Search

1. To investigate the participation of guanylyl cyclase in the muscarinic regulation of the cardiac L-type calcium current (ICa), we examined the effects of three guanylyl cyclase inhibitors, 1H-[1,2,4]oxidiazo-lo[4,3-a]quinoxaline-1-one (ODQ), 6-anilino-5,8-quinolinedione (LY 83583), and methylene blue (MBlue), on the beta-adrenoceptor; muscarinic receptor and nitric oxide (NO) regulation of ICa and on the muscarinic activated potassium current I(K,ACh), in frog atrial and ventricular myocytes. 2. ODQ (10 microM) and LY 83583 (30 microM) antagonized the inhibitory effect of an NO-donor (S-nitroso-N-acetylpenicillamine, SNAP, 1 microM) on the isoprenaline (Iso)-stimulated ICa which was consistent with their inhibitory action on guanylyl cyclase. However, MBlue (30 microM) had no effect under similar conditions. 3. In the absence of SNAP, LY 83583 (30 microM) potentiated the stimulations of ICa by either Iso (20 nM), forskolin (0.2 microM) or intracellular cyclic AMP (5-10 microM). ODQ (10 microM) had no effect under these conditions, while MBlue (30 microM) inhibited the Iso-stimulated ICa. 4. LY 83583 and MBlue, but not ODQ, reduced the inhibitory effect of up to 10 microM acetylcholine (ACh) on ICa. 5. MBlue, but not LY 83583 and ODQ, antagonized the activation of I(K,ACh) by ACh in the presence of intracellular GTP, and this inhibition was weakened when I(K,ACh) was activated by intracellular GTPgammaS. 6. The potentiating effect of LY 83583 on Iso-stimulated ICa was absent in the presence of either DL-dithiothreitol (DTT, 100 microM) or a combination of superoxide dismutase (150 u ml(-1)) and catalase (100 u ml(-1)). 7. All together, our data demonstrate that, among the three compounds tested, only ODQ acts in a manner which is consistent with its inhibitory action on the NO-sensitive guanylyl cyclase. The two other compounds produced severe side effects which may involve superoxide anion generation in the case of LY 83583 and alteration of beta-adrenoceptor and muscarinic receptor-coupling mechanisms in the case of M Blue.

1. The whole-cell patch-clamp was used for studying the effects of various beta1- and beta2-adrenoceptor agonists and antagonists on the L-type Ca current (Ica) in frog ventricular myocytes. 2. Dose-response curves for the effects of isoprenaline (non selective beta-agonist), salbutamol (beta2-agonist), dobutamine (beta1-agonist) on ICa were obtained in the absence and presence of various concentrations of ICI 118551 (beta2-antagonist), metoprolol (beta1-antagonist) and xamoterol (partial beta1-agonist) to derive EC50 (i.e. the concentration of beta-agonist at which the response was 50% of the maximum) and Emax (the maximal response) values by use of a Michaelis equation. Schild regression analysis was performed to examine whether the antagonists were competitive and to determine the equilibrium dissociation constant (K(B)) for the antagonist-receptor complex. 3. Isoprenaline increased ICa with an EC50 of 20.0 nM and an Emax of 597%. ICI 118551 and metoprolol competitively antagonized the effect of isoprenaline with a K(B) of 3.80 nM and 207 nM, respectively. 4. Salbutamol increased ICa with an EC50 of 290 nM and an Emax of 512%. ICI 118551 and metoprolol competitively antagonized the effect of salbutamol with a K(B) of 1.77 nM and 456 nM, respectively. 5. Dobutamine increased ICa with an EC50 of 2.40 microM and an Emax of 265%. ICI 118551 and metoprolol competitively antagonized the effect of dobutamine with a K(B) of 2.84 nM and 609 nM, respectively. 6. Xamoterol had no stimulating effect on ICa. However, xamoterol competitively antagonized the stimulating effects of isoprenaline, salbutamol and dobutamine on ICa with a K(B) of 58-64 nM. 7. We conclude that a single population of receptors is involved in the beta-adrenoceptor-mediated regulation of ICa in frog ventricular myocytes. The pharmacological pattern of the response of ICa to the different beta-adrenoceptor agonists and antagonists tested suggests that these receptors are of the beta2-subtype.

EHNA (Erythro-9-[2-hydroxy-3-nonyl]adenine) is a wellknown inhibitor of adenosine deaminase. Recently, EHNA was shown to block the activity of purified soluble cGMPstimulated phosphodiesterase (PDE2) from frog, human, and porcine heart with an apparent Ki value of approximately 1 microM and with negligible effects on Ca2+/calmodulin PDE (PDE1), cGMP-inhibited PDE (PDE3), and low Km cAMP-specific PDE (PDE4) (Méry, P.F., C. Pavoine, F. Pecker, and R. Fischmeister. 1995. Mol. Pharmacol. 48:121-130; Podzuweit, T., P. Nennstiel, and A. Muller. 1995. Cell. Signalling. 7:733- 738). To investigate the role of PDE2 in the regulation of cardiac L-type Ca2+ current (ICa), we have examined the effect of EHNA on ICa in freshly isolated human atrial myocytes. Extracellular application of 0.1-10 microM EHNA induced an increase in the amplitude of basal ICa ( approximately 80% at 1 microM) without modification of the current-voltage or inactivation curves. The maximal stimulatory effect of EHNA on ICa was comparable in amplitude with the maximal effect of isoprenaline (1 microM), and the two effects were not additive. The effect of EHNA was not a result of adenosine deaminase inhibition, since 2'-deoxycoformycin (1-30 microM), another adenosine deaminase inhibitor with no effect on PDE2, or adenosine (1-10 microM) did not increase ICa. In the absence of intracellular GTP, the substrate of guanylyl cyclase, EHNA did not increase ICa. However, under similar conditions, intracellular perfusion with 0.5 microM cGMP produced an 80% increase in ICa. As opposed to human cardiomyocytes, EHNA (1-10 microM) did not modify ICa in isolated rat ventricular and atrial myocytes. We conclude that basal ICa is controlled by PDE2 activity in human atrial myocytes. Both PDE2 and PDE3 may contribute to keep the cyclic nucleotides concentrations at minimum in the absence of adenylyl and/or guanylyl cyclase stimulation.

The regulation of cardiac L-type Ca2- current (Ica) and contraction by dihydropyridine antagonists and beta-adrenergic receptor agonists has been the subject of numerous studies over the last decade. However, little is known on the crosstalk between these two regulatory pathways. For instance, a fundamental question that remains unanswered is: does activation of the beta-adrenergic receptors modify the sensitivity of the myocardium to dihydropyridine agonists? To answer this question, we examined in the present study how activation of the beta-adrenergic receptors modifies the effects of nifedipine on the mechanical and energetic parameters of the isolated perfused rat heart. Activation of the beta-adrenergic receptors was achieved by perfusing the hearts with isoprenaline, a non-selective beta-adrenergic receptor agonist, and could be reduced by atenolol, a beta-adrenergic receptor antagonist. To examine possible alterations during hypertension in the sensitivity of the hearts to the drug, tested, the study was performed in both normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive animals (SHR). While 0.1 microM nifedipine reduced left ventricular pressure (LVP) by 36% and 34% in WKY and SHR rats, respectively, under basal conditions, its effects became negligible in both groups of rats after stimulation of the hearts with 0.1 microM isoprenaline. Addition of 1 microM atenolol in the presence of isoprenaline restored the inhibitory effect of nifedipine to control values in both WKY and SHR rats. Additional experiments were performed in isolated ventricular myocytes from WKY rats using the whole-cell patch-clamp technique. The inhibitory effects of 0.1 to 1 microM nifedipine were significantly larger on basal Ica than after the current had been previously elevated by 0.1 microM isoprenaline. Addition of 1 microM atenolol in the presence of isoprenaline partially restored the inhibitory effect of nifedipine on Ica. Our results demonstrate a reduced sensitivity of the heart muscle to nifedipine during activation of beta 1-adrenergic receptors. This effect is partly explained by a reduced inhibitory effect of nifedipine on Ic during activation of cAMP-dependent phosphorylation.

