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Cholinergic antagonism of β-adrenergic stimulated action potentials and adenylate cyclase activity in rabbit ventricular cardiomyocytes
European Journal of Pharmacology, 155 (1988) 101-108 Elsevier
101
EJP 50456
Cholinergic antagonism of fl-adrenergic stimulated action potentials and adenylate cyclase activity in rabbit ventricular cardiomyocytes Julie M. W a t s o n , Stephen M. Vogel 1, D a v i d J. Cotterell 2 a n d M a g a r i t a L. D u b o c o v i c h * Department of Pharmacology, Northwestern Universi(v Medical School Chicago, IL 60611, U.S.A.
Received 7 June 1988, accepted 12 July 1988
The cholinergic antagonism of/3-adrenergic stimulation was examined by measuring adenylate cyclase activity and calcium-mediated action potentials in isolated ventricular cardiomyocytes of adult rabbits. The fl-adrenoceptor agonist isoproterenol and the direct adenylate cyclase activator forskolin increased adenylate cyclase activity in homogenates of the myocyctes. The cholinergic agonist carbachol (10 nM-100/~M) inhibited in a concentration dependent manner basal, isoproterenol-stimulated and forskolin-stimulated adenylate cyclase activity. The carbachol effect on basal adenylate cyclase activity was antagonized by atropine (10 ~M). In parallel experiments using intact cardiomyocytes. calcium action potentials were elicited by intracellular depolarizing current pulses in partially depolarized preparations. These action potentials were prolonged by isoproterenol, forskolin and dibutyryl cyclic AMP. Acetylcholine reversibly inhibited the prolongation of the action potential induced by isoproterenol and forskolin but not dibutyryl cyclic AMP. These results suggest that cholinergic agonists modulate the increase in the calcium current elicited by isoproterenol and forskolin in isolated ventricular cardiomyocytes by inhibiting adenylate cyclase activity.
C a 2+
action potentials; Cardiomyocytes; Adenylate cyclase activity; Muscarinic acetylcholine receptors; fl-Adrenoceptors; (Rabbit)
1. Introduction
Activation of fi-adrenoceptors and muscarinic acetylcholine receptors in the heart leads to opposite physiological and biochemical responses, t31and fi2-adrenoceptors and M-1 and M-2 muscarinic acetylcholine receptors have been char-
1 Present address: Department of Pharmacology, University of College of Medicine, Chicago, IL 60612, U.S.A. 2 Present address: Glaxo Group Research, Ltd., Clinical Research Division, Greenford Road, Middx, UB60HE. Great Britain. * To whom all correspondence should be addressed: Department of Pharmacology, Northwestern University, 303 East Chicago Avenue, Chicago, IL 60611, U.S.A.
acterized in the heart (Minneman et al., 1979; Buxton and Brunton, 1985; Fields et al., 1978; Watson et al., 1983). Muscarinic acetylcholine receptor agonists have been reported to antagonize fl-adrenergic effects on cardic contractility, the maximum upstroke velocity and duration of calcium-dependent slow action potentials, and activity of adenylate cyclase in the heart (Schwegler et al., 1976), heart muscle homogenates (Watanabe et al., 1978; Jakobs et al., 1979) and embryonic ventricular muscle (Linden et al., 1982). The data from such multicellular preparations suggest that one mechanism by which acetylcholine receptor agonists exert their antiadrenergic effects in the heart may be by modulating adenylate cyclase activity in the ventricular cardiomyocyte.
0014-2999/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
102 Direct analysis of the mechanism of fi-adrenergic and muscarinic interaction in a preparation of ventricular cardiomyocytes isolated from other heart cell types is needed. Recently, it has been reported that while both 131- and/92-adrenoceptors exist in heart muscle, only the fll-adrenoceptor is found on the cardiomyocyte (Buxton and Brunton, 1985). ill-, but not /~2-, adrenoceptor activation has been shown to increase calcium accumulation by the sarcoplasmic reticulum in the rabbit ventricular myocyte (Cotterell et al., 1985); this calcium influx appears to be secondary to increases in cyclic A M P accumulation, mediated by filadrenoceptors. Likewise, recent evidence suggests that while M-2 muscarinic receptors are negatively linked to adenylate cyclcase activity, M-1 muscarinic receptors may instead be linked to phospatidylinositol turnover (Watson et al., 1983; Brown and Brown, 1984). Analysis of acetylcholine receptor and ,8-adrenoceptor interaction in isolated cells also permits study of whether the opposite physiological effects mediated by these receptors occur within the same cell; this is not possible in studies carried out in homogenates of heterogenous membranes and multicellular preparations. The use of isolated myocytes has several advantages for electrophysiological studies. First, the isolated cells are free from influences of autonomic innervation. Thus, effects of agents used here such as forskolin and dibutyryl cyclic A M P can be considered to be direct actions on the myocytes. Secondly, stable microelectrode impalements are possible in isolated myocytes because of the weak contractile force; this permits multiple drug applications to be compared in a single cell. Thirdly, planned studies using voltage and patch clamp methods will be possible in isolated myocytes. Fourth, correlation between biochemical and physiological consequences of receptor activation is possible in isolated myocytes, as the present experiments will show. The present study investigated the effect of activation of muscarinic receptors and /~-adrenoceptors on the calcium action potential and on adenylate cyclase activity in isolated ventricular cardiomyocytes. We report here that acetylcholine receptor agonists appear to exert antiadrenergic
effects on the calcium action potential in isolated ventricular cardiomyocytes by inhibiting adenylate cyclase activity.
2. Materials and methods
2.1. Cardiomyoo'te preparation Rabbit ventricular myocytes were prepared as previously described (Rajs et al., 1978). Briefly, male New Zealand white rabbits (2.5-3 kg) were decapitated and thoractomized. The heart with a large portion of the aorta intact was quickly removed and washed with ice cold saline. The aorta was cannulated and the heart perfused retrogradely at 10 m l / m i n per g wet weight of heart muscle for 30 s with calcium free Krebs buffer to wash out blood remaining in the heart (Rajs et al., 1978). Thereafter the heart was continuously perfused with a calcium free Krebs solution gassed with 95% 02-5% CO~ at 3 7 ° C containing 0.144% Type II collagenase during 30-40 min. The perfused ventricles were cut with scissors into small pieces. If further digestion was required, the pieces of ventricles were incubated for an additional 10 min in Krebs solution containing 0.144% collagenase at 3 7 ° C in a shaking waterbath. The resulting cell suspension was filtered through 250 ~ m nylon gauze and centrifuged at 40 ×,g for 1 rain. The cells were washed three times with the standard calcium free Krebs solution, were layered on 4% bovine serum albumin (fraction V Sigma) and the suspensions centrifuged at 25 >( eg for 1 min. The dispersed cells were resuspended in IV bovine serum albumin. After 15 min, the cells were resuspended in Krebs solution containing 2.6 mM CaC12. Isolated cells were examined microscopically and viability assessed by noting shape and trypan blue exclusion.
2.2. Adenylate cyclase activi O' Adenylate cyclase activity was assayed in homogenates of the isolated myocytes by measuring the conversion of a-[)2p]ATP to [32p]cyclic AMP according to the method of Solomon (1979) with modifications as described by Minneman et al.
