvius and R. Fischmeister

METHODSPatch-clamp studies with frog ventricular myocytesVentricular cells were enzymatically dispersed from frog (Ranaesculenta) heart with a combination of collagenase (Yakult, Japan)and trypsin (Sigma) as described previously (Fischmeister &Hartzell, 1986). Frogs were decapitated and double pithed. Theisolated cells were stored in standard Ringer solution and kept at4 °C until use (2-48 h after dissociation). Rod-shaped Ca2-tolerant frog ventricular myocytes (250-400 ,um long) were sealedat both ends to two patch-clamp pipettes (resistance, 1P0-1P5 MQi)and whole-cell recording conditions were established for bothelectrodes using two independent patch-clamp amplifiers (RK400;Bio-Logic, Claix, France). One electrode (El) was under voltage-clamp conditions to measure ICa (Fig. 1). ICa was measured bydepolarizing the cell every 8 s to 0 mV for 200 ms from a holdingpotential of -80 mV. K+ currents were blocked by replacing all K+ions with intracellular and extracellular Cs+ (Fischmeister &Hartzell, 1986) and the fast Nae current was blocked bytetrodotoxin. ICa was measured as the difference between peakinward current and the current at the end of the 200 ms pulse(Fischmeister & Hartzell, 1986). The other electrode (E2) wasunder current-clamp conditions (at zero current) to allow themeasurement of membrane potential at the most remote part ofthe cell (Fig. 1). The voltage signal measured by E2 (V2) wascompared with the clamp potential (Vi) provided by the voltagestimulator using an ordinary electronic negative feedback system.This allowed the adjustment of the voltage applied to El in orderto reduce the difference between V2 and V,. Under theseconditions, even when the peak ICa amplitude reached 3 nA, this

difference was < 3 mV (Fig. 1). All experiments were performed atroom temperature (19-26 °C).

Microperfusion of single cardiac myocytesAfter the breaking of the patch membrane at the tips of bothelectrodes and establishment of whole-cell recording conditions,El and E2 were moved separately so that the cell was positionedtransversally at the mouth of two adjacent capillaries (squaresection, 400 x 400 sm) made out of Plexiglass and separated by anintermediate wall -5 ,um thick (Fig. 1). The cell was thus exposedto two different solutions (SI, S2). Mixing of the solutions at the cellmembrane was minimized by (i) pressing the cell against the wall,(ii) positioning the tips of El and E2 inside the mouth of thecapillaries, and (iii) pressure ejection of the solutions out of thecapillaries at a linear velocity of -2 cm s l.

Solutions and drugsThe control Ringer solution contained (mM): 107 NaCl, 10 Hepes,20 CsCl, 4 NaHCO3, 0-8 NaH2PO4, 18 MgCl2, 1-8 CaCl2, 5D-glucose, 5 sodium pyruvate, 3 x 10-4 tetrodotoxin; pH 7*4,adjusted with NaOH. Ca2+-free solution was obtained by simplyomitting CaCl2 from the above solution (no CaP+ buffer added).Patch electrodes were filled with control internal solution whichcontained (mM): 119-8 CsCl, 5 EGTA (acid form), 4 MgCl2, 5creatine phosphate (disodium salt), 3.1 Na2ATP, 0-42 Na2GTP,0-062 CaCl2 (pCa 8 5), 10 Hepes; pH 7-1, adjusted with CsOH.Isoprenaline was prepared as stock solutions of 1 mm in 5 mMascorbic acid. Forskolin was prepared as stock solutions of 10 mMin 96% ethanol and each solution tested contained a similaramount of ethanol (< 03 %). Acetylcholine was prepared as stock

0

Current clamp

~~~S2 _ p E2

mv

-80

0

pA

-500

100 ms

Figure 1. Schematic diagram of the double-barrelled microperfusion of single frog ventricularmyocytesA frog ventricular myocyte was sealed at both ends to two patch electrodes, El and E2, and whole-cellrecording conditions were established for both electrodes. El and E2 were under voltage-clamp andcurrent-clamp conditions, respectively. The individual traces on the right indicate the current measuredby El (bottom) and the voltage measured by E2 (top) in response to a 0 mV command potential appliedduring 200 ms to El (dashed line) from a holding potential of -80 mV. (For further details, see Methods.)

J Physiol. 491.3670

Muscarinic inhibition oj

solutions of 1 or 10 mm in distilled water. Tetrodotoxin was fromLatoxan (Rosans, France) and all other drugs were from Sigma.

All values are given as the means + the standard error of the mean(S.E.M.).