Muscarinic agonists regulate the L-type calcium current in isolated cardiac myocytes. The second messengers pathways involved in this regulation are discussed briefly, with particular emphasis on the involvement of cAMP and cGMP pathways.

1. A fast perfusion system was used to analyse the kinetics of the response of L-type calcium current (ICa) to rapid applications and washouts of the dihydropyridine antagonist nifedipine in whole-cell patch-clamped frog ventricular myocytes. 2. Both the inhibition of ICa induced by nifedipine and the recovery from inhibition upon washout of the drug behaved as mono-exponential functions of time. 3. During application or washout of 100 nM nifedipine, only the peak amplitude of ICa varied but not its time course of activation or inactivation. 4. The rate constant of the onset of ICa inhibition increased with the concentration of nifedipine. However, the time course of the recovery from inhibition was independent of drug concentration. 5. Both rate constants were strongly sensitive to the holding potential but insensitive to the test potential. 6. Using simple rate equations and a one-binding-site analysis it was possible to determine the rate constants for association (k1) and dissociation (k-1) and the equilibrium dissociation constant (KD) of the reaction between nifedipine and Ca2+ channels. KD values for nifedipine were identical to IC50 values obtained from classical steady-state experiments. 7. With depolarized holding potentials, KD decreased strongly due to a large reduction in k-1 and a modest increase in k1. Assuming that these changes result from the distribution of Ca2+ channels between resting and inactivated states, a low-affinity binding to the resting state (R) and a high-affinity binding to the inactivated state (I) were obtained with the binding constants: k1R = 1.0 x 10(6) M-1 S-1, k-1R = 0.077 S-1, and KDR = 77 nM for the resting state; k1I = 4.47 x 10(6) M-1 S-1, k-1I = 7.7 x 10(-4) S-1, and KDI = 0.17 nM for the inactivated state. 8. Rapid application/washout experiments provide a unique way to determine, in an intact cell and in a relatively short period (2-4 min), the binding rate constants and the KD value of the reaction between a dihydropyridine antagonist and the Ca2+ channels.

In the heart, the parasympathetic neurotransmitter acetylcholine (ACh) reduces the force of contraction. Although the effect of ACh can be partly explained by an inhibition of adenylyl cyclase, some of the effects of ACh may also be mediated via stimulation of nitric oxide synthase (NOS) and production of guanosine 3', 5'-cycle monophosphate (cGMP). NOS inhibitors can prevent the negative chronotropic effect of ACh on spontaneously beating cardiomyocytes and suppress the inhibition of the L-type calcium current (ICa) by ACh in sinoatrial myocytes. This pathway may be relevant not only to the chronotropic effect of ACh but also to its inotropic effect, because ACh, NO, and cGMP regulate the force of contraction and ICa in the cardiac ventricle. Here we report the effects of L-arginine (L-Arg), the substrate of NOS, and NG-monomethyl-L-arginine (L-NMMA) and NG-nitro-L-arginine (L-NNA), two NOS inhibitors, on muscarinic effects in the cardiac ventricle. We found that L-Arg, L-NMMA, and L-NNA have no effect on the muscarinic inhibition of ICa in isolated frog myocytes. In addition, these compounds have no significant effects on basal ICa or beta-adrenergic stimulation of ICa. L-Arg and its analogues did not change the negative inotropic effect of ACh in frog ventricular fibers. Basal active tension and the positive inotropic effect of isoproterenol, a beta-adrenergic agonist, also were unaffected. We conclude that NOS in not involved in muscarinic inhibition of ICa in isolated from ventricular myocytes or the negative inotropic effect of ACh in the frog ventricle.

1. The effects of acetylcholine (ACh) on the L-type Ca2+ current (ICa) stimulated by isoprenaline (Iso) or forskolin (Fsk) were examined in frog ventricular myocytes using the whole-cell patch-clamp technique and a double capillary for extracellular microperfusion. 2. The exposure of one half of the cell to 1 microM Iso produced a half-maximal increase in ICa since a subsequent application of Iso to the other half induced an additional effect of nearly the same amplitude. Similarly, addition of 1 microM ACh to only one half of a cell exposed to Iso on both halves reduced the effect of Iso by only approximately 50%. 3. When 10 microM Iso or 30 microM Fsk were applied to a Ca(2+)-free solution on one half of the cell, ICa was increased in the remote part of the cell where adenylyl cyclase activity was not stimulated. However, addition of ACh (3-10 microM) 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 the stimulatory effects of these drugs. 4. Our data demonstrate that ACh regulates ICa by acting at a site proximal to adenylyl cyclase in frog ventricular cells. We conclude that the muscarinic regulation of ICa does not involve any additional cAMP-independent mechanisms occurring downstream from cAMP generation.

The role of cAMP subcellular compartmentation in the progress of beta-adrenergic stimulation of cardiac L-type calcium current (ICa) was investigated by using a method based on the use of whole-cell patch-clamp recording and a double capillary for extracellular microperfusion. Frog ventricular cells were sealed at both ends to two patch-clamp pipettes and positioned approximately halfway between the mouths of two capillaries that were separated by a 5-micron thin wall. ICa could be inhibited in one half or the other by omitting Ca2+ from one solution or the other. Exposing half of the cell to a saturating concentration of isoprenaline (ISO, 1 microM) produced a nonmaximal increase in ICa (347 +/- 70%; n = 4) since a subsequent application of ISO to the other part induced an additional effect of nearly similar amplitude to reach a 673 +/- 130% increase. However, half-cell exposure to forskolin (FSK, 30 microM) induced a maximal stimulation of ICa (561 +/- 55%; n = 4). This effect was not the result of adenylyl cyclase activation due to FSK diffusion in the nonexposed part of the cell. To determine the distant effects of ISO and FSK on ICa, the drugs were applied in a zero-Ca solution. Adding Ca2+ to the drug-containing solutions allowed us to record the local effect of the drugs. Dose-response curves for the local and distant effects of ISO and FSK on ICa were used as an index of cAMP concentration changes near the sarcolemma. We found that ISO induced a 40-fold, but FSK induced only a 4-fold, higher cAMP concentration close to the Ca2+ channels, in the part of the cell exposed to the drugs, than it did in the rest of the cell. cAMP compartmentation was greatly reduced after inhibition of phosphodiesterase activity with 3-isobutyl-methylxanthine, suggesting the colocalization of enzymes involved in the cAMP cascade. We conclude that beta-adrenergic receptors are functionally coupled to nearby Ca2+ channels via local elevations of cAMP.

Hormonal regulation of cardiac inotropism is often correlated with modification of the L-type Ca-channel current. Among several regulatory pathways that control Ca-channel activity, the best described one is the cAMP cascade. Cyclic AMP-dependent phosphorylation of the Ca-channel results in an increase of the mean open probability of the individual Ca-channels and, thus, of the macroscopic Ca current. Modulation of cAMP concentration can take place at the level of adenylyl cyclases or cAMP phosphodiesterases. Of major interest is the fact that the activity of two different forms of phosphodiesterases is controlled by the level of intracellular cGMP. Thus, cAMP metabolism is intimately associated with cGMP metabolism, and both determine the degree of cAMP-dependent phosphorylation of cardiac Ca-channels. This brief discussion will focus on these two levels of control and their relative importance in the cAMP-dependent regulation of myocardial Ca-channels.

Early studies in whole heart indicated that cGMP antagonized the positive inotropic effects of catecholamines and cAMP. Since the L-type Ca2+ channel current (ICa) plays a predominant role in the initiation and development of cardiac electrical and contractile activities, regulation of ICa by cGMP pathways has received much attention over the last ten years. Patch-clamp measurements of ICa in isolated cardiac myocytes reveal at least three different cGMP effectors that may participate to different degrees in different animal species and cardiac tissues in the regulation of ICa by cGMP. In frog ventricular myocytes, cGMP inhibits ICa by stimulation of a cGMP-stimulated cAMP phosphodiesterase (PDE2), whereas in rat ventricular myocytes, cGMP predominantly inhibits ICa via a mechanism involving activation of a cGMP-dependent protein kinase (cGMP-PK). In guinea pig, frog and human cardiomyocytes, cGMP can also stimulate ICa via an inhibition of a cGMP-inhibited cAMP phosphodiesterase (PDE3). This effect is most predominant in human atrial myocytes and appears readily during an activation of the soluble guanylate cyclase activity by low concentrations of nitric oxide (NO)-donors. Biochemical characterization of the endogenous phosphodiesterases and cGMP-PK in purified cardiac myocytes provide further evidence in support of these mechanisms of cGMP action on ICa. However, the regulation of cGMP levels by a variety of agents is not always consistent with their effects on contractility. In particular, the participation of cGMP and NO pathways in the regulation of cardiac ICa and contractility by acetylcholine is still questionable.