103
(1979). Myocytes were homogenized by hand, centrifuged for 5 min at 35000 × g and resuspended to a concentration of 6.6 × 105 cells per ml in 2 mM HEPES buffer containing 2 mM EGTA, pH 7.5 at 4°C. The reaction mixture contained 1-2 million cmp of ce-[32P]ATP (New England Nuclear), 50 mM HEPES (pH 7.5), 0.05 mM cyclic AMP, 1 mM MgC12, 0.25 mM ATP, 1 mM theophylline, 0.1 m g / m l creatine kinase, 10 mM phosphocreatine, 50 mM GTP, 50 mM NaC1, and appropriate drugs. The reaction was initiated by addition of an aliquot of homogenates and incubated for 10 rain at 30 o C. The reaction was terminated by the addition of 100/~1 of 1% sodium lauryl sulfate and 5 mM ATP in 50 mM Tris buffer. Samples were then boiled for 10 min to improve column flow during separation. The [32p]cyclic AMP was separated by sequential chromatography on Dowex A G 50 WX4 and alumina columns. Recovery was monitored by addition of [3H]cyclic AMP (10 nCi) to each sample prior to chromatography. 32p and 3H content of final elute were determined simultaneously by liquid scintillation spectrometry. 32p content was corrected for recovery and adenylate cyclase activity expressed as pmol cyclic AMP f o r m e d / m i n per mg protein. Protein concentration of the homogenates was determined by the method of Bradford (1976).
digitized using an analog to digital converter and stored on a computer for subsequent off line analysis. Action potential amplitude and duration (ms) at 100% repolarization were measured in control conditions and in the presence of drugs. Drugs were added to a bath volume of 2 ml (35 + I ° C ) at a superfusion rate of 6 ml/min.
3. Results
3.1. Dual control of adenylate cyclase activity' by" fl-adrenoceptor and muscarinic receptor activation Adenylate cyclase activity was used as a functional m e a s u r e m e n t in h o m o g e n a t e s of cardiomyocytes of fl-adrenoceptor and muscarinic receptor activation. The basal adenylate cyclase activity in homogenates of the cardiomyocyctes was 35 _+ 2 pmol [32p]cyclic AMP f o r m e d / m i n per mg protein when assayed in the presence of the phosphodiesterase inhibitor theophylline (table 1). The muscarinic acetylcholine receptor agonist carbachol inhibited adenylate cyclase activity in the cardiomyocytes in a dose dependent manner (fig. 1). The maximal inhibition of adenylate cyclase activity produced by carbachol was 36 _+ 4.7% (n = 4) below basal at 100 ~M (fig. 1~ table 1) with an IC~0 of 100 nM. The inhibiting effect of carbachol was completely antagonized by
2.3. Calcium action potentials Parallel electrophysiological experiments were conducted in intact cardiomyocytes. Myocytes were suspended in a buffer containing 2 mM CaC1 z and 20 mM KC1. This K + concentration partially depolarized the membrane to a resting potential of - 4 0 mV. Under these conditions an intracellular depolarizing pulse elicits an action potential whose inward current is carried by calcium ions. Myocytes were impaled with a conventional microelectrode filled with 3 M KC1 and with a resistance of 40-60 M~2. Each slow action potential was elicited by an intracellular depolarizing current pulse delivered through the microelectrode by means of a bridge circuit. The frequency of stimulation was 0.33 Hz. Action potentials were
TABLE 1 Effect of carbachol on adenylate cyclase activity in homogenates of rabbit vertricular cardiomyocytes. Values represent the mean_+ S.E.M. of three to five determinations performed in duplicate. ~ P < 0.05; h p < 0.005 when compared to corresponding control using Student's t-test. Adenylate cyclase activity (pmol c A M P / min per mg protein) Control Basal Isoproterenol (10 p,M) Forskolin (10/~M)
% inhibition
Carbachol (100 ~M)
35+_ 2
21_+ 2 h
36_+4.7
120_+ 7
92_+ 8 '~
24_+4.3
458_+32
351 +26 "
20+3.1
104
p~M) to a maximum of 24 _+ 4.4% (n = 5) (table 1, fig. 1). Forskolin (10/~M), a direct activator of adenylate cyclase, stimulated a 12.7 fold increase in adenylate cyclase activity in the cardiomyocytes (table 1). The forskolin-induced adenylate cyclase activity was also inhibited by carbachol to a maximum of 20_+ 3.1%, n = 3 (table 1, fig. 1).
I00"
E
H ~
90"
! oo.
3.2. Dual control of calcium action potential duration by fl-adrenoceptor and muscarinic receptor actil~ation
70:
60-
r"~P
,
11
!
I
i
8
7
6
5
4
- L~
lCAI~BACHOI_I IMI
Fig. 1. Muscarinic inhibition of adenylate cyclase activity in homogenates of ventricular cardiomyocytes. Cardiomyocytes were incubated with increasing concentrations of carbachol, in the absence (~), and in the presence of either 10 /~M isoproterenol (v) or 10 p~M forskolin (A). Ordinate: adenylate cyclase activity expressed as percent of activity in the absence of carbachol. Adenylate cyclase activity in controls was 35_+2 pmol [~2]PcAMP formed/min per mg protein, in the presence of 10 /~M isoproterenol was 120_+7 [32P]cAMP formed/min per mg protein, and in the presence of 10 /~M forskolin was 458 _+32 [32p]cAMP formed/min per mg protein (see table 1). Abscissa: negative log molar concentration of carbachol. Values represent the means_+S.E.M, of four determinations performed in duplicate. Ordinate: adenylate cyclase activity (% maximum).
10 /~M atropine, suggesting activation of a muscarinic receptor. The fl-adrenoceptor agonist isoproterenol stimulated in a dose-dependent manner basal adenylate cyclase activity in the homogenates of cardiomyocyctes (fig. 2). The maximum stimulation of adenylate cyclase activity by isoproterenol was observed at 100 /~M, and the half-maximal stimulatory concentration was 300 nM. The effect of isoproterenol was competitively antagonized by propranolol (300 nM) (fig. 2). The dissociation constant (K B) for propranolol calculated from the Schild equation was 3.1 nM. This value is in fairly good agreement with K B values for propranolol calculated by other methods in guinea pig ventricle (Engel et al., 1981). Carbachol inhibited adenylate cyclase activity stimulated by isoproterenol (10
Parallel electrophysiological experiments were conducted to assess the functional interaction of acetylcholine and agents which elevated cyclic AMP. Acetylcholine (10 ~M) did not affect the calcium action potential elicited by an intracellular depolarizing pulse in the absence of other drugs (data not shown). However, when the action potential duration was prolonged by agents which also stimulate adenylate cyclase, acetylcholine attenuated the prolongation. Figure 3A, B presents original records of acetylcholine's action in the presence of forskolin and isoproterenol. Table 2 I00
,)
; S
,C
0¸
~./
I 9
I 8
[ 7
I 6
L 5
1 4
I 3
Fig. 2. fl-Adrenoceptor stimulation of adenylate cyclase in homogenates of ventricular cardiomyocytes. Cardiomyocyte homogenates were incubated with increasing concentrations of isoproterenol in the absence (filled circle) or presence (open circle) of 300 nM propranolol. Values represent the means_+ S.E.M. of three determinations performed in duplicate. Ordinate: adenylate cyclase activity expressed in pmol [3~P] cAMP formed/min per mg protein. Abscissa: negative log molar concentration of isoproterennl.
105
summarizes the quantiative effects of acetylcholine on action potential duration (100% repolarization) in 4 myocytes stimulated by the presence of forskolin, various concentrations of isoproterenol, or IBMX. Figure 3A illustrates the effects of isoproterenol (33 nM) on the calcium action potential in the presence and absence of acetylcholine in a single
a. CONTROL
A
b. ISOPROT (53 riM)
~
c. ISOPROT plus Ach d. ISOPROT d
\\ ira. L - - . .
J -~
-~
m . . . . . . . . .
