Pharmacological characterization of the receptors involved in the beta-adrenoceptor-mediated stimulation of the L-type Ca2+ current in frog ventricular myocytes.

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.

Muscarinic regulation of the L-type calcium current in isolated cardiac myocytes.

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.

Effect of slow calcium channel blockers on the electromechanical activity of frog myocardium in the presence of epinephrine.

The calcium channels blockers fenihidine (3.5 X 10(-5) mol/l), ryosidine (10(-5) mol/l), D-600 (10(-5) mol/l) and Mn ions (2 X 10(-3) mol/l or 5 X 10(-3) mol/l) block contraction force and shorten the duration of action potentials of the frog myocardial ventricle strand under normal conditions. When contraction force and the duration of action potentials were restored by epinephrine (10(-5) mol/l), these agents were unable to suppress these parameters. The increase in both contraction force and the duration of action potentials induced by epinephrine were blocked by acetylcholine. Recording by voltage clamp of inward calcium current (Ica) of the frog atrial trabeculae it was found that fenihidine decreases Ica activated by epinephrine to a smaller extent than observed at normal conditions. Let as assume that epinephrine increases Ica by means of increasing number of calcium channels so these data support the proposed existence of as many as two calcium channel fractions in frog myocardium, which differ in the sensitivity to calcium channels blockers.

Effect of oxidative phosphorylation uncoupler FCCP and F1F0-ATPase inhibitor oligomycin on the electromechanical activity of human myocardium.

PURPOSE: The purpose of the study was to determine the influence of oxidative phosphorylation uncoupler FCCP (carbonyl cyanide p-trifluoromethoxy-phenylhydrazone) and F1F0-ATPase inhibitor oligomycin on the parameters of electromechanical activity in human myocardium. MATERIAL AND METHODS: The experiments were performed on isolated human ventricle strips from patients undergoing cardiac corrective open heart surgery. Effect of investigative agents was registered using conventional method of registration of cardiac electromechanical activity. RESULTS: FCCP (10(-9), 10(-8), 10(-7), 10(-6) mol/L) caused the gradual reduction of contraction force (P). The maximal decrement of P (to 8.3 +/- 3.1% (n=5) vs control), was achieved at 10(-6) mol/L FCCP concentration. The duration of action potential at 50% of repolarization (AP50) was decreased only at 10(-7) and 10(-6) mol/L FCCP concentrations, i.e. to 94.3 +/- 1.9% and 55.5 +/- 3.1% (n=4), respectively, vs control. Oligomycin (2 x 10(-5) mol/L) alone decreased P only to 77.8 +/- 5.1% (n=5) and slightly reduced AP50 to 94.2 +/- 6.2% (n=4) vs control. Application of FCCP on top of oligomycin decreased P at the smaller extent than under the action of FCCP alone: the highest concentration of FCCP (10(-6) mol/L) reduced P to 21.1 +/- 4.5% (n=5) vs effect of oligomycin. The duration of AP50 was also less shortened after application of FCCP in the presence of oligomycin. The highest concentration of FCCP (10(-6) mol/L) reduced AP50 to 73.5 +/- 10.1% (n=4) vs effect of oligomycin. CONCLUSIONS: In conclusion, our data show that the inhibition of F1F0-ATPase reduces the impairment of electromechanical activity caused by oxidative phosphorylation uncoupler FCCP in human myocardium.

Changes of beta2-adrenergic stimulation induced by hyperosmosis in human atrium.

PURPOSE: The purpose of the present study was to determine whether extracellular osmotic pressure modulates beta2-adrenergic stimulation of the contraction force and L-type Ca2+ current in human atrial myocytes. MATERIAL AND METHODS: Experiments were performed on human atrial trabeculae and myocytes isolated from the right atrium. The concentration dependent effect of salbutamol (SAL), a beta2-adrenoreceptor agonist, on peak tension (P) and L-type calcium current (ICaL) under isoosmolar (345 mOsm) and hyperosmolar (405 or 525 mOsm was achieved by adding of mannitol) conditions was studied. RESULTS: Salbutamol (10 nmol/L-10 micromol/L) added to the control solution increased P by 180.6 +/- 45.8% over control with a half-stimulation constant EC50 = 27 +/- 6 nmol/L. Under isoosmolar conditions SAL (0.1/10(3)nmol/L) increased ICaL by 182.3 +/- 19.8% over control with an EC50 2.9 +/- 0.9 nmol/L. In hyperosmolar solutions the same concentrations of SAL increased P and ICaL by 57.2 +/- 12.6% and 217.2 +/- 70.5% over control with EC50 = 640 +/- 260 nmol/L and 12 +/- 5 nmol/L respectively. CONCLUSIONS: These results indicated that hyperosmolarity reduced the effect of beta2-adrenergic stimulation, i.e. the dose-response curve of salbutamol on L-type calcium current was shifted to the higher concentration range and maximal increase in contraction force was diminished in human atrial cells.