Recently, an inhibitor of adenosine deaminase, erythro-9-(2-hydroxyl-3-nonyl)adenine (EHNA), was shown to selectively block the activity of purified cGMP-stimulated phosphodiesterase (PDE) (cGS-PDE, or PDE2) in human and porcine heart [J. Mol. Cell. Cardiol. 24 (Suppl. V):102 (1992)]. Because cGS-PDE was found to mediate the cGMP-induced inhibition of L-type Ca2+ current (Ica) in frog ventricular cells, we tested the effects of EHNA in this preparation. Ica was measured using the whole-cell patch-clamp technique and a perfusing pipette. EHNA (0.3-30 microM) had no significant effect on either basal Ica or isoprenaline (1 nM)- or cAMP (10 microM)-elevated Ica. However, EHNA dose-dependently (IC50 approximately 3 microM) reversed the inhibitory effect of cGMP on cAMP-stimulated Ica. EHNA (30 microM) also blocked the inhibitory effect of NO donors, such as sodium nitroprusside (1 mM) and 3-morpholinosydnonimine (30 microM), on isoprenaline-stimulated Ica. In addition, EHNA dose-dependently (IC50 approximately 4-5 microM) inhibited the cGMP-induced stimulation of PDE activity in frog ventricle particulate fraction, as well as purified soluble cGS-PDE. However, EHNA (up to 30 microM) did not modify the activities of three other purified soluble PDE isoforms. Moreover, EHNA did not change the Ka (40 nM) for cGMP activation of cGS-PDE, which suggests that EHNA does not inhibit cGS-PDE by displacing cGMP from the allosteric regulator site. Because adenosine did not mimic the effects of EHNA on Ica or PDE activity, it is unlikely that the effects of EHNA are due to adenosine deaminase inhibition. We conclude that EHNA acts primarily to inhibit cGS-PDE in intact cardiac myocytes. This compound should be useful in evaluating the physiological role of cGS-PDE in various tissues.

Cardiac Ca2+ current (ICa) was shown to be regulated by cGMP in a number of different species. Recently, we found that the NO-donor SIN-1 (3-morpholino-sydnonimine) exerts a dual regulation of ICa in frog ventricular myocytes via an accumulation of cGMP. To examine whether NO also regulates Ca2+ channels in human heart, we investigated the effects of SIN-1 on ICa in isolated human atrial myocytes. An extracellular application of SIN-1 produced a profound stimulatory effect on basal ICa at concentrations > 1 pM. Indeed, 10 pM SIN-1 induced a approximately 35% increase in ICa. The stimulatory effect of SIN-1 was maximal at 1 nM (approximately 2-fold increase in ICa) and was comparable with the effect of a saturating concentration (1 microM) of isoprenaline, a beta-adrenergic agonist. Increasing the concentration of SIN-1 to 1-100 microM reduced the stimulatory effect in two thirds of the cells. The stimulatory effect of SIN-1 was not mimicked by SIN-1C, the cleavage product of SIN-1 produced after liberation of NO. This suggests that NO mediates the effects of SIN-1 on ICa. Because, in frog heart, the stimulatory effect of SIN-1 on ICa was found to be due to cGMP-induced inhibition of cGMP-inhibited phosphodiesterase (cGI-PDE), we compared the effects of SIN-1 and milrinone, a cGI-PDE selective inhibitor, on ICa in human. Milrinone (10 microM) induced a strong stimulation of ICa (approximately 150%), demonstrating that cGI-PDE controls the amplitude of basal ICa in this tissue. In the presence of milrinone, SIN-1 (0.1-1 nM) had no stimulatory effect on ICa, suggesting that the effects of SIN-1 and MIL were not additive. We conclude that NO may stimulate ICa in human atrial myocytes via inhibition of the cGI-PDE.

The ability of acetylcholine (ACh) to inhibit beta-agonist stimulated calcium current was compared to its ability to activate the inwardly rectifying potassium current IK(ACh) in frog atrial myocytes. As suggested by previous studies, ACh inhibited the calcium current at concentrations (EC50 = 8 nM) significantly lower than those required for the activation of IK(ACh) (EC50 = 101 nM). The pharmacological profiles of the two responses suggest that despite the differences in agonist sensitivity, both are mediated by the same (m2) type of muscarinic receptors. Intracellular application of GDP beta S, an inhibitor of G protein function, completely abolished both responses, implying that both actions of ACh are coupled to effectors by G proteins. In contrast, intracellular application of pertussis toxin (PTX) shifted to higher concentrations (EC50 = 170 nM) but did not abolish inhibition of the calcium current by ACh even though the block of the IK(ACh) response was complete. Increasingly large PTX concentrations and/or prolonged PTX treatments revealed a limiting, PTX-resistant inhibitory component that appears to be mediated by a PTX-insensitive G protein distinct from that mediating IK(ACh). For the PTX-sensitive components, the different agonist dependencies of IK(ACh) activation and calcium current inhibition may imply that different G proteins mediate each response although alternate possibilities involving the same G protein either functionally sequestered and/or differentially affected by interactions with effectors, can not be ruled out.

The effects of the nitric oxide (NO) donor 3-morpholino-sydnonimine (SIN-1) on the L-type Ca2+ current (ICa) were examined in frog ventricular myocytes under basal and phosphorylated conditions. SIN-1 was found to exert insignificant effects on basal ICa but to induce a biphasic action on stimulated ICa. Indeed, in the nanomolar range of concentrations (0.1-10 nM), SIN-1 induced a pronounced (approximately 40%) stimulation of ICa elevated by a non-maximal concentration of forskolin (0.3 microM). However, the stimulatory effects of SIN-1 on ICa were not additive with those of maximal concentrations (10 microM) of forskolin or intracellular cAMP. In contrast, at higher concentrations (100 nM to 1 mM), SIN-1 strongly reduced ICa (by up to 85%) which had been previously stimulated by cAMP, forskolin, or isoprenaline. All the effects of SIN-1 appeared to be mediated by the liberation of NO since they were suppressed by methylene blue and LY83583 and were not mimicked by SIN-1C, a metabolite of SIN-1. The stimulatory or inhibitory effects of SIN-1 were absent, respectively, in the presence of milrinone (10 microM) or when the hydrolysis-resistant cAMP analog 8-bromo-cAMP was used instead of cAMP to stimulate ICa. In addition to its effects on ICa, SIN-1 induced a dose-dependent stimulation of guanylyl cyclase activity in the cytosolic and membrane fractions of frog ventricle. The membrane form of guanylyl cyclase displayed a higher sensitivity to SIN-1 than the cytosolic form, which correlated with SIN-1 sensitivity of ICa. Our data suggest that the activatory and inhibitory effects of NO donors on ICa result from an inhibition of the cGMP-inhibited cAMP-phosphodiesterase and an activation of the cGMP-stimulated cAMP-phosphodiesterase, respectively, both linked to the activation of guanylyl cyclase, possibly a membrane form of the enzyme.