~_, . . . .
a. CONTROL
B
cell (also see table 2, cell 3, 4). Results similar to those presented in fig. 3A were obtained in five out of five preparations. In these experiments, each concentration of isoproterenol was tested in the same cell following wash out of the previous drugs. Trace (a) in fig. 3A shows an action potential elicited under control conditions. Isoproterenol (33 nM) caused a steady state enhancement of the amplitude and duration of the action potential within 5 min (fig. 3A, trace b). The addition of 10 # M acetylchohne in the continued presence of isoproterenol nearly reversed the effects of the B-adrenoceptor agonist (fig. 3A, trace c). To demonstrate the reversibility of the acetylcholine effect, acetylcholine was removed from the bath in the continued presence of isoproterenol. As can be seen in fig. 3A, trace (d), isoproterenol retained its prolonging action following wash out of acetylcholine. Forskolin (3 I~M), a direct activator of adenylate cyclase, exerted effects on the calcium action potential similar to those of isoproterenol. In three separate experiments, one of which is illustrated in fig. 3B (also, see table 2, cell 4), acetylcholine substantially diminished the action potential dura-
b. FORSKOLIN
GO0 ms
a. CONTROL
• C e
b. d b - c A M P
~a~l~l~.~,~
1
~
C, d h - c A M P plus Ach
Fig. 3. Cholinergic effects on calcium mediated action potentials augmented by agents that elevate the cyclic AMP level. (A) Cholinergic inhibition of the effect of isoproterenol. Trace a: control; trace b:'33 nM isoproterenol, trace c: acetylcholine (10 ~tM) in the presence of isoproterenol (33 nM); trace d: washout of acetylcholine with a solution containing isoproterenol (33 nM). (Similar results to those illustrated here were obtained in four additional myocytes from four different rabbit hearts.) (B) Cholinergic inhibition of the effect of forskolin, an activator of adenylate cyclase. Trace a: control; trace b: steady-state effect of 3 p,M forskolin (at 10 min); trace c: acetylcholine (10 #M) in the presence of forskolin (3 #M); trace d: washout in drug-free solution for 15 min. (Similar results were obtained in two additional myocytes from two hearts.) (C) Absence of an effect of acetylcholine on calcium action potentials augmented by dibutyryl cyclic AMP. Trace a: control; trace b: 2 mM dibutyryl cyclic AMP after 10 min; trace c: acetylcholine (10 /~M) plus dibutyryl cyclic A M P (1 mM). (Similar observations were made in two other cells from two hearts.) External K + ion concentration was 20 mM throughout. Horizontal full scale: 600 ms in (A) and (B) and 250 ms in (C); vertical full scale: 100 mV in all panels. Tick mark indicates 0 mV level. Stimulus artifact (from intracellular depolarizing current pulse) appears at the beginning of each trace.
106 TABLE 2 Effect of acetylcholine (ACh) on slow action potentials (APs) stimulated by the addition of various positive inotropic agents in isolated rabbit ventricular myocytes. Action potential duration (at 100% repolarization) is given as mean value of 5-20 action potentials. Standard error of mean values varied from 0.8-4 ms in the control category, 0.9-6 ms in the Addition category (with the exception of cell 4, forskolin, where it was 11 ms) and 2-8 ms in the Addition plus ACh category. Only steady-state effects of drugs are given, a Statistically significant increase in action potential duration over that of control (P < 0.01) due to the addition of a positive inotropic agent, b Statistically significant decline in action potential duration compared to the Addition category (P < 0.01 ). ~No significant change in action potential duration in comparison to Addition category (P > 0.1). d This column gives the relative effect of ACh on the action potential stimulated by the various positive inotropic agents (as listed in second column). Note the lack of an effect of ACh on action potentials stimulated by cyclic AMP. Cell
Positive inotropic
Action potential duration (ms)
number
agent added
Control
Addition
1 2 3 4 4 4 5 6
IBMX (1 mM) IBMX (1 mM) Isoproterenol (33 nM) Isoproterenol (50 nM) Isoproterenol (33 nM) Forskoline (2.5 p.M) Dibutyryl-cAMP (1 mM) cAMP (intracellularly applied)
60 55 79 84 110 106 130 78
103 100 118 274 292 473 164 104
% Decline in Addition
stimulated AP d
plus ACh
tion in the presence of forskolin. Wash out of forskolin and acetylcholine caused the action potential to return to control levels. Methylisobutylxanthine (IBMX), a phosphodiesterase inhibitor, also caused a prolongation of the calcium action potential which was attenuated by acetylcholine (table 2, cell 1 and 2). The ability of acetylcholine to inhibit the effects of IBMX, which is not a stimulator of adenylate cyclase, suggests that acetylcholine may interfere with the effects of accumulated cyclic AMP. This notion was tested in an experiment in which acetylcholine was added to the bath in the presence of dibutyryl cyclic AMP, a cyclic A M P analogue. This experiment is illustrated in fig. 3C (see also table 2, cell 5). Trace (a) depicts a control calcium-mediated action potential recorded from an isolated myocyte. Trace (b) shows the prolonging effect of dibutyryl cyclic A M P at 10 min. No shortening of the action potential duration was observed 3 rain following the addition of acetylcholine (trace c). The slight further increase in action potential duration in the presence of acetylcholine suggests that the effect of dibutyryl cyclic A M P may not quite have reached a steady state level in trace (b). This is consistent with the extremely slow time
'' ~' ~ " ~' ~ ~ "
57 83 94 207 164 310 164 112
h b b h b b ~ •
44.7 17.(I 20.3 24.5 43.8 34.5 0.0 7.7
course of dibutyryl cyclic A M P compared to isoproterenol and forskolin. The effect of acetylcholine on the action potential prolonged by the intracellular application of cyclic A M P was examined in an additional myocyte. To do this, a microelectrode was filled with 1 M of the Na ~ salt of cyclic AMP. Cyclic A M P was injected into the myocyte by a 30-s 1 nA hyperpolarizing pulse. The slow action potential was elicited at a constant rate at all times, including during the period of microinjection. The action potential that was recorded immediately following the period of injection was markedly prolonged in comparison to the control (see table 2, cell 6). In addition, the action potential duration remained above that of control for several minutes after the end of the injection. Following the return of the action potential duration to the control level, acetylcholine (10 txM) was added to the solution superfusing the myocyte for 5 rain (this time is sufficient for acetylcholine to act in the presence of isoproterenol, forskolin or IBMX). A repeat injection of cyclic AMP was made in order to determine whether acetylcholine could prevent the effect of cyclic AMP. As shown in table 2 (cell 6), an identical iontophoretic applica-
107 tion of cyclic AMP caused nearly the same degree of prolongation in the presence of acetylcholine as in its absence. Hence, it is concluded that acetylcholine does not attenuate the effects of cyclic AMP per se or those of its analogue, dibutyryl cyclic AMP. Thus, it would appear that another explanation for the ability of acetylcholine to inhibit the effects of methylxanthines must be sought.