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

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.

Longitudinal distribution of Na+ and Ca2+ channels and beta-adrenoceptors on the sarcolemmal membrane of frog cardiomyocytes.

1. The distribution of L-type Ca2+ and tetrodotoxin-sensitive Na+ channels and of beta-adrenergic receptors was examined in frog ventricular myocytes using the whole-cell patch-clamp technique and a double capillary for extracellular microperfusion. 2. Rod-shaped cells (250-300 microns long) were sealed at both ends to two patch-clamp pipettes and positioned transversally at different positions between the mouths of two microcapillaries separated by a thin wall. A combination of nifedipine (1 microM) and tetrodotoxin (0.3 microM) (blocking solution) was added to one capillary in order to inhibit macroscopic Ca2+ and Na+ currents (Ica and INa, respectively) in the part of the cell exposed to this capillary. 3. Moving the cell in 10-20 microns steps from the control capillary to the capillary containing the blocking solution induced step decreases in Ica and INa amplitudes. Complete block of both currents occurred when the entire cell was exposed to the blocking solution. 4. Each step decrease in current was due to the loss of activity of the functional Ca2+ and Na+ channels present in the slice of sarcolemmal membrane newly exposed to the blocking solution. These step current changes allowed longitudinal mapping of current density for Ca2+ and Na+ channels on the sarcolemmal membrane. 5. Addition of a submaximal concentration of isoprenaline (10 nM) to the control capillary induced a local increase in Ica which enabled examination of the distribution of functional beta-adrenergic receptors as well. 6. Our results demonstrate that Ca2+ and Na+ channels and beta-adrenergic receptors are equally and essentially uniformly distributed on the sarcolemmal of frog ventricular myocytes.

cAMP compartmentation is responsible for a local activation of cardiac Ca2+ channels by beta-adrenergic agonists.

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.

Intracellular Na+ modulates the cAMP-dependent regulation of ion channels in the heart.

The cAMP-dependent regulation of ion channels was studied by using the whole-cell configuration of the patch clamp technique. In isolated cardiac ventricular myocytes, the beta-adrenergically regulated Cl- current (ICl) exhibited an unusual dependence on Na+, such that replacement of extracellular Na+ with compounds such as tetramethylammonium, choline, Tris, or N-methyl-D-glucamine resulted in a reduction in current amplitude without changing the reversal potential. Replacement of extracellular Na+ with tetramethylammonium also reduced the magnitude of the beta-adrenergically enhanced Ca2+ current and delayed rectifier K+ current, suggesting that removal of Na+ was affecting the cAMP pathway that regulates all three currents. Replacement of extracellular Na+ also reduced ICl that was stimulated by (i) direct activation of adenylate cyclase with forskolin, (ii) inhibition of phosphodiesterase with 3-isobutyl-1-methylxanthine, (iii) exposure to the membrane-permeable cAMP derivative 8-bromoadenosine 3',5'-cyclic monophosphate, or (iv) direct phosphorylation of the channel with protein kinase A catalytic subunit. This suggests that the Na+ dependence is at a point beyond the activation of protein kinase A. The Na+ dependence of ICl regulation could not be explained by changes in intracellular Ca2+. However, the sensitivity of the ICl to changes in extracellular Na+ depended significantly on the intracellular Na+ concentration, suggesting that intracellular Na+ plays an important role in the cAMP-dependent regulation of ion channels.

Beta-2 adrenergic activation of L-type Ca++ current in cardiac myocytes.