RESULTSThe double-barrelled microperfusion system described inFig. 1 allowed partial exposure of a cardiac myocyte todifferent drugs. When the cell was positioned transversallyat the mouth of the two adjacent capillaries, approximatelyhalf-way between the two capillaries, one half of the cellwas exposed to Si and the other half to S2. ICa could beinhibited in one region or the other by omitting Ca2+ ions(no Ca2+ buffer added) from Si or S2. When this was done,whole-cell Ic. amplitude was reduced by 51-1 + 2-2%(n = 4). In each experiment, this reductioh in Ica was usedto quantify the portion of the cell membrane exposed toeach solution assuming a homogeneous distribution of Ca2+channels on the cell membrane. As shown previously(Jurevicius & Fischmeister, 1996), the exposure of one halfof the cell to a large concentration of Iso (1 #M) produced anon-maximal stimulation of Ica (Fig. 2). Indeed, a

e cardiac Ca2" current 671

subsequent application of Iso to the other half of the cellinduced an additional effect of nearly similar amplitude.We now show that ACh also had different effects on Ica'depending on whether the muscarinic agonist was presentin one or in both halves of the cell. Indeed, addition of 1 ,UMACh to Si totally antagonized the stimulatory effect of Isoadded to Si but had little effect on the stimulation inducedby the presence of Iso in S2 (Fig. 2). This was observed infour similar experiments, the results of which can besummarized as follows. When the entire cell was exposed to1/M Iso, ICa was increased by 882 + 54% over its controlamplitude (147-7 + 48-8 pA; n = 4). In these cells, additionof ACh to only one half of the cell reduced ICa to 463 + 55%above control. This was somewhat smaller than the degreeof ICa stimulation observed in the absence of ACh, when Isowas present in one half of the cell only (529 + 36%).However, when both drugs were applied to both sides of thecell, as in conventional experiments, ACh reduced the Iso-stimulated ICa to 12 + 8% above its control amplitude.

The above experiments indicate that ACh is unable toproduce a complete inhibition of Iso-stimulated ICa whenonly one half of the cell is exposed to ACh. This may

pA-500

I 1 ,MAChS2 0 Ca2+ 1 /M Iso

S1 1 /SM ISO

1 uM ACh

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ca

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Time (min)15

Figure 2. Half versus whole-cell exposure to Iso and AChAs indicated by the diagram, the cell was positioned approximately half-way between SI and S2. In thisand subsequent figures, the current traces shown at the top were obtained at the times indicated by thecorresponding letters in the bottom graph. SI and S2 initially contained control Ringer solution. During a

short period, Ca2P ions were omitted from S2 (O Ca2+) and ICa rapidly decreased by 44%. This indicatesthat 44% of the total cell surface was exposed to S2, the rest of the cell being exposed to SI. Afterreintroduction of Ca2P ions into S2, Iso (1 uM) was added to St or to both S1 and S2 during the periodsindicated. Subsequently, ACh (1 uM) was added to S1 or to both S1 and S2, together with Iso.

1*2 -

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0-8 -

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0-4 -

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J Physiol. 491.3

J Jurevic'ius and R. Fischmeister

suggest that activation of only one half of the total numberof muscarinic receptors is insufficient to fully antagonizethe response of ICa generated by the activation of the totalnumber of /3-adrenergic receptors. Alternatively, it mayindicate that ACh inhibits ICa by acting locally at a siteproximal to cAMP generation. If the latter hypothesis iscorrect, ACh should exert an effect on Ica only if AChactivates a population of muscarinic receptors which areclose to the population of adenylyl cyclases activated by Iso.To test this hypothesis, ACh was applied alternately on oneor the other half of a cell while only one half of the cell wasexposed to Iso. In addition, Iso was applied in a Ca2+-freesolution (Fig. 3) so that the only Ca2+ channels generating acurrent were those not exposed to Iso. To fully eliminateresidual contamination between solutions, propranolol(1 /,M) was added to the distant part of the cell. Underthese conditions, the increase in ICa reflects the 'distant'effect of the drug (Jurevicius & Fischmeister, 1996). A10 lM concentration of Iso was used to produce a cleardistant effect on ICa (Jurevicius & Fischmeister, 1996).Adding Ca2+ ions to the Iso-containing solutions for shortperiods elicited an additional increase in Ica' whichreflected the local effect of the drug (Jurevicius &Fischmeister, 1996). Distant and local effects of 10 /zM Iso

on Ica corresponded, respectively, to a 494 + 68 and a350 + 79% (n = 7) increase in Ica over the basal level of Icameasured independently in each half of the cell. Figure 3shows that the addition of 10 uM ACh to the part of the cellnot exposed to Iso had absolutely no effect on either thedistant or the local effect of Iso (353 + 67 and 474 + 70%,respectively; n = 7). However, addition of a 10-fold lowerconcentration of ACh (1 /M) to the part of the cell exposedto Iso fully blocked both the local (25 + 17%) and distanteffects of Iso (20 + 4%; n = 7). Thus, to exert an effect onIcw ACh needs to be added to the part of the cell exposedto Iso.