4. Discussion
The results presented here demonstrate that a acetylcholine receptor agonist can antagonize the increase in adenylate cyclase activity resulting from the addition of forskolin and isoproterenol to homogenates of isolated ventricular cardiomyocytes. Parallel experiments in intact isolated cardiomyocytes showed that increases in the calcium action potential duration induced by these agents are also antagonized by an acetylcholine receptor agonist. Acetylcholine had no effect, however, on the action of extracellular dibutyryl cyclic AMP. These results suggest that acetylcholine receptor agonists modulate the increase in calcium current elicited by isoproterenol and forskolin by inhibiting a step prior to the formation of cyclic AMP, most likely adenylate cyclase activity. These results confirm previous findings with multicellular ventricular preparations (Schwegler et al., 1976; Watanabe et al., 1978; Jakobs et al., 1979; Linden et al., 1982; Wahler and Sperelakis, 1986) and extent those findings to a myocardial preparation free of nerve terminals. This provides certainty that the observed effects are direct actions on the cardiomyocyte and not the result of transmitter release from nerve terminals. Our results are also consistent with the report of Vickroy et al. (1985) demonstrating the opposing action of cholinergic and fl-adrenergic agents on cyclic AMP accumulation in isolated rat cardiomyocytes. One aspect of the data deserving comment is the ability of acetylcholine to antagonize the effect of the phosphodiesterase inhibitor IBMX, which presumably mimics the addition of cyclic AMP to the cell, while being ineffective against the action of dibutyryl cyclic AMP on the calcium action
potential. However, acetylcholine receptor agonists are capable of attenuating the increase in cyclic AMP affected by phosphodiesterase inhibitors. In our assay, basal levels of adenylate cyclase activity were defined as those measured in the presence of 1 mM of the phosphodiesterase inhibitor theophylline, and carbachol inhibited basal levels of adenylate cyclase activity. Linden et al. (1982) demonstrated a direct effect of acetylcholine receptor agonists on the accumulation of cyclic AMP stimulated by IBMX. This evidence, in combination with the data on dibutryl cyclic AMP, suggests that while acetylcholine may interfere with the accumulation of cyclic AMP, it does not interfere with the effects of accumulated cyclic AMP. Whereas isproterenol concentrations of 10-100 nM produced only slight increases in adenylate cyclase activity (fig. 2), there were marked effects on action potentials (fig. 4). This finding may be best explained from the perspective of spare receptors (or an amplification process) for the agonist. That is, only a low occupancy of receptors would be needed for a maximal physiological response. A similar concept also seems to apply to full muscarinic receptor agonists. For example, in human auricular preparations, the receptor occupancy curve is shifted in the direction of lower concentrations by nearly two orders of magnitude relative to the concentration-effect curve for the inhibition of contractions, and by one order of magnitude in relation to the concentration-effect curve for adenylate cyclase inhibition; these discrepancies were not seen with the partial agonist, pilocarpine (Delhaye et al., 1984). The data presented here provide evidence that cholinergic and /3-adrenoceptors are present on and interact within the same cell since the effect of one agent in an isolated myocyte is influenced by the presence of the other. The functional interaction on adenylate cyclase activity of/3-adrenoceptors and muscarinic acetylcholine receptors present on the cardiomyocyte is similar to the interaction of the /3-adrenoceptor and D-2 dopamine receptor in the isolated mammotroph of the pituitary intermediate lobe (Cote et al., 1982). In this system, the D-2 dopamine receptor is linked to the inhibitory GTP-binding protein, N,, while
108
the /~-adrenoceptor is linked to the stimulatory GTP-binding protein, N~. Both N~ and N~ are linked to the catalytic subunit of adenylate cyclase. There is evidence that in the cardiomyocyte, muscarinic receptors are linked to Ni; Hazeki and Ui (1981) showed that in adult rat cardiomyocytes that pertussis toxin eliminated the inhibitory effect of carbachol on cyclic AMP accumulation stimulated by isoproterenol. The results presented here confirm and extend previous findings regarding muscarinic acetylcholine receptor and fl-adrenoceptor antagonism in the heart and in the isolated ventricular cardiomyocyte. These studies also demonstrate the usefulness of the isolated cardiomyocyte as a model for the study of integrative receptor interactions.
Acknowledgments This work was supported by the American Heart Association Grant 783 (with funds contributed by the Chicago Heart Association) to M.L.D. and Chicago Heart Association Grant S-84 to S.M.V. We wish to thank Dr. Toshio Narahashi, in whose laboratory the electrophysiological experiments were conducted, for his generosity.
References Bradford, M.M., 1976, A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein binding, Anal. Biochem. 72, 248. Brown, J.H. and S.L. Brown, 1984, Agonists differentiate muscarinic receptors that inhibit cyclic A M P formation form those that stimulate phosphoinositide metabolism, J. Biol. Chem. 259, 3777. Buxton, I.L.O. and L.L. Brunton, 1985, Direct analysis of ,8-adrenergic receptor subtypes on intact adult ventricular myocytes of the rat, Circ. Res. 56, 126. Cote, T.E., C.W. Grewe and J.W. Kebabian, 1982, Guanyl nucleotides participate in the beta-adrenergic stimulation of adenylate cyclase activity in the intermediate lobe of the rat pituitary gland, Endocrinology 110, 805. Cotterell, D.J., J.M. Watson and M.L. Dubocovich, 1985, Beta-adrenoceptor activation increases calcium-uptake into ventricular myocytes, Fed. Proc. 44, 1249. Delhaye, M., J.M. De Smet, G. Taton, P. De Neef, J.C. Camus, J. Fontaine, M. Waelbroeck, P. Robberecht and J. Chris-
tophe, 1984, A comparison between muscarinic receptor occupancy, adenylate cyclase inhibition, and inotropic response in h u m a n heart, N a u n y n - S c h m i e d e b . Arch. Pharmacol. 325, 170. Engel, G., D. Hoyer, B. Berthold, and H. Wagner, 1981, (_+)[125Iodo]cyanopindolol, a new ligand for ,8-adrenoceptors in guinea pig, Naunyn-Schmiedeb. Arch. Pharmacol. 317, 277. Fields, J.Z., W.R. Roeske, E. Morkin and H.I. Yamamura, 1978, Cardiac muscarinic receptors. Biochemical identification and characterization, J. Biol. Chem. 253, 3251. Hazeki, O. and M. Ui, 1981, Modification by islet-activating protein receptor-mediated regulation of cyclic A M P accumulation in isolated rat heart cells, J. Biol. Chem. 256, 2856. Jakobs, K.H., K. Aktories and G. Schuhz, 1979, GTP-dependent inhibition of cardiac adenylate cyclase by muscarinic chotinergic agonists. Naunyn-Schmiedeb. Arch. Pharmacol. 310. 113. Linden, J., S. Vogel and N. Sperelakis, 1982, Sensitivity of Ca-dependent slow action potentials to methacholine is induced by phosphodiesterase inhibitors in embwonic chick ventricles. J. Pharmacol. Exp. Ther. 222, 383. Minneman, K.P., L.R. Hegstrand and B. Molinoff, 1979, The pharmacological specificity of ill- and ,8:-adrenergic receptors in rat heart and lung in vitro, Mol. Pharmacol. 16, 21. Rajs. J., G.B. Sundby, N. Danell, G. Tornling, P. Biberfeld and S. Jakobsson, 1978, A rapid method for the isolation of viable cardiac myocytes from adult rat, Exp. ('ell Res. 115, 183. Schwegler, M., K. Reitter, G. Shieber and R. Jacob, 1976, Noncompetitive catecholamine-antagonism of acetylcholine in the sympathectomized mammalian ventricular myocardiurn, Basic Res. Cardiol. 71,407. Solomon, Y., 1979, Adenylate cyclase assay, Adv. Cycl. Nucl. Res. 10, 35. Vickroy, T.W., J.J. Bahl, H.I. Y a m a m u r a and W.R. Roeske, 1985, Muscarinic cholinergic receptor-mediated regulation of isoproterenol-stimulated c A M P formation in rat cardiomyocytes, Fed. Proc. 44, 1481. Wahler, G.M. and N. Sperelakis, 1986, Cholinergic attenuation of the electrophysiological effects of forskolin, J. Cvcl. Nucl. Prot. Phosphor. Res. 11, 1. Watanabe, A.M., M.M. McConnaughey, R.A. Strawbridge. J.W. Fleming, L.R. Jones and H.R. Besch, Jr., 1978, Muscarinic cholinergic receptor modulation of ,8-adrenergic receptor affinity for catecholamines, J. Biol. Chem. 253, 4833. Watson, M., H.I. Y a m a m u r a and W.R. Roeske, 1983, A unique regulatory profile and regional distribution of )H-pirenzepine binding in the rat provide evidence for distinct M I and M 2 muscarinic receptor subtypes, Life Sci. 32, 3001.