A fast perfusion system was used to analyze the kinetics of the response of L-type calcium current (ICa) to rapid exposures to beta-adrenergic or dihydropyridine agonists in whole-cell patch-clamped frog ventricular myocytes. The perfusion system was based on the lateral motion of an array of plastic capillary tubes from which solutions flowed at a velocity of approximately 5 cm/s. Movement from one capillary to the adjacent one occurred in < 20 ms and complete exchange of extracellular solution was achieved in < 50 ms as demonstrated by the block of ICa by fastflow application of Cd during a depolarizing pulse. Fastflow applications of increasing concentrations of isoprenaline (Iso) led to a dose-dependent stimulation of ICa at [Iso] > 1 nM. The response of ICa to Iso always started after a delay of several seconds. The delay duration decreased as [Iso] increased, and was typically approximately 3 s at 10 microM Iso. The rising phase of ICa increase was monophasic and independent of [Iso] > 100 nM. For short applications of Iso (8.8 s), half maximal and maximal stimulation of ICa occurred approximately 20 s and approximately 40 s after the beginning of Iso application, respectively. When Iso was applied during a depolarizing pulse (with Ba as the charge carrier), IBa never increased during that pulse. The kinetics of the ICa response to Iso were not affected by varying the voltage clamp protocols or the ionic composition of intracellular and extracellular solutions. In comparison with the effects of Iso, the stimulatory effect of the dihydropyridine agonist (-)Bay K 8644 on ICa was approximately 15 times faster: delay, half-time to maximal and time to maximal responses were 15 times shorter with (-)Bay K 8644 than with Iso. It is concluded that frog ventricular myocytes respond slowly to a quick application of beta-adrenergic agonists.

Fast-flow perfusion and flash photolysis of caged compounds were used to study the activation kinetics of L-type calcium current (ICa) in frog cardiac myocytes. Rapid exposure to isoproterenol (Iso) for 1 s or approximately 1 min produced similar kinetics of increase in ICa with an initial lag period of approximately 3 s, followed by a monophasic rise in current with a half-time of approximately 20 s. Epinephrine, as well as caged Iso, produced increases with similar kinetics. The fact that ICa increased significantly even after short Iso applications suggests that agonist binding to the receptor is rapid and that the increase in ICa is independent of free agonist. To dissect the kinetic contributions of various steps in the cAMP-phosphorylation cascade, the kinetics of the responses to caged cAMP and caged GTP gamma S and fast perfusion of forskolin, acetylcholine, and propranolol were compared. The response to caged cAMP exhibited no lag period, but otherwise increased at a rate similar to that produced by Iso and reached a peak at approximately 40 s after flash photolysis. This suggests that the lag period itself is due to a step before cAMP accumulation, but that activation of protein kinase and phosphorylation of the calcium channel are relatively slow. A lag period was also observed when ICa was stimulated by flash photolysis of caged GTP gamma S and when adenylyl cyclase was activated directly by rapid perfusion with forskolin. The lag period observed with forskolin may be due to slow binding of forskolin. The lag period was not due to the time required for cAMP to reach a threshold concentration, because a similar lag was observed in response to Iso in cells having ICa previously stimulated submaximally by internal perfusion with a low concentration of cAMP. These results suggest that the lag period can be attributed to a step associated with activation of adenylyl cyclase and cAMP accumulation.

1. The whole-cell patch clamp and intracellular perfusion techniques were used for studying the effects of atropine and other muscarinic acetylcholine receptor (mAChR) antagonists on the L-type calcium currents (ICa) in frog and rat ventricular myocytes, and on the mAChR-activated K+ current (IK(ACh)) in frog atrial myocytes. 2. In frog ventricular myocytes, atropine (0.1 nM to 1 microM) reversed the inhibitory effect of acetylcholine (ACh, 1 nM) on ICa previously stimulated by isoprenaline (Iso, 2 microM), a beta-adrenergic agonist. However, in the concomitant presence of Iso, ACh and atropine, ICa was > 50% larger than in Iso alone. 3. The effects of atropine were then examined in the absence of mAChR agonists. After a preliminary stimulation of ICa with Iso (0.1 or 2 microM), atropine induced a dose-dependent stimulation of ICa. EC50 (i.e. the concentration of atropine at which the response was 50% of the maximum) and Emax (i.e. maximal stimulation of ICa expressed as percentage increase in ICa with respect to the level in Iso alone) were respectively 0.6 nM and 35%. The stimulatory effect of atropine on ICa was not voltage dependent. 4. Atropine (1 microM) had no effect on frog ICa (i) under basal conditions, (ii) upon stimulation of ICa by the dihydropyridine agonist (-)-Bay K 8644 (1 microM), or (iii) when ICa had been previously stimulated by intracellular perfusion with cyclic AMP (3 microM). However, atropine increased ICa after a stimulation by forskolin (0.3 microM). Therefore, an increased adenylyl cyclase activity was required for atropine to produce its stimulatory effect on ICa. 5. The order of potency of mAChR antagonists to reverse the inhibitory effect of ACh on Iso elevated ICa in frog ventricle was atropine > AF-DX 116 >> pirenzepine. In the absence of ACh, mAChR antagonists produced their stimulatory effect on Iso elevated ICa with the same order of potency. 6. Intracellular substitution of Gpp(NH)p (5'-guanylylimidiphosphate) for GTP (420 microM) induced a strong inhibition of frog ICa in the presence of Iso (2 microM). This effect was attributed earlier to the spontaneous and irreversible activation of the GTP-binding regulatory protein (G protein), Gi, responsible for adenylyl cyclase inhibition. Atropine (1 microM) slowed down by a factor of 2 the rate of ICa inhibition induced by Gpp(NH)p. 7. In frog atrial myocytes, intracellular perfusion with 1 mM Gpp(NH)p induces spontaneous activation of IK(ACh). This effect was attributed earlier to the spontaneous and irreversible activation of the G protein, GK.(ABSTRACT TRUNCATED AT 400 WORDS)

The cardiac L-type Ca2+ channel has served as a model for ion channel regulation for over a decade. The Ca2+ current is increased by beta-adrenoceptor stimulation and this effect is inhibited by muscarinic acetylcholine receptor stimulation. It is well established that beta-adrenoceptor stimulation increases this current largely by cAMP-dependent phosphorylation but recently data have been presented that suggest that this channel may also be regulated directly by G proteins. This review by Criss Hartzell and Rodolphe Fischmeister evaluates evidence for this second regulatory pathway and concludes that, although G proteins affect cardiac Ca2+ channels in bilayers and excised patches, there is little evidence that this pathway is physiologically significant.

We have recently reported that glucagon activated the L-type Ca2+ channel current in frog ventricular myocytes and showed that this was linked to the inhibition of a membrane-bound low-Km cAMP phosphodiesterase (PDE) (Méry, P. F., Brechler, V., Pavoine, C., Pecker, F., and Fischmeister, R. (1990) Nature 345, 158-161). We show here that the inhibition of membrane-bound PDE activity by glucagon depends on guanine nucleotides, a reproducible inhibition of 40% being obtained with 0.1 microM glucagon in the presence of 10 microM GTP, with GTP greater than GTP gamma S, while GDP and ATP gamma S were without effect. Glucagon had no effect on the cytosolic low-Km cAMP PDE, assayed with or without 10 microM GTP. Glucagon inhibition of membrane-bound PDE activity was not affected by pretreatment of the ventricle particulate fraction with cholera toxin. However, it was abolished after pertussis toxin pretreatment. Mastoparan, a wasp venom peptide known to activate G(i)/G(o) proteins directly, mimicked the effect of glucagon. PDE inhibition by glucagon was additive with the inhibition induced by Ro 20-1724, but was prevented by milrinone. This was correlated with an increase by glucagon of cAMP levels in frog ventricular cells which was not additive with the increase in cAMP due to milrinone. We conclude that glucagon specifically inhibits the cGMP-inhibited, milrinone-sensitive PDE (CGI-PDE). Insensitivity of adenylylcyclase to glucagon and inhibition by the peptide of a low-Km cAMP PDE were not restricted to frog heart, but also occurred in mouse and guinea pig heart. These results confirm that two mechanisms mediate the action of glucagon in heart: one is the activation of adenylylcyclase through Gs, and the other relies on the inhibition of the membrane-bound low-Km CGI-PDE, via a pertussis toxin-sensitive G-protein.