Similar experiments were performed using Fsk instead ofIso to stimulate Ica. Forskolin produces a 10-fold morehomogeneous distribution of intracellular cAMP than Iso(Jurevicius & Fischmeister, 1996). Thus, when a saturatingconcentration of Fsk is applied locally to one half of the cell,local and remote Ca2+ channels are equally and maximallyactivated (Jurevicius & Fischmeister, 1996). Figure 4 showssuch an experiment in which Fsk (30 /M) was applied tohalf of the cell, first in a Ca2+-free solution and then, forshort periods, in a Ca2+-containing solution. Forskolinproduced strong distant and local effects on ICa even in thepresence of ACh (3/M) in the remote part of the cell.

.........r

100 ms0 Ca2+ 0 Ca2+ 0 Ca2+ 0 Ca2+ 0 Ca2+

S2 Io10FM ISO 10 #M Iso + 1 /ZMACh

----------1p--------------------------------------------rS1 1 /icm propranolol

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0 -

C

-_p

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d f-_

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0 5 10 15 20 25Time (min)

Figure 3. ACh inhibits the local but not the distant effect of IsoAs indicated by the diagram, the cell was positioned approximately half-way between St and S2. Sicontained propranolol (1 /M) throughout the entire experiment. After 2-3 min, Ca2' ions were omittedfrom S2 (O Ca2+) and ICa rapidly decreased by 52%. Addition of Iso (10 SM) to 82 induced a net increase inICa' which was not prevented by the addition of acetylcholine (10 /M) to St. However, addition of 1 uMACh to S2 together with Iso totally eliminated the distant effect of the drug. When a steady state wasachieved, Ca2+ ions were briefly reintroduced into S2, together with Iso alone or in the presence of ACh, toevaluate the local effect of Iso on Ica-

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Muscarinic inhibition oj

Indeed, with 3 /M ACh added to the part of the cell notexposed to Fsk, 30 UM Fsk increased ICa by 528 + 55%locally and by 534 + 21 % (n = 7) distantly over the basallevel of ICa measured independently in each half of the cell.However, when ACh was added to the part of the cellexposed to Fsk, then the effects of Fsk on local and distantCa2+ channels were dramatically reduced (168 + 69 and66 + 27 %, respectively; n = 4; Fig. 4).

DISCUSSIONUsing a double-barrelled microperfusion technique inwhole-cell patch-clamped frog ventricular myocytes, wehave shown in this study that (i) ACh had no significanteffect on ICa when applied to the part of the cell notexposed to Iso or Fsk, whereas (ii) in the same cells, AChexerted profound inhibitory effects on ICa when applied tothe part of the cell exposed to these drugs. Thus, weconclude that ACh regulates Ic. by acting exclusively at asite proximal to adenylyl cyclase.

This conclusion in itself is not new. Indeed, we have shownpreviously in frog ventricular myocytes that AChantagonizes the effects of Iso (Fischmeister & Hartzell,1986) or Fsk (Hartzell & Fischmeister 1987; Fischmeister &

b

pA

-500

a

100 ms

f cardiac Ca2" current 673

Shrier, 1989) on ICa but has no effect on the current elevatedby intracellular perfusion with cAMP (Fischmeister &Hartzell, 1986). Similar observations have been made inguinea-pig cardiac myocytes (Hescheler et al. 1986) and infrog atrial myocytes (Nakajima, Wu, Irisawa & Giles, 1990)and had already led the authors to the conclusion that theinhibitory action of ACh on ICa took place at a siteproximal to the production of cAMP. However, in the lightof recent findings obtained in rat (Balligand et al. 1995),guinea-pig (Levi, Alloatti, Penna & Gallo, 1994; Herzig etat. 1995), rabbit (Han et al. 1994, 1995) and cat (Wang &Lipsius, 1995) cardiomyocytes, two alternative cAMP-independent mechanisms have been proposed to participatein the muscarinic regulation of cardiac ICa. Thesemechanisms are (i) an activation of protein phosphatase(Herzig et al. 1995), and (ii) an activation of NO synthaseleading to an accumulation of cGMP (Mubagwa,Shirayama, Moreau & Pappano, 1993; Levi et al. 1994; Hanet al. 1994, 1995; Wang & Lipsius, 1995). Both mechanismsshould regulate ICa by acting at steps located downstreamfrom cAMP generation. Indeed, exogenous applications ofNO (Mery, Pavoine, Belhassen, Pecker & Fischmeister,1993), cGMP (Hartzell, 1988; Lohmann, Fischmeister &Walter, 1991), or protein phosphatases (Kameyama,

d

0 Ca2+ 0 Ca2+ 0 Ca2+

S2 < 30,uM Fsk 30.uMFsk+3.uM ACh------------------------------------------------------_.