101
EJP 50456
Cholinergic antagonism of fl-adrenergic stimulated action potentials and adenylate cyclase activity in rabbit ventricular cardiomyocytes Julie M. W a t s o n , Stephen M. Vogel 1, D a v i d J. Cotterell 2 a n d M a g a r i t a L. D u b o c o v i c h * Department of Pharmacology, Northwestern Universi(v Medical School Chicago, IL 60611, U.S.A.
Received 7 June 1988, accepted 12 July 1988
The cholinergic antagonism of/3-adrenergic stimulation was examined by measuring adenylate cyclase activity and calcium-mediated action potentials in isolated ventricular cardiomyocytes of adult rabbits. The fl-adrenoceptor agonist isoproterenol and the direct adenylate cyclase activator forskolin increased adenylate cyclase activity in homogenates of the myocyctes. The cholinergic agonist carbachol (10 nM-100/~M) inhibited in a concentration dependent manner basal, isoproterenol-stimulated and forskolin-stimulated adenylate cyclase activity. The carbachol effect on basal adenylate cyclase activity was antagonized by atropine (10 ~M). In parallel experiments using intact cardiomyocytes. calcium action potentials were elicited by intracellular depolarizing current pulses in partially depolarized preparations. These action potentials were prolonged by isoproterenol, forskolin and dibutyryl cyclic AMP. Acetylcholine reversibly inhibited the prolongation of the action potential induced by isoproterenol and forskolin but not dibutyryl cyclic AMP. These results suggest that cholinergic agonists modulate the increase in the calcium current elicited by isoproterenol and forskolin in isolated ventricular cardiomyocytes by inhibiting adenylate cyclase activity.
C a 2+
action potentials; Cardiomyocytes; Adenylate cyclase activity; Muscarinic acetylcholine receptors; fl-Adrenoceptors; (Rabbit)
1. Introduction
Activation of fi-adrenoceptors and muscarinic acetylcholine receptors in the heart leads to opposite physiological and biochemical responses, t31and fi2-adrenoceptors and M-1 and M-2 muscarinic acetylcholine receptors have been char-
1 Present address: Department of Pharmacology, University of College of Medicine, Chicago, IL 60612, U.S.A. 2 Present address: Glaxo Group Research, Ltd., Clinical Research Division, Greenford Road, Middx, UB60HE. Great Britain. * To whom all correspondence should be addressed: Department of Pharmacology, Northwestern University, 303 East Chicago Avenue, Chicago, IL 60611, U.S.A.
acterized in the heart (Minneman et al., 1979; Buxton and Brunton, 1985; Fields et al., 1978; Watson et al., 1983). Muscarinic acetylcholine receptor agonists have been reported to antagonize fl-adrenergic effects on cardic contractility, the maximum upstroke velocity and duration of calcium-dependent slow action potentials, and activity of adenylate cyclase in the heart (Schwegler et al., 1976), heart muscle homogenates (Watanabe et al., 1978; Jakobs et al., 1979) and embryonic ventricular muscle (Linden et al., 1982). The data from such multicellular preparations suggest that one mechanism by which acetylcholine receptor agonists exert their antiadrenergic effects in the heart may be by modulating adenylate cyclase activity in the ventricular cardiomyocyte.
0014-2999/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
102 Direct analysis of the mechanism of fi-adrenergic and muscarinic interaction in a preparation of ventricular cardiomyocytes isolated from other heart cell types is needed. Recently, it has been reported that while both 131- and/92-adrenoceptors exist in heart muscle, only the fll-adrenoceptor is found on the cardiomyocyte (Buxton and Brunton, 1985). ill-, but not /~2-, adrenoceptor activation has been shown to increase calcium accumulation by the sarcoplasmic reticulum in the rabbit ventricular myocyte (Cotterell et al., 1985); this calcium influx appears to be secondary to increases in cyclic A M P accumulation, mediated by filadrenoceptors. Likewise, recent evidence suggests that while M-2 muscarinic receptors are negatively linked to adenylate cyclcase activity, M-1 muscarinic receptors may instead be linked to phospatidylinositol turnover (Watson et al., 1983; Brown and Brown, 1984). Analysis of acetylcholine receptor and ,8-adrenoceptor interaction in isolated cells also permits study of whether the opposite physiological effects mediated by these receptors occur within the same cell; this is not possible in studies carried out in homogenates of heterogenous membranes and multicellular preparations. The use of isolated myocytes has several advantages for electrophysiological studies. First, the isolated cells are free from influences of autonomic innervation. Thus, effects of agents used here such as forskolin and dibutyryl cyclic A M P can be considered to be direct actions on the myocytes. Secondly, stable microelectrode impalements are possible in isolated myocytes because of the weak contractile force; this permits multiple drug applications to be compared in a single cell. Thirdly, planned studies using voltage and patch clamp methods will be possible in isolated myocytes. Fourth, correlation between biochemical and physiological consequences of receptor activation is possible in isolated myocytes, as the present experiments will show. The present study investigated the effect of activation of muscarinic receptors and /~-adrenoceptors on the calcium action potential and on adenylate cyclase activity in isolated ventricular cardiomyocytes. We report here that acetylcholine receptor agonists appear to exert antiadrenergic
effects on the calcium action potential in isolated ventricular cardiomyocytes by inhibiting adenylate cyclase activity.
2. Materials and methods
2.1. Cardiomyoo'te preparation Rabbit ventricular myocytes were prepared as previously described (Rajs et al., 1978). Briefly, male New Zealand white rabbits (2.5-3 kg) were decapitated and thoractomized. The heart with a large portion of the aorta intact was quickly removed and washed with ice cold saline. The aorta was cannulated and the heart perfused retrogradely at 10 m l / m i n per g wet weight of heart muscle for 30 s with calcium free Krebs buffer to wash out blood remaining in the heart (Rajs et al., 1978). Thereafter the heart was continuously perfused with a calcium free Krebs solution gassed with 95% 02-5% CO~ at 3 7 ° C containing 0.144% Type II collagenase during 30-40 min. The perfused ventricles were cut with scissors into small pieces. If further digestion was required, the pieces of ventricles were incubated for an additional 10 min in Krebs solution containing 0.144% collagenase at 3 7 ° C in a shaking waterbath. The resulting cell suspension was filtered through 250 ~ m nylon gauze and centrifuged at 40 ×,g for 1 rain. The cells were washed three times with the standard calcium free Krebs solution, were layered on 4% bovine serum albumin (fraction V Sigma) and the suspensions centrifuged at 25 >( eg for 1 min. The dispersed cells were resuspended in IV bovine serum albumin. After 15 min, the cells were resuspended in Krebs solution containing 2.6 mM CaC12. Isolated cells were examined microscopically and viability assessed by noting shape and trypan blue exclusion.
2.2. Adenylate cyclase activi O' Adenylate cyclase activity was assayed in homogenates of the isolated myocytes by measuring the conversion of a-[)2p]ATP to [32p]cyclic AMP according to the method of Solomon (1979) with modifications as described by Minneman et al.