A new and inexpensive system allowing rapid and synchronized changes of solutions around a membrane patch or a cell under voltage-clamp conditions is described. Four plastic capillary tubings (OD 640 microns; ID 430 microns) were glued together horizontally and attached to a coil of a commercially available loudspeaker. Servo-control of the position of the coil allowed the mouth of any of the capillaries to be positioned near the pipette tip within 6 ms. A high flow speed of the test solution was crucial to achieve rapid solution exchange. At a flow speed of 5 cm/s, complete exchange of the external environment of a frog ventricular cell was achieved within 20-30 ms. The time course of solution change was found to be 3-5 times faster at the tip of an open patch pipette. To preserve the physical integrity of the cell, the cell was usually perfused by a control capillary at a slow velocity (0.2-0.4 cm/s) and test solutions flowing out of adjacent capillaries at high velocity (4-5 cm/s) were applied to the cell only for short periods. Determination of the three-dimensional contamination profile around the mouth of the control capillary allowed the optimal conditions for the use of the system to be established and possible sources of contamination to be avoided between adjacent capillaries with unmatched flow speeds. Successive and multiple changes in external solutions could be easily synchronized with voltage-clamp depolarizations to examine the time course of the effect of drugs on voltage-operated ion channels.(ABSTRACT TRUNCATED AT 250 WORDS)

The positive inotropic effect of the sympathetic nervous system on the heart is partly mediated by an increase in the voltage-gated Ca2+ current (ICa). This increase is generally attributed to beta-adrenergic receptor-stimulated cyclic AMP-dependent phosphorylation of the Ca2+ channel. It has been suggested that cAMP-dependent phosphorylation cannot explain all the effects of beta-adrenergic agonists on ICa and that a parallel membrane-delimited pathway involving the 'direct' action of the G protein Gs also stimulates ICa. A precedent exists for such a membrane-delimited pathway in the activation of a K+ channel by acetylcholine in heart. A membrane-delimited pathway for stimulation of ICa might be important in rapid beat-to-beat regulation of contraction by the sympathetic nervous system, because isoproterenol may produce a biphasic increase in ICa with the rapid phase (tau = 150 ms) putatively mediated by the direct pathway and the slow phase (tau = 35 s) by cAMP-dependent phosphorylation. Here we report that in frog, rat, and guinea pig ventricular myocytes ICa increases slowly and monophasically in response to isoproterenol. The increase is completely blocked by inhibitors of cAMP-dependent phosphorylation. Furthermore, the time course of the increase in ICa closely parallels the increase in contractile force produced by sympathetic nerve stimulation. These data refute earlier suggestions that regulation of Ca2+ channels by the sympathetic nervous system involves or requires a direct G-protein pathway.

Regulation of cardiac contraction by neurotransmitters and hormones is often correlated with regulation of the L-type Ca2(+)-channel current (ICa) through the opposite actions of two second messengers, cyclic AMP and cyclic GMP. While cyclic AMP stimulation of ICa is mediated by the activation of cyclic AMP-dependent protein kinase, inhibition of ICa by cyclic GMP in frog heart is largely mediated by activation of cyclic AMP phosphodiesterase. The present patch-clamp study reveals that, in rat ventricular cells, cyclic GMP can also regulate ICa via activation of endogenous cyclic GMP-dependent protein kinase (cGMP-PK). Indeed, the effect of cyclic GMP on ICa was mimicked by intracellular perfusion with the proteolytic active fragment of purified cGMP-PK. Moreover, cGMP-PK immunoreactivity was detected in pure rat ventricular myocytes by using a specific polyclonal antibody. These results demonstrate a dual mechanism for the inhibitory action of cyclic GMP in heart, as well as a physiological role for cGMP-PK in the control of mammalian heart function.

At least four different isoforms of phosphodiesterases (PDEs) are responsible for the hydrolysis of cAMP in cardiac cells. However, their distribution, localization and functional coupling to physiological effectors (such as ion channels, contractile proteins, etc.) vary significantly among various animal species and cardiac tissues. Because the activity of cardiac Ca2+ channels is strongly regulated by cAMP-dependent phosphorylation, Ca(2+)-channel current (ICa) measured in isolated cardiac myocytes may be used as a probe for studying cAMP metabolism. When the activity of adenylyl cyclase is bypassed by intracellular perfusion with submaximal concentrations of cAMP, effects of specific PDE inhibitors on ICa amplitude are mainly determined by their effects on PDE activity. This approach can be used to evaluate in vivo the functional coupling of various PDE isozymes to Ca2+ channels and their differential participation in the hormonal regulation of ICa and cardiac function. Combined with in vitro biochemical studies, such an experimental approach has permitted the discovery of hormonal inhibition of PDE activity in cardiac myocytes.

The diterpene forskolin is widely known for its ability to directly activate adenylyl cyclase and consequently increase intracellular cAMP. In cardiac cells, one result is a cAMP-mediated increase in the L-type Ca2(+)-channel current (ICa). However, forskolin was also shown recently to affect a number of ionic channels in noncardiac cells by mechanisms that do not involve activation of adenylyl cyclase. The present study reveals such an effect of forskolin on cardiac Ca2+ channels. Indeed, under appropriate conditions, forskolin was found to cause an inhibition of ICa. Although the stimulation of adenylyl cyclase and ICa requires micromolar concentrations of forskolin, the inhibitory effect of forskolin was observed in the nanomolar range of concentrations, i.e., 2-3 orders of magnitude lower. This high affinity forskolin inhibition of ICa was observed when ICa was previously enhanced via a cAMP-dependent pathway, but not when ICa was at its basal level or when the current was elevated by the dihydropyridine Bay K 8644. The inhibitory effect occurred at a site of action remote from adenylyl cyclase, because forskolin similarly inhibited ICa that had been previously elevated by isoprenaline (a beta-adrenergic agonist) or directly by intracellular perfusion with cAMP. Under these conditions, forskolin was inhibitory when applied to either side of the cell membrane, but only in its lipid-soluble form. The inhibitory effect of forskolin appeared to be independent of membrane potential and was not accompanied by a change in the time constants of ICa activation and inactivation. This may indicate that forskolin mainly reduces the number of functional Ca2+ channels without changing the gating of individual channels. However, the reduction in ICa amplitude was not equally distributed among the different exponential components that constitute ICa, which suggests that forskolin also modifies the resting state of the channels. This novel high affinity forskolin inhibition of ICa may take place at some step in the pathway between cAMP and Ca2+ channel phosphorylation and/or at Ca2+ channels only after they have been phosphorylated.

The voltage-gated Ca2+ current (ICa) in cardiac myocytes is regulated by cAMP-dependent phosphorylation. Although the regulation of ICa via mechanisms involving modulation of cAMP synthesis is well understood, the regulation of cAMP degradation has been less thoroughly investigated. The goal of the present study was to investigate the participation of different subclasses of cAMP phosphodiesterase (PDE) in regulating cAMP-dependent phosphorylation of Ca2+ channels in frog ventricular myocytes. Cardiomyocytes were isolated enzymatically and mechanically and were patch-clamped using the whole-cell configuration of the patch-clamp technique. The effects of various low-Km cAMP PDE inhibitors on ICa were examined. None of the inhibitors tested [milrinone, indolidan, 1-methyl 3-isobutyl xanthine (MIX), rolipram, or Ro 20-1724] were able to elevate ICa in the absence of elevated cAMP, although they all increased ICa in the presence of submaximal levels of cAMP. This result suggests that these compounds do not act directly on Ca2+ channels but rather modulate cAMP degradation. Half-maximal effects were observed with 1.4 microM milrinone and 3.4 microM MIX. Milrinone was effective when applied from either the extracellular or intracellular surface, whereas MIX was effective only when applied from the extracellular solution. In the presence of internal cGMP, which stimulates the cGMP-stimulated PDE, the low-Km cAMP PDE inhibitors had no effect on ICa, whereas high concentrations of MIX, which inhibit the cGMP-stimulated PDE, increased ICa. This would support the hypothesis that cGMP-stimulated PDE either has a much stronger capacity to hydrolyze cAMP or is more efficiently coupled to Ca2+ channels than the low-Km cAMP PDEs.