1 > _

1-2 -

- 1.0-c

c 0-8-

o 0-6-E= 04-

o 0-2-

0-0 -

0

3/M ACh

C

d

afb~~ ~ ~ ~ ~ ~

5 10Time (min)

15 20

Figure 4. ACh inhibits the local but not the distant effect of FskAs indicated by the diagram, the cell was positioned approximately half-way between Si and S2. S1contained ACh (3/SM) throughout the entire experiment. After 2-3 min, Ca2+ ions were omitted from S2(O CaP+) and ICa rapidly decreased by 52%. Addition of Fsk (30 /SM) to S2 induced a net increase in ICa'which was not prevented by the presence of ACh in SI. However, addition of ACh to S2 together with Fskeliminated the distant effect of Fsk. When a steady state was achieved, Ca2P ions were brieflyreintroduced into S2, together with Fsk alone or in the presence of ACh, to evaluate the local effect of Fskon Ica-

J Phy8iol.491.3

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J Jurevicius and R. Fischmeister

Hescheler, Mieskes & Trautwein, 1986) inhibit ICa incardiomyocytes intracellularly dialysed with cAMP. Thus,one would expect ACh to also inhibit ICa in myocytesdialysed with cAMP, but this was not the case (Fischmeister& Hartzell, 1986; Hescheler et al. 1986; Nakajima et al.1990). While the reasons for this discrepancy are not clear,the possibility exists that perfusing the patch pipette with asolution containing cAMP may obviate cAMP-independentactions of ACh. Indeed, intracellular perfusion withexogenous cAMP differs from an elevation of cAMP by Isoor Fsk. In the latter, cAMP is elevated by an endogenousproduction which takes place at the sarcolemmal membrane,whereas in the first case, cAMP concentration is buffered inthe cytoplasm by continuous dialysis of the cell with freshsolution.

For this reason, we have re-examined the effects of AChunder conditions in which cAMP concentration is elevatedby an endogenous production. However, to separate thecAMP-independent effects of ACh from its classical andstrong action on adenylyl cyclase activity, we used a double-barrelled perfusion technique. This technique allowed theseparation of the surface of a cardiac myocyte into twodistinct areas independently superfused with differentsolutions. This technique was used in a recent study toanalyse the local and distant effects of Iso and Fsk in frogventricular cells (Jurevicius & Fischmeister, 1996). In thatstudy, we found that in a portion of a cell not exposed toIso or Fsk, the Ca2P channel activity can be increased whenthe adenylyl cyclase is activated by Iso or Fsk in the otherpart of the cell. This is due mainly to passive diffusion ofcAMP from one side of the cell to the other. However,cAMP may diffuse to different extents depending onwhether Iso or Fsk is used to activate the cyclase(Jurevicius & Fischmeister, 1996). Also, ACh inhibition ofICa differed markedly depending on whether Iso or Fsk was

used to stimulate the current (Fischmeister & Shrier, 1989).For these reasons, we compared the effect of ACh on boththe local and distant effects of Iso and Fsk on IcaWhile our experimental approach was designed tospecifically dissect the cAMP-independent effects of AChfrom the overall effects of the drug, we found that ACh wasunable to inhibit the distant effect of Iso or Fsk on ICawhen applied far from the sites of action of Iso and Fsk.Although these experiments will have to be extended tomammalian cardiac myocytes, we conclude that in frogheart the muscarinic regulation of ICa does not involve theparticipation of mechanisms occurring downstream fromcAMP generation, such as activation of protein phosphatase,phosphodiesterase, or NO synthase activities. However, thepossibility remains that such mechanisms exist in the closevicinity of adenylyl cyclase units where they would notaffect Ca2+ channel activity per se but might affectprocesses prior to cAMP generation (e.g. phosphorylation/dephosphorylation of proteins involved in adenylyl cyclase

activity) or prior to cAMP delivery for cAMP-dependentphosphorylation of Ca2P channels (e.g. localized phospho-diesterase activity).