103
(1979). Myocytes were homogenized by hand, centrifuged for 5 min at 35000 × g and resuspended to a concentration of 6.6 × 105 cells per ml in 2 mM HEPES buffer containing 2 mM EGTA, pH 7.5 at 4°C. The reaction mixture contained 1-2 million cmp of ce-[32P]ATP (New England Nuclear), 50 mM HEPES (pH 7.5), 0.05 mM cyclic AMP, 1 mM MgC12, 0.25 mM ATP, 1 mM theophylline, 0.1 m g / m l creatine kinase, 10 mM phosphocreatine, 50 mM GTP, 50 mM NaC1, and appropriate drugs. The reaction was initiated by addition of an aliquot of homogenates and incubated for 10 rain at 30 o C. The reaction was terminated by the addition of 100/~1 of 1% sodium lauryl sulfate and 5 mM ATP in 50 mM Tris buffer. Samples were then boiled for 10 min to improve column flow during separation. The [32p]cyclic AMP was separated by sequential chromatography on Dowex A G 50 WX4 and alumina columns. Recovery was monitored by addition of [3H]cyclic AMP (10 nCi) to each sample prior to chromatography. 32p and 3H content of final elute were determined simultaneously by liquid scintillation spectrometry. 32p content was corrected for recovery and adenylate cyclase activity expressed as pmol cyclic AMP f o r m e d / m i n per mg protein. Protein concentration of the homogenates was determined by the method of Bradford (1976).
digitized using an analog to digital converter and stored on a computer for subsequent off line analysis. Action potential amplitude and duration (ms) at 100% repolarization were measured in control conditions and in the presence of drugs. Drugs were added to a bath volume of 2 ml (35 + I ° C ) at a superfusion rate of 6 ml/min.
3. Results
3.1. Dual control of adenylate cyclase activity' by" fl-adrenoceptor and muscarinic receptor activation Adenylate cyclase activity was used as a functional m e a s u r e m e n t in h o m o g e n a t e s of cardiomyocytes of fl-adrenoceptor and muscarinic receptor activation. The basal adenylate cyclase activity in homogenates of the cardiomyocyctes was 35 _+ 2 pmol [32p]cyclic AMP f o r m e d / m i n per mg protein when assayed in the presence of the phosphodiesterase inhibitor theophylline (table 1). The muscarinic acetylcholine receptor agonist carbachol inhibited adenylate cyclase activity in the cardiomyocytes in a dose dependent manner (fig. 1). The maximal inhibition of adenylate cyclase activity produced by carbachol was 36 _+ 4.7% (n = 4) below basal at 100 ~M (fig. 1~ table 1) with an IC~0 of 100 nM. The inhibiting effect of carbachol was completely antagonized by
2.3. Calcium action potentials Parallel electrophysiological experiments were conducted in intact cardiomyocytes. Myocytes were suspended in a buffer containing 2 mM CaC1 z and 20 mM KC1. This K + concentration partially depolarized the membrane to a resting potential of - 4 0 mV. Under these conditions an intracellular depolarizing pulse elicits an action potential whose inward current is carried by calcium ions. Myocytes were impaled with a conventional microelectrode filled with 3 M KC1 and with a resistance of 40-60 M~2. Each slow action potential was elicited by an intracellular depolarizing current pulse delivered through the microelectrode by means of a bridge circuit. The frequency of stimulation was 0.33 Hz. Action potentials were
TABLE 1 Effect of carbachol on adenylate cyclase activity in homogenates of rabbit vertricular cardiomyocytes. Values represent the mean_+ S.E.M. of three to five determinations performed in duplicate. ~ P < 0.05; h p < 0.005 when compared to corresponding control using Student's t-test. Adenylate cyclase activity (pmol c A M P / min per mg protein) Control Basal Isoproterenol (10 p,M) Forskolin (10/~M)
% inhibition
Carbachol (100 ~M)
35+_ 2
21_+ 2 h
36_+4.7
120_+ 7
92_+ 8 '~
24_+4.3
458_+32
351 +26 "
20+3.1
104
p~M) to a maximum of 24 _+ 4.4% (n = 5) (table 1, fig. 1). Forskolin (10/~M), a direct activator of adenylate cyclase, stimulated a 12.7 fold increase in adenylate cyclase activity in the cardiomyocytes (table 1). The forskolin-induced adenylate cyclase activity was also inhibited by carbachol to a maximum of 20_+ 3.1%, n = 3 (table 1, fig. 1).
I00"
E
H ~
90"
! oo.
3.2. Dual control of calcium action potential duration by fl-adrenoceptor and muscarinic receptor actil~ation
70:
60-
r"~P
,
11
!
I
i
8
7
6
5
4
- L~
lCAI~BACHOI_I IMI
Fig. 1. Muscarinic inhibition of adenylate cyclase activity in homogenates of ventricular cardiomyocytes. Cardiomyocytes were incubated with increasing concentrations of carbachol, in the absence (~), and in the presence of either 10 /~M isoproterenol (v) or 10 p~M forskolin (A). Ordinate: adenylate cyclase activity expressed as percent of activity in the absence of carbachol. Adenylate cyclase activity in controls was 35_+2 pmol [~2]PcAMP formed/min per mg protein, in the presence of 10 /~M isoproterenol was 120_+7 [32P]cAMP formed/min per mg protein, and in the presence of 10 /~M forskolin was 458 _+32 [32p]cAMP formed/min per mg protein (see table 1). Abscissa: negative log molar concentration of carbachol. Values represent the means_+S.E.M, of four determinations performed in duplicate. Ordinate: adenylate cyclase activity (% maximum).
10 /~M atropine, suggesting activation of a muscarinic receptor. The fl-adrenoceptor agonist isoproterenol stimulated in a dose-dependent manner basal adenylate cyclase activity in the homogenates of cardiomyocyctes (fig. 2). The maximum stimulation of adenylate cyclase activity by isoproterenol was observed at 100 /~M, and the half-maximal stimulatory concentration was 300 nM. The effect of isoproterenol was competitively antagonized by propranolol (300 nM) (fig. 2). The dissociation constant (K B) for propranolol calculated from the Schild equation was 3.1 nM. This value is in fairly good agreement with K B values for propranolol calculated by other methods in guinea pig ventricle (Engel et al., 1981). Carbachol inhibited adenylate cyclase activity stimulated by isoproterenol (10
Parallel electrophysiological experiments were conducted to assess the functional interaction of acetylcholine and agents which elevated cyclic AMP. Acetylcholine (10 ~M) did not affect the calcium action potential elicited by an intracellular depolarizing pulse in the absence of other drugs (data not shown). However, when the action potential duration was prolonged by agents which also stimulate adenylate cyclase, acetylcholine attenuated the prolongation. Figure 3A, B presents original records of acetylcholine's action in the presence of forskolin and isoproterenol. Table 2 I00
,)
; S
,C
0¸
~./
I 9
I 8
[ 7
I 6
L 5
1 4
I 3
Fig. 2. fl-Adrenoceptor stimulation of adenylate cyclase in homogenates of ventricular cardiomyocytes. Cardiomyocyte homogenates were incubated with increasing concentrations of isoproterenol in the absence (filled circle) or presence (open circle) of 300 nM propranolol. Values represent the means_+ S.E.M. of three determinations performed in duplicate. Ordinate: adenylate cyclase activity expressed in pmol [3~P] cAMP formed/min per mg protein. Abscissa: negative log molar concentration of isoproterennl.
105
summarizes the quantiative effects of acetylcholine on action potential duration (100% repolarization) in 4 myocytes stimulated by the presence of forskolin, various concentrations of isoproterenol, or IBMX. Figure 3A illustrates the effects of isoproterenol (33 nM) on the calcium action potential in the presence and absence of acetylcholine in a single
a. CONTROL
A
b. ISOPROT (53 riM)
~
c. ISOPROT plus Ach d. ISOPROT d
\\ ira. L - - . .
J -~
-~
m . . . . . . . . .