Glucagon exerts positive inotropic and chronotropic effects in the heart. Like its glycogenolytic effect in liver cells, the cardiac effects of glucagon are often correlated with adenylyl cyclase stimulation. Therefore, cyclic AMP-dependent phosphorylation of L-type Ca2+ channels might be involved in the inotropic effect of glucagon. There have been no reports, however, of the effects of glucagon on the cardiac Ca2+ current (ICa). Also, the physiological effects of glucagon could involve mechanisms other than stimulation of adenylyl cyclase. Here we show that glucagon enhances ICa in frog and rat ventricular myocytes. The effect of glucagon in rats resulted from a stimulation of adenylyl cyclase. In frogs, however, the effect of glucagon on ICa was smaller and occurred at a concentration tenfold lower than in rats, and adenylyl cyclase was not modified. In addition, cAMP potentiated the effect of glucagon on ICa in frog ventricle, which correlated with the observed inhibition by glucagon of low-Km cAMP phosphodiesterase activity. Therefore, this is an example of a hormone that affects cardiac function in a similar way to a variety of synthetic cardiotonic compounds, such as milrinone and Ro-20-1724. Inhibition of phosphodiesterase activity by glucagon may be essential in animals in which glucagon increases cardiac contractility but does not effectively stimulate adenylyl cyclase.

The validity of a Hodgkin-Huxley type voltage-dependent inactivation of slow inward Ca current (Isi) was tested in frog heart using a computer simulation. The time course of Isi was calculated during the development of a frog atrial action potential (AP). With a time constant of inactivation (tauf) of 55 ms at a membrane potential (Em) of -15 mV, the variation of Isi was biphasic: after a transient increase followed by a decrease to zero, Isi partially "reactivated" (at the beginning of the AP repolarization phase) and then fully deactivated. The "reactivation" phase of Isi developed whether tauf was an increasing, decreasing, U-shaped, or bell-shaped function of Em. The addition of an independent and slower process responsible for the recovery from inactivation only partly suppressed the "reactivation" phase. However, until now there was no experimental evidence supporting such a biphasic variation of Isi during AP repolarization. Thus our results indicate that the Hodgkin-Huxley type model of the voltage-dependence of Isi-inactivation process may not correctly represent the actual behavior of frog cardiac muscle.

The effects of extracellular application of cicletanine on the voltage-sensitive calcium current (ICa) was studied in isolated cells from frog ventricle. Myocytes were isolated by enzymatic dissociation and ICa was measured using the whole-cell configuration of the patch-clamp technique modified to permit intracellular perfusion with various substances. Cicletanine (10 to 100 microM) had no effect on control ICa. However, when ICa was enhanced by superfusion of the cell with saturating doses of beta-adrenergic agonist (isoprenaline, 2 microM) or by intracellular perfusion with maximal doses of cAMP (20 microM), cicletanine exerted a dual effect on ICa. At 10 microM, cicletanine generally induced a transient or sustained stimulation of ICa (5 to 40%), while 100 microM of the drug generally reduced ICa. The effects of cicletanine were reversible and not voltage-dependent. These results suggest that cicletanine affects ICa by acting on a mechanism occurring after cAMP synthesis, by enhancing cAMP concentration (e.g. through an inhibition of cAMP phosphodiesterase) or facilitating cAMP-dependent phosphorylation of the Ca channels.

The effect of external application of synthetic atrial natriuretic factor (ANF) on calcium current (ICa) was studied in single cells isolated from frog ventricle using the whole-cell patch-clamp technique. Rat atriopeptin III (APIII) and 3-28 ANF rat (rANF) had negligible effects on basal ICa at concentrations up to 200 nM. However, when ICa was increased by isoprenaline, both peptides had significant inhibitory effects. rANF (3 nM) decreased isoprenaline-elevated ICa by an average of 33% after 3-5 minutes. APIII was slightly less effective than rANF. The effects of rANF and APIII were dose-dependent in a complex manner: one stimulatory and two different inhibitory effects were observed, one being responsible for an irreversible rundown of ICa. The effects of ANF were not blocked by atropine and desensitization of the cells to isoprenaline did not play a significant role in the response to ANF. When ICa was elevated by intracellular perfusion with cyclic adenosine 3',5'-monophosphate, added to the patch electrode solution or using a perfused pipette, rANF or APIII had less inhibitory effect, and no rundown of ICa was observed. It is proposed that adenylate cyclase may be one of several mechanisms by which ANF regulates ICa.

The effect of intracellular perfusion with cyclic GMP (cGMP) on Ca current (Ica) was investigated in Cs-loaded isolated cells from guinea pig ventricle using the whole-cell patch-clamp technique and a perfused patch pipette. cGMP (5 microM) strongly reduced Ica which had been elevated by intracellular perfusion with 50 microM of either cyclic AMP (cAMP) or its hydrolysis-resistant analog 8-Bromo-cAMP. In addition, cGMP prevented the stimulation of Ica by IBMX, a phosphodiesterase inhibitor. The membrane permeant cGMP analog 8-Bromo-cGMP (100 microM), when applied outside the cell, also antagonized the stimulatory effect of IBMX on Ica. It is concluded that cGMP inhibits Ica in guinea pig ventricular cells by a mechanism different from the activation of a cGMP-stimulated phosphodiesterase recently found in frog ventricular cells.

1. Calcium currents (ICa) were measured in single cells isolated from frog ventricle using the whole-cell patch-clamp technique and a perfused pipette. The dose-dependent stimulatory effects of isoprenaline (Iso, 0.1-100 microM) and forskolin (Fo. 0.1-50 microM) on ICa were determined in the presence and absence of acetylcholine (ACh, 10 microM) and/or threshold concentrations of Fo (0.2 microM) and Iso (0.05 microM), respectively. EC50 (i.e. concentration of Iso or Fo at which the response was 50% of the maximum) and Emax (i.e. maximal stimulation of Ica expressed as percentage increase in ICa with respect to control) were measured under each condition. 2. ACh increased EC50 for the stimulatory action of Iso on ICa from 0.84 to 3.72 microM while it reduced Emax from 658 to 185%. Thus, ACh mainly reduced the efficacy of Iso to stimulate ICa. 3. ACh increased EC50 for the stimulatory action of Fo on ICa from 2.06 to 10.26 microM but only slightly reduced Emax from 893 to 778%. Thus, ACh mainly reduced the potency of Fo to stimulate ICa. 4. Intracellular perfusion with 100 microM of hydrolysis-resistant GTP analogues, GTP-gamma-S [guanosine-5'-O-(3-thiotriphosphate)] and Gpp (NH)p (5'-guanylylimido-diphosphate), had no effect on basal ICa but reduced by greater than 50% the stimulatory effect of 2 microM-Iso on ICa. 5. In the presence of Gpp(NH)p or GTP-gamma-S, Fo (3 microM) reversibly increased ICa by 490%, as compared to a 717% increase in control (GTP) intracellular solution. Although ACh could still inhibit Fo-stimulated ICa, the degree of inhibition was significantly smaller than in the presence of GTP. 6. Extracellular perfusion with low concentrations of a combination of Iso (33 nM) and Fo (330 nM) enhanced ICa to a much greater extent than did either agent alone at 3 times higher concentrations. Thus, low concentrations of Iso and Fo appear to increase ICa in a synergistic fashion. 7. ICa stimulated by a combination of Iso and Fo appeared to be more resistant to inhibition by ACh than when stimulated by either alone. It was the efficacy, rather than the potency, of ACh to inhibit ICa that was reduced upon dual stimulation of ICa. 8. In the presence of 0.2 microM-Fo, EC50 and Emax for the effects of Iso on ICa were 0.27 microM and 619%, respectively. By comparison with the effects of Iso alone, Fo reduced EC50 approximately 3 times with no significant change in maximal stimulation.(ABSTRACT TRUNCATED AT 400 WORDS)

The variation in the concentration of a diffusing substance inside a cylindrical cell submitted to a time-dependent flux at the sarcolemmal membrane was studied theoretically. An application was derived to estimate the local modifications of intracellular free Ca2+ concentration ([CA2+]i) induced by the slow inward Ca2+ current (ICa) in frog heart. During a O mV voltage clamp depolarization, [Ca2+]i at the inner side of the membrane rises earlier and faster than [Ca2+]i at the center of the cell. The binding of intracellular Ca2+ to specific sites enhances the deviation between the two concentrations and may generate an accumulation-depletion process of Ca2+ near the membrane. However, it also decreases the overall [Ca2+]i. The relatively slow diffusion of sarcoplasmic Ca2+ does not significantly affect the kinetics of ICa through a modification in the Ca2+ gradient across the membrane.