BALLIGAND, J.-L., KOBZIK, L., HAN, X. Q., KAYE, D. M., BELHASSEN,L., OHARA, D. S., KELLY, R. A., SMITH, T. W. & MICHEL, T. (1995).Nitric oxide-dependent parasympathetic signaling is due toactivation of constitutive endothelial (type III) nitric oxide synthasein cardiac myocytes. Journal of Biological Chemistry 270,14582-14586.

FISCHMEISTER, R. & HARTZELL, H. C. (1986). Mechanism of action ofacetylcholine on calcium current in single cells from frog ventricle.Journal of Physiology 376, 183-202.

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FISCHMEISTER, R. & SHRIER, A. (1989). Interactive effects ofisoprenaline, forskolin and acetylcholine on Ca2P current in frogventricular myocytes. Journal of Physiology 417, 213-239.

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HARTZELL, H. C. (1988). Regulation of cardiac ion channels bycatecholamines, acetylcholine and 2nd messenger systems. Progressin Biophysics and Molecular Biology 52, 165-247.

HARTZELL, H. C. & FISCHMEISTER, R. (1987). Effect of forskolin andacetylcholine on calcium current in single isolated cardiac myocytes.Molecular Pharmacology 32, 639-645.

HERZIG, S., MEIER, A., PFEIFFER, M. & NEUMANN, J. (1995).Stimulation of protein phosphatases as a mechanism of themuscarinic-receptor-mediated inhibition of cardiac L-type Ca2+channels. Pfluigers Archiv 429, 531-538.

HESCHELER, J., KAMEYAMA, M. & TRAUTWEIN, W. (1986). On themechanism of muscarinic inhibition of the cardiac Ca current.Pfluigers Archiv 407, 182-189.

JUREVICIUS, J. & FISCHMEISTER, R. (1996). cAMP compartmentationis responsible for a local activation of cardiac Ca2+ channels by/J-adrenergic agonists. Proceedings of the National Academy ofSciences of the USA 93, 295-299.

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LEVI, R. C., ALLOATTI, G., PENNA, C. & GALLO, M. P. (1994).Guanylate-cyclase-mediated inhibition of cardiac ICa by carbacholand sodium nitroprusside. Pfluigers Archiv 426, 419-426.

LINDEMANN, J. P. & WATANABE, A. M. (1991). Mechanisms ofadrenergic and cholinergic regulation of myocardial contractility. InPhysiology and Pathophysiology of the Heart, 2nd edn, ed.SPERELAKIS, N., pp. 423-452. Kluwer Academic Publishers,Boston.

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MERY, P.-F., PAVOINE, C., BELHASSEN, L., PECKER, F. &FISCHMEISTER, R. (1993). Nitric oxide regulates cardiac Cacurrent. Involvement of cGMP-inhibited and cGMP-stimulatedphosphodiesterases through guanylyl cyclase activation. Journial ofBiological Chemistry 268, 26286-26295.

MUBAGWA, K., SHIRAYAMA, T., MOREAU, M. & PAPPANO, A. J. (1993).Effects of PDE inhibitors and carbachol on the L-type Ca current inguinea-pig ventricular myocytes. American Journal of Physiology,Heart and Circulatory Physiology 264, H1353-1363.

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PETIT-JACQUES, J., Bois, P., BESCOND, J. & LENFANT, J. (1993).Mechanism of muscarinic control of the high-threshold calciumcurrent in rabbit sino-atrial node myocytes. Pfluigers Archiv 423,21-27.

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AcknowledgementsWe thank Patrick Lechene and A. Navalinskas for skilful technicalassistance, F. Lefebvre and I. Ribaud for preparation of the cells,and Drs P.-F. Mery, V. Veksler, J.-L. Mazet and A. V. Skeberdis forhelpful discussions. This work was supported by a grant from theAssociation Francaise contre les Myopathies and the Fondationpour la Recherche Medicale. J. Jurevicius was supported by afellowship from the Ministere de l'Enseignement Superieur et de laRecherche.

Author's permanent addressJ. Jurevicius: Laboratory of Membrane Biophysics, Institute ofCardiology, Kaunas Medical Academy, Kaunas 3007, Lithuania.

Received 4 December 1995; accepted 23 January 1996.