~_, . . . .
a. CONTROL
B
cell (also see table 2, cell 3, 4). Results similar to those presented in fig. 3A were obtained in five out of five preparations. In these experiments, each concentration of isoproterenol was tested in the same cell following wash out of the previous drugs. Trace (a) in fig. 3A shows an action potential elicited under control conditions. Isoproterenol (33 nM) caused a steady state enhancement of the amplitude and duration of the action potential within 5 min (fig. 3A, trace b). The addition of 10 # M acetylchohne in the continued presence of isoproterenol nearly reversed the effects of the B-adrenoceptor agonist (fig. 3A, trace c). To demonstrate the reversibility of the acetylcholine effect, acetylcholine was removed from the bath in the continued presence of isoproterenol. As can be seen in fig. 3A, trace (d), isoproterenol retained its prolonging action following wash out of acetylcholine. Forskolin (3 I~M), a direct activator of adenylate cyclase, exerted effects on the calcium action potential similar to those of isoproterenol. In three separate experiments, one of which is illustrated in fig. 3B (also, see table 2, cell 4), acetylcholine substantially diminished the action potential dura-
b. FORSKOLIN
GO0 ms
a. CONTROL
• C e
b. d b - c A M P
~a~l~l~.~,~
1
~
C, d h - c A M P plus Ach
Fig. 3. Cholinergic effects on calcium mediated action potentials augmented by agents that elevate the cyclic AMP level. (A) Cholinergic inhibition of the effect of isoproterenol. Trace a: control; trace b:'33 nM isoproterenol, trace c: acetylcholine (10 ~tM) in the presence of isoproterenol (33 nM); trace d: washout of acetylcholine with a solution containing isoproterenol (33 nM). (Similar results to those illustrated here were obtained in four additional myocytes from four different rabbit hearts.) (B) Cholinergic inhibition of the effect of forskolin, an activator of adenylate cyclase. Trace a: control; trace b: steady-state effect of 3 p,M forskolin (at 10 min); trace c: acetylcholine (10 #M) in the presence of forskolin (3 #M); trace d: washout in drug-free solution for 15 min. (Similar results were obtained in two additional myocytes from two hearts.) (C) Absence of an effect of acetylcholine on calcium action potentials augmented by dibutyryl cyclic AMP. Trace a: control; trace b: 2 mM dibutyryl cyclic AMP after 10 min; trace c: acetylcholine (10 /~M) plus dibutyryl cyclic A M P (1 mM). (Similar observations were made in two other cells from two hearts.) External K + ion concentration was 20 mM throughout. Horizontal full scale: 600 ms in (A) and (B) and 250 ms in (C); vertical full scale: 100 mV in all panels. Tick mark indicates 0 mV level. Stimulus artifact (from intracellular depolarizing current pulse) appears at the beginning of each trace.
106 TABLE 2 Effect of acetylcholine (ACh) on slow action potentials (APs) stimulated by the addition of various positive inotropic agents in isolated rabbit ventricular myocytes. Action potential duration (at 100% repolarization) is given as mean value of 5-20 action potentials. Standard error of mean values varied from 0.8-4 ms in the control category, 0.9-6 ms in the Addition category (with the exception of cell 4, forskolin, where it was 11 ms) and 2-8 ms in the Addition plus ACh category. Only steady-state effects of drugs are given, a Statistically significant increase in action potential duration over that of control (P < 0.01) due to the addition of a positive inotropic agent, b Statistically significant decline in action potential duration compared to the Addition category (P < 0.01 ). ~No significant change in action potential duration in comparison to Addition category (P > 0.1). d This column gives the relative effect of ACh on the action potential stimulated by the various positive inotropic agents (as listed in second column). Note the lack of an effect of ACh on action potentials stimulated by cyclic AMP. Cell
Positive inotropic
Action potential duration (ms)
number
agent added
Control
Addition
1 2 3 4 4 4 5 6
IBMX (1 mM) IBMX (1 mM) Isoproterenol (33 nM) Isoproterenol (50 nM) Isoproterenol (33 nM) Forskoline (2.5 p.M) Dibutyryl-cAMP (1 mM) cAMP (intracellularly applied)
60 55 79 84 110 106 130 78
103 100 118 274 292 473 164 104
% Decline in Addition
stimulated AP d
plus ACh
tion in the presence of forskolin. Wash out of forskolin and acetylcholine caused the action potential to return to control levels. Methylisobutylxanthine (IBMX), a phosphodiesterase inhibitor, also caused a prolongation of the calcium action potential which was attenuated by acetylcholine (table 2, cell 1 and 2). The ability of acetylcholine to inhibit the effects of IBMX, which is not a stimulator of adenylate cyclase, suggests that acetylcholine may interfere with the effects of accumulated cyclic AMP. This notion was tested in an experiment in which acetylcholine was added to the bath in the presence of dibutyryl cyclic AMP, a cyclic A M P analogue. This experiment is illustrated in fig. 3C (see also table 2, cell 5). Trace (a) depicts a control calcium-mediated action potential recorded from an isolated myocyte. Trace (b) shows the prolonging effect of dibutyryl cyclic A M P at 10 min. No shortening of the action potential duration was observed 3 rain following the addition of acetylcholine (trace c). The slight further increase in action potential duration in the presence of acetylcholine suggests that the effect of dibutyryl cyclic A M P may not quite have reached a steady state level in trace (b). This is consistent with the extremely slow time
'' ~' ~ " ~' ~ ~ "
57 83 94 207 164 310 164 112
h b b h b b ~ •
44.7 17.(I 20.3 24.5 43.8 34.5 0.0 7.7
course of dibutyryl cyclic A M P compared to isoproterenol and forskolin. The effect of acetylcholine on the action potential prolonged by the intracellular application of cyclic A M P was examined in an additional myocyte. To do this, a microelectrode was filled with 1 M of the Na ~ salt of cyclic AMP. Cyclic A M P was injected into the myocyte by a 30-s 1 nA hyperpolarizing pulse. The slow action potential was elicited at a constant rate at all times, including during the period of microinjection. The action potential that was recorded immediately following the period of injection was markedly prolonged in comparison to the control (see table 2, cell 6). In addition, the action potential duration remained above that of control for several minutes after the end of the injection. Following the return of the action potential duration to the control level, acetylcholine (10 txM) was added to the solution superfusing the myocyte for 5 rain (this time is sufficient for acetylcholine to act in the presence of isoproterenol, forskolin or IBMX). A repeat injection of cyclic AMP was made in order to determine whether acetylcholine could prevent the effect of cyclic AMP. As shown in table 2 (cell 6), an identical iontophoretic applica-
107 tion of cyclic AMP caused nearly the same degree of prolongation in the presence of acetylcholine as in its absence. Hence, it is concluded that acetylcholine does not attenuate the effects of cyclic AMP per se or those of its analogue, dibutyryl cyclic AMP. Thus, it would appear that another explanation for the ability of acetylcholine to inhibit the effects of methylxanthines must be sought.