Single-channel currents were recorded with the cell-attached patch-clamp technique from small clusters (2-20 cells) of spontaneously beating 7-d embryo ventricle cells. Because the preparation was rhythmically active, the trans-patch potential varied with the action potential (AP). The total current through the patch membrane was the patch action current (AC). ACs and APs could be recorded simultaneously, with two electrodes, or sequentially with one electrode. Channel activity, which varied depending on the number and type of channels in the patch, was present during normal cell firing. This method can reveal the kinetics and magnitudes of the specific currents that contributed to the AP, under conditions that reflect not only the time and voltage dependence of the channels, but also environmental factors that may influence channel behavior during the AP.

1. The effects of the current (iex) generated by the Na-Ca exchange mechanism have been investigated on the electrical activity of a cardiac cell using the model of MC ALLISTER et al. (1975) for the Purkinje fibre action potential (AP). 2. The reversal potential and the steady-state value of iex were described by the same equations as for the squid axon (MULLINS, 1976). The maximal intensity and the time constant of iex were extrapolated from experiments using frog hearts. 3. A compact program written with the Adams method in Fortran IV (PLANT, 1979) allowed a minicomputer to be used. 4. The addition of iex to the previous model induced a prolongation of the AP and a reduction of the duration of the diastolic phase. 5. Increasing the maximal steady-state amplitude of iex may lead to early after depolarizations and oscillatory behaviour. These effects can be prevented by adequate changes in the amplitude of some potassium outward currents. 6. It is concluded that : (i) alterations of the Na-Ca exchange, e.g., by cellular Na loading, should induce variations of both AP repolarization and diastolic phase durations and, consequently, alter the beating rate ; (ii) iex could interfere with the outward currents whose characteristics should be reconsidered.

Abrupt exposure of 90- to 130-micron diameter chick embryonic myocardial cell aggregates to 10 mM caffeine has been shown to induce a transient inward current. In the present study, we recorded a similar current in small cell clusters (less than 10 cells) in which access of caffeine to each of the cells was rapid. The resulting inward current consisted of a single peak, which decayed exponentially (predominant time constant 335 +/- 130 ms at -40 mV) and had a peak amplitude of up to 15.5 microA/cm2. The caffeine-induced current persisted when the slow inward current was abolished by a 30-s pretreatment with 2 microM D 600 and could be observed at potentials where the fast sodium channels were fully inactivated. The current-voltage relation of the caffeine response was linear between -110 and -40 mV, giving an extrapolated voltage intercept of +12 mV. However, the inward current did not diminish or reverse with further depolarization. A substantial inward current occurred at potentials up to +60 mV, which is more positive than the reversal potential of the tetrodotoxin-sensitive inward current. We conclude that the caffeine-induced current is mediated in part by electrogenic Na+-Ca2+ exchange.

Ca current inactivation was investigated in frog atrial muscle under voltage-clamp conditions. To inhibit the outward currents, experiments were performed on Cs-loaded fibers and in 20 mM Cs (K-free) Ringer with 4-AP added. Inactivation, produced by a conditioning pulse, was measured by reducing the current during a subsequent test pulse. The extent of inactivation increased initially with prepulse amplitude and then decreased as the prepulse potential became progressively positive. Relative inactivation follows a U-shaped curve. When Sr was substituted for Ca, both the degree and the rate of inactivation decreased. Relative inactivation appeared to be linearly related to the amount of divalent cations (Ca and Sr) carried into the cell during the prepulse. Elevating Ca enhanced peak current and accelerated its decline. Elevating Mg decreased peak current and slowed its decline. An application of Na-free (LiCl) solution resulted in a somewhat smaller but faster inactivating current. Adrenaline increased and D600 decreased the maximal Ca conductance with little alteration in the inactivation rate; Co decreased both peak current and the rate of inactivation. Enhancement of the outward currents, reduced driving force, and intracellular surface charge screening do not adequately account for the above results. Evidence was considered that Ca entry mediates most of Ca current inactivation in frog atrial fibers. Removal from inactivation was also investigated in normal-Ca, Ca-rich, and Sr solutions. Recovery after partial inactivation by high depolarization was biphasic. Recovery was slowed by 10 Ca and accelerated by 1.8 Sr, whereas opposite effects have been shown on activation.

Ca currents (ICa) were measured by whole-cell patch clamp in single cells isolated from frog ventricle in which K currents were blocked with intracellular (120 mM) and extracellular (20 mM) Cs. Inward currents elicited by depolarizing voltage steps from a holding potential of -80 mV were blocked completely by 0.5 mM-Cd. The quality of the voltage clamp was assessed using two patch electrodes on a single cell. One electrode was used in the voltage-clamp mode to measure membrane currents and the other in current-clamp to measure membrane potential. Ca currents as large as 2 nA were well clamped in cells as long as 210 micron. Acetylcholine (ACh) had no effect on ICa in the absence of beta-adrenergic stimulation but reduced to control levels ICa elevated by isoprenaline. Nanomolar concentrations of ACh were able to reduce significantly ICa elevated by 2 microM-isoprenaline. ACh had no effect on the shape of the I-V curve, on the reactivation (recovery from inactivation), or on the inactivation of ICa. Although isoprenaline increased ICa by an average of 6.5-fold, it had no effect on the shape of the I-V curve or on the inactivation at test potentials negative to +40 mV. However, isoprenaline slowed the half-reactivation time from a control value of 120 +/- 10 ms (mean +/- S.D.) to 153 +/- 12 ms at -80 mV. The effect of cyclic AMP on ICa was investigated using two patch electrodes, one filled with cyclic AMP. Maximal effects of cyclic AMP were observed with 5 microM-cyclic AMP in the pipette. Maximal ICa was recorded several minutes after breaking the patch with the second electrode. After removing the cyclic-AMP-containing electrode, ICa declined to control levels after approximately 10 min. 5 microM-cyclic AMP in the patch electrode increased ICa by an average of 6.9-fold, but had no effect on the shape of the I-V curve or on inactivation. Cyclic AMP had a slowing effect on reactivation (half-reactivation time = 155 +/- 24 ms) similar to that of isoprenaline. ACh (1-10 microM) did not reduce ICa elevated with cyclic AMP (0.1-20 microM-cyclic AMP in the pipette). With low concentrations of cyclic AMP in the pipette (0.1 microM), isoprenaline augmented ICa, but with 5 microM-cyclic AMP in the pipette, isoprenaline was incapable of increasing ICa further. These results suggest that the decrease of ICa produced by ACh can be explained solely by decreases in cyclic AMP levels.

The effects of transient alterations in Nao were investigated under voltage clamp conditions in frog heart cells previously loaded with Cs. Tetrodotoxin and Cs were used to inhibit Na and K currents. On applying a Na-poor solution (39.2 mM), an outward current was generated during both depolarizations and hyperpolarizations. The current amplitude described a U-shaped function of the membrane potential. On reapplying the standard solution after 15 min equilibration, an inward current was then induced that exhibited a bell-shaped function of the membrane potential. Current amplitude was sensitive to the external Ca concentration. Increasing pHi by 10 mM NH4Cl enhanced this current, while the internal acidification that occurred on switching back to the control solution greatly reduced it. Variations in the amplitude of this current during repetitive stimulations or long pauses are best explained by subsequent alterations in Nai and pHi; no evidence for a time dependence was found. This current was inhibited by La3+, Co2+, and D600, and was sensitive to adriamycin, quinidine, and disopyramide; lidocaine, another local anesthetic, and nifedipine had no effect. These observations extend previous work on intact heart cells and sarcolemmal vesicles. They suggest that the Na-Ca exchange may generate a current that is outward when Ca ions are moving into the cell.

With two independent patch electrodes sealed to small clusters of electrically coupled chick embryo cardiac cells, we have measured four parameters: true seal and patch resistance, channel conductance, and membrane potential. One electrode was in the cell-attached mode, and recorded current flowing in parallel through the membrane patch and seal. The second electrode, sealed on a different cell in the cluster, was in the whole cell recording configuration, and served to record or control the membrane potential of the cluster. We fit the four measured parameters to a simple electrical model to reveal errors not usually recognized in the patch-clamp technique. Among these are the following: (1) The apparent seal resistance, determined by changing the potential in a patch electrode, may be a poor estimate of true seal resistance, since it includes the parallel combination of seal- and patch-resistance. (2) Patch resistance may be influenced by the electrode filling solution, and is often much lower than is usually assumed. (3) With a small cell preparation that has an input resistance in the gigaohm range, measurements of single-channel conductance using a cell-attached patch electrode may be inaccurate because cell membrane potential does not remain constant as electrode potential is varied.