4. Discussion
The results presented here demonstrate that a acetylcholine receptor agonist can antagonize the increase in adenylate cyclase activity resulting from the addition of forskolin and isoproterenol to homogenates of isolated ventricular cardiomyocytes. Parallel experiments in intact isolated cardiomyocytes showed that increases in the calcium action potential duration induced by these agents are also antagonized by an acetylcholine receptor agonist. Acetylcholine had no effect, however, on the action of extracellular dibutyryl cyclic AMP. These results suggest that acetylcholine receptor agonists modulate the increase in calcium current elicited by isoproterenol and forskolin by inhibiting a step prior to the formation of cyclic AMP, most likely adenylate cyclase activity. These results confirm previous findings with multicellular ventricular preparations (Schwegler et al., 1976; Watanabe et al., 1978; Jakobs et al., 1979; Linden et al., 1982; Wahler and Sperelakis, 1986) and extent those findings to a myocardial preparation free of nerve terminals. This provides certainty that the observed effects are direct actions on the cardiomyocyte and not the result of transmitter release from nerve terminals. Our results are also consistent with the report of Vickroy et al. (1985) demonstrating the opposing action of cholinergic and fl-adrenergic agents on cyclic AMP accumulation in isolated rat cardiomyocytes. One aspect of the data deserving comment is the ability of acetylcholine to antagonize the effect of the phosphodiesterase inhibitor IBMX, which presumably mimics the addition of cyclic AMP to the cell, while being ineffective against the action of dibutyryl cyclic AMP on the calcium action
potential. However, acetylcholine receptor agonists are capable of attenuating the increase in cyclic AMP affected by phosphodiesterase inhibitors. In our assay, basal levels of adenylate cyclase activity were defined as those measured in the presence of 1 mM of the phosphodiesterase inhibitor theophylline, and carbachol inhibited basal levels of adenylate cyclase activity. Linden et al. (1982) demonstrated a direct effect of acetylcholine receptor agonists on the accumulation of cyclic AMP stimulated by IBMX. This evidence, in combination with the data on dibutryl cyclic AMP, suggests that while acetylcholine may interfere with the accumulation of cyclic AMP, it does not interfere with the effects of accumulated cyclic AMP. Whereas isproterenol concentrations of 10-100 nM produced only slight increases in adenylate cyclase activity (fig. 2), there were marked effects on action potentials (fig. 4). This finding may be best explained from the perspective of spare receptors (or an amplification process) for the agonist. That is, only a low occupancy of receptors would be needed for a maximal physiological response. A similar concept also seems to apply to full muscarinic receptor agonists. For example, in human auricular preparations, the receptor occupancy curve is shifted in the direction of lower concentrations by nearly two orders of magnitude relative to the concentration-effect curve for the inhibition of contractions, and by one order of magnitude in relation to the concentration-effect curve for adenylate cyclase inhibition; these discrepancies were not seen with the partial agonist, pilocarpine (Delhaye et al., 1984). The data presented here provide evidence that cholinergic and /3-adrenoceptors are present on and interact within the same cell since the effect of one agent in an isolated myocyte is influenced by the presence of the other. The functional interaction on adenylate cyclase activity of/3-adrenoceptors and muscarinic acetylcholine receptors present on the cardiomyocyte is similar to the interaction of the /3-adrenoceptor and D-2 dopamine receptor in the isolated mammotroph of the pituitary intermediate lobe (Cote et al., 1982). In this system, the D-2 dopamine receptor is linked to the inhibitory GTP-binding protein, N,, while
108
the /~-adrenoceptor is linked to the stimulatory GTP-binding protein, N~. Both N~ and N~ are linked to the catalytic subunit of adenylate cyclase. There is evidence that in the cardiomyocyte, muscarinic receptors are linked to Ni; Hazeki and Ui (1981) showed that in adult rat cardiomyocytes that pertussis toxin eliminated the inhibitory effect of carbachol on cyclic AMP accumulation stimulated by isoproterenol. The results presented here confirm and extend previous findings regarding muscarinic acetylcholine receptor and fl-adrenoceptor antagonism in the heart and in the isolated ventricular cardiomyocyte. These studies also demonstrate the usefulness of the isolated cardiomyocyte as a model for the study of integrative receptor interactions.
Acknowledgments This work was supported by the American Heart Association Grant 783 (with funds contributed by the Chicago Heart Association) to M.L.D. and Chicago Heart Association Grant S-84 to S.M.V. We wish to thank Dr. Toshio Narahashi, in whose laboratory the electrophysiological experiments were conducted, for his generosity.
References Bradford, M.M., 1976, A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein binding, Anal. Biochem. 72, 248. Brown, J.H. and S.L. Brown, 1984, Agonists differentiate muscarinic receptors that inhibit cyclic A M P formation form those that stimulate phosphoinositide metabolism, J. Biol. Chem. 259, 3777. Buxton, I.L.O. and L.L. Brunton, 1985, Direct analysis of ,8-adrenergic receptor subtypes on intact adult ventricular myocytes of the rat, Circ. Res. 56, 126. Cote, T.E., C.W. Grewe and J.W. Kebabian, 1982, Guanyl nucleotides participate in the beta-adrenergic stimulation of adenylate cyclase activity in the intermediate lobe of the rat pituitary gland, Endocrinology 110, 805. Cotterell, D.J., J.M. Watson and M.L. Dubocovich, 1985, Beta-adrenoceptor activation increases calcium-uptake into ventricular myocytes, Fed. Proc. 44, 1249. Delhaye, M., J.M. De Smet, G. Taton, P. De Neef, J.C. Camus, J. Fontaine, M. Waelbroeck, P. Robberecht and J. Chris-
tophe, 1984, A comparison between muscarinic receptor occupancy, adenylate cyclase inhibition, and inotropic response in h u m a n heart, N a u n y n - S c h m i e d e b . Arch. Pharmacol. 325, 170. Engel, G., D. Hoyer, B. Berthold, and H. Wagner, 1981, (_+)[125Iodo]cyanopindolol, a new ligand for ,8-adrenoceptors in guinea pig, Naunyn-Schmiedeb. Arch. Pharmacol. 317, 277. Fields, J.Z., W.R. Roeske, E. Morkin and H.I. Yamamura, 1978, Cardiac muscarinic receptors. Biochemical identification and characterization, J. Biol. Chem. 253, 3251. Hazeki, O. and M. Ui, 1981, Modification by islet-activating protein receptor-mediated regulation of cyclic A M P accumulation in isolated rat heart cells, J. Biol. Chem. 256, 2856. Jakobs, K.H., K. Aktories and G. Schuhz, 1979, GTP-dependent inhibition of cardiac adenylate cyclase by muscarinic chotinergic agonists. Naunyn-Schmiedeb. Arch. Pharmacol. 310. 113. Linden, J., S. Vogel and N. Sperelakis, 1982, Sensitivity of Ca-dependent slow action potentials to methacholine is induced by phosphodiesterase inhibitors in embwonic chick ventricles. J. Pharmacol. Exp. Ther. 222, 383. Minneman, K.P., L.R. Hegstrand and B. Molinoff, 1979, The pharmacological specificity of ill- and ,8:-adrenergic receptors in rat heart and lung in vitro, Mol. Pharmacol. 16, 21. Rajs. J., G.B. Sundby, N. Danell, G. Tornling, P. Biberfeld and S. Jakobsson, 1978, A rapid method for the isolation of viable cardiac myocytes from adult rat, Exp. ('ell Res. 115, 183. Schwegler, M., K. Reitter, G. Shieber and R. Jacob, 1976, Noncompetitive catecholamine-antagonism of acetylcholine in the sympathectomized mammalian ventricular myocardiurn, Basic Res. Cardiol. 71,407. Solomon, Y., 1979, Adenylate cyclase assay, Adv. Cycl. Nucl. Res. 10, 35. Vickroy, T.W., J.J. Bahl, H.I. Y a m a m u r a and W.R. Roeske, 1985, Muscarinic cholinergic receptor-mediated regulation of isoproterenol-stimulated c A M P formation in rat cardiomyocytes, Fed. Proc. 44, 1481. Wahler, G.M. and N. Sperelakis, 1986, Cholinergic attenuation of the electrophysiological effects of forskolin, J. Cvcl. Nucl. Prot. Phosphor. Res. 11, 1. Watanabe, A.M., M.M. McConnaughey, R.A. Strawbridge. J.W. Fleming, L.R. Jones and H.R. Besch, Jr., 1978, Muscarinic cholinergic receptor modulation of ,8-adrenergic receptor affinity for catecholamines, J. Biol. Chem. 253, 4833. Watson, M., H.I. Y a m a m u r a and W.R. Roeske, 1983, A unique regulatory profile and regional distribution of )H-pirenzepine binding in the rat provide evidence for distinct M I and M 2 muscarinic receptor subtypes, Life Sci. 32, 3001.