The effect of extracellular and intracellular application of forskolin on the voltage-sensitive calcium current, ICa, was studied in myocytes isolated from frog ventricle. Myocytes were isolated by enzymatic dissociation, and ICa was measured using the whole-cell configuration of the patch clamp technique modified to permit intracellular perfusion of various substances. Intracellular perfusion with forskolin (0.1 to 10 microM) had a negligible effect on ICa: ICa was increased 15 +/- 13% (mean +/- SE; N = 5). In contrast, superfusion of the cell with forskolin increased ICa significantly. The EC50 for the forskolin effect was 0.4 microM. A maximal 4.5-fold increase in ICa occurred with 3 microM forskolin. This is somewhat less than the maximal response to isoprenaline seen in this same series of experiments. The effects of forskolin, isoprenaline, and intracellular cAMP were not additive. In contrast, the effects of isoprenaline or intracellular cAMP and calcium channel agonists, such as Sandoz (+)202-791, were additive. This supports the hypothesis that the positive inotropic effects of forskolin are at least partly mediated by an increase in intracellular cAMP and a stimulation of ICa. The effects of forskolin were antagonized by acetylcholine (1 microM) or intracellular perfusion with cGMP. Acetylcholine on the average decreased forskolin-stimulated ICa 57 +/- 11% (N = 17). The relevance of these results to the suggestion that acetylcholine acts by mechanisms other than inhibition of adenylate cyclase is discussed.

The slow inward Ca2+ current, ICa, is fundamental in the initiation of cardiac contraction and neurohormonal regulation of cardiac function. It is increased by beta-adrenergic agonists, which stimulate synthesis of cyclic AMP (cAMP) and cAMP-dependent phosphorylation. The neurotransmitter acetylcholine reduces ICa by an unknown mechanism. There is strong evidence that acetylcholine reduces ICa by decreasing adenylate cyclase activity, but cGMP has also been implicated as ACh stimulates cGMP accumulation and activates cGMP-dependent protein kinase. Application of cGMP decreases contractile force, decreases Ca flux, shortens the duration of action potentials and inhibits Ca-dependent action potentials. Other studies, however, have concluded that cGMP levels do not correlate with contractile force and that cGMP has no effect on ICa. We have therefore examined the effects of intracellular perfusion of cGMP on ICa using isolated, voltage-clamped cells from frog ventricle. We find that cGMP has negligible effects on basal ICa, but greatly decreases the ICa that had been elevated by beta-adrenergic agonists or by intracellular perfusion with cAMP. The decrease of ICa is mediated by cAMP hydrolysis via a cGMP-stimulated cyclic nucleotide phosphodiesterase.

1. Ca2+ currents were measured in single cells isolated from frog ventricle using the whole-cell patch clamp technique and a perfused pipette. K+ currents were blocked with intracellular (120 mM) and extracellular (20 mM) Cs+. 2. A single type of Ca2+ current (ICa) was found in these cells. The current activated at voltages positive to -30 mV, exhibited a symmetrical current-voltage relationship with a peak at 0 mV, and was slowly inactivating with Ba2+ as charge carrier. 3. Large variations in ICa amplitude were observed from cell to cell (ICa at 0 mV = 293.1 +/- 283.3 pA; N = 152). These variations were not due simply to differences in cell membrane area, which was estimated by cell membrane capacitance (Cm), because the density of Ca2+ current (dICa = ICa/Cm) also varied significantly from cell to cell (1.3-28 pA/pF at 0 mV; mean +/- S.D. = 4.49 +/- 3.96; N = 152). 4. The inactivation curve of ICa was a complex function of membrane potential. 200 ms pre-pulses to voltages between -60 and +20 mV progressively inactivated ICa elicited by a subsequent test pulse with half-maximal inactivation occurring for pre-pulses to approximately -40 mV. With pre-pulses positive to +20 mV, ICa elicited by the test pulse became progressively larger. The degree of inactivation induced by a 200 ms depolarization to potentials more positive than +20 mV varied significantly from cell to cell, while no such variations were observed in the negative range of membrane potentials. 5. The time course of reactivation (i.e. removal from inactivation) of ICa at -80 mV often exhibited an overshoot. The amplitude of the overshoot varied between 100% (i.e. no overshoot) and approximately 180% in eighty-one cells. 6. The degree of inactivation at positive potentials (+100 mV) and the amplitude of the overshoot were strongly correlated with the Ca2+ current density. The overshoot was more pronounced, the reactivation was faster, and the inactivation at positive potentials was less in cells with lower ICa density. 7. Increasing the stimulation frequency from 0.125 to 2 Hz induced a positive staircase of ICa in cells with ICa density less than 2 pA/pF and a negative staircase in cells with ICa density greater than 3 pA/pF. 8. Perfusing the patch pipette with 5 mM-BAPTA instead of EGTA reduced the amplitude of the overshoot and slightly slowed the inactivation kinetics. Replacing extracellular Ca2+ ions by Ba2+ ions completely suppressed the overshoot.(ABSTRACT TRUNCATED AT 400 WORDS)

1. The effect of intracellular perfusion with cyclic AMP and cyclic GMP on Ca2+ current (ICa) was studied in single cells isolated from frog ventricle using the whole-cell patch-clamp technique and a perfused pipette. 2. Intracellular perfusion with cyclic GMP (0.1-20 microM) had no effect on the basal ICa. However, when ICa was increased by isoprenaline or by intracellular perfusion with cyclic AMP, perfusion with cyclic GMP (20 microM) reduced ICa by an average of 67%. The effect of cyclic GMP on ICa elevated by cyclic AMP was reversible. A half-maximal effect of cyclic GMP was observed at 0.6 microM. Cyclic GMP had no significant effect on the shape of the ICa current-voltage relationship. 3. The effect of cyclic GMP was specific to the 3',5' form; 2',3'-cyclic GMP had no effect. 4. The effect of cyclic GMP was apparently not mediated by stimulation of cyclic-GMP-dependent protein kinase because 8-bromo-cyclic GMP, a very potent activator of the protein kinase, was without effect. 5. Cyclic GMP had no effect on ICa elevated by the non-hydrolysable 8-bromo-cyclic AMP. The effect of cyclic GMP on cyclic-AMP-elevated ICa was partially blocked by the phosphodiesterase inhibitor, methylisobutylxanthine. Thus, it was hypothesized that the effect of cyclic GMP was mediated by hydrolysis of cyclic AMP as a result of a stimulation of a cyclic nucleotide phosphodiesterase by cyclic GMP. 6. The dose-response curve for cyclic AMP on ICa was well fitted by the Michaelis equation with a K50 (i.e. concentration of cyclic AMP at which response is 50% of the maximum) of 0.7 microM and a maximal 11-fold stimulation of ICa. Cyclic GMP shifted the curve one log unit to the right and decreased the maximal stimulation to 8.6-fold. Thus, the effect of cyclic GMP appeared uncompetitive. 7. The products of cyclic AMP and cyclic GMP hydrolysis, 5'-AMP and 5'-GMP, had no effect on ICa. Furthermore, strong buffering of intracellular pH did not reduce the effect of cyclic GMP. 8. It is proposed that cyclic-GMP-stimulation of a cyclic nucleotide phosphodiesterase may be one of several mechanisms by which acetylcholine regulates ICa.

The effects of cardiac glycosides (1 microM ouabain, 50 microM dihydrooubain, 1 microM strophanthidin) on Ca current (ICa) were investigated on Cs-loaded single frog ventricular cells using the whole-cell patch-clamp technique (9). Cardiac glycosides exert both inhibitory and stimulatory effects on ICa in 20 Cs Ringer solution, but have only a stimulatory effect in 0 Cs, when the Na,K pump is blocked. The inhibitory response seems related to the inhibition of the Na,K pump by glycosides. The stimulatory effect on ICa may contribute to the positive inotropic effect of cardiac glycosides.