The whole-cell patch-clamp and intracellular perfusion techniques were used for studying the effects of a beta-2 adrenergic receptor activation on the L-type Ca current (ICa) in frog ventricular myocytes. The beta-2 adrenergic agonist zinterol increased ICa in a concentration-dependent manner with an EC50 (i.e., the concentration of zinterol at which the response was 50% of the maximum) of 2.2 nM. The effect of zinterol was essentially independent of the membrane potential. The stimulatory effect of zinterol was competitively antagonized by ICI 118,551, a beta-2 adrenergic antagonist. The maximal stimulatory effect of zinterol was comparable in amplitude to the effect of a saturating concentration (1 or 10 microM) of isoprenaline, a nonselective beta adrenergic agonist. Moreover, 3-isobutyl-1-methylxanthine (100 microM), a nonselective phosphodiesterase inhibitor, or forskolin (10 microM), a direct activator of adenylyl cyclase, had no additive effects in the presence of 0.1 microM zinterol. Zinterol had a long lasting action on frog ICa because after washout of the drug, ICa returned to basal level with a time constant of 17 min. An application of acetylcholine (1 microM) during this recovery phase promptly reduced ICa back to its basal level suggesting a persistent activation of adenylyl cyclase due to a slow dissociation rate constant of zinterol from its receptor. Zinterol also increased ICa in rat ventricular and human atrial myocytes, and the maximal effect was obtained at 10 and 1 microM, respectively. In all three preparations, intracellular perfusion with 20 microM PKI(15-22), a highly selective peptide inhibitor of cAMP-dependent protein kinase, completely antagonized the stimulatory effect of zinterol on ICa. We conclude that beta-2 adrenergic receptor activation produces a strong increase in ICa in frog, rat and human cardiac myocytes which is due to stimulation of adenylyl cyclase and activation of cAMP-dependent phosphorylation.

Influence of action potential duration and resting potential on effects of quinidine, lidocaine and ethmozin on Vmax in guinea-pig papillary muscles.

The effects of class I antiarrhythmic drugs (quinidine, lidocaine, ethmozin) on the maximum upstroke velocity (Vmax) of the action potential (AP) of guinea-pig papillary muscles were investigated in the presence and absence of nifedipine and a potassium-free solution. Nifedipine (10 microM) decreased AP duration from 208 +/- 13 ms to 148 +/- 17 ms (P less than 0.05, n = 4), but did not influence resting potential and Vmax. Removal of K+ from the perfusate increase the membrane potential from -86 +/- 4 mV to -103 +/- 7 mV (P less than 0.05, n = 4). Quinidine (20 microM) lidocaine (40 microM) and ethmozin (2 microM) decreased Vmax. Nifedipine and K(+)-free solution elevated Vmax when depressed by lidocaine and ethmozin. At 2 Hz rate of stimulation, Vmax increased from 69.5 +/- 12.0% to 76.0 +/- 10.6% and 98.5 +/- 3.2% (P less than 0.05, n = 7) for lidocaine and from 33.9 +/- 13.9% to 41.3 +/- 14.4% and 75.3 +/- 10.3% (P less than 0.05, n = 6) for ethmozin, compared to the control, when nifedipine or K(+)-solution was used, respectively. Nifedipine induced a slight decrease and potassium-free solution, a slight increase of Vmax in the case with quinidine from 49.9 +/- 11.8% to 48.8 +/- 7.2% and 52.7 +/- 6.7 (P greater than 0.05, n = 7), respectively. The time constant of recovery (tau r) from use-dependent block of Vmax decreased in K(+)-free solution containing lidocaine and ethmozin, but not quinidine. The guarded receptor hypothesis was used to stimulate the effects of these drugs. Our estimates of the drug affinities for activated, inactivated, and rested channels were: for quinidine 1.28 x 10(5), 1.15 x 10(4), 1.0 x 10(3); for lidocaine 1.7 x 10(4), 1.88 x 10(5) 2.5 x 10(1); and for ethmozin 1.5 x 10(6), 3.0 x 10(6), 1.5 x 10(4), respectively. The results suggest that the role of AP duration on lidocaine and ethmozin effectiveness is reduced when resting potential decreases and that removal of K+ from a perfusate containing. quinidine had a small effect on Vmax, despite a marked increase in AP duration and resting potential.

Regulation of myocardial calcium channels by cyclic AMP metabolism.

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.

Local response of L-type Ca(2+) current to nitric oxide in frog ventricular myocytes.

1. The regulation of L-type Ca(2+) current (I(Ca)) by the two nitric oxide (NO) donors sodium nitroprusside (SNP, 1 microM to 1 mM) and (+/-)-S-nitroso-N-acetylpenicillamine (SNAP, 3 or 10 microM) was investigated in frog ventricular myocytes using double voltage clamp and double-barrelled microperfusion techniques. 2. SNP and SNAP depressed the isoprenaline (ISO, 10-100 nM)- or forskolin (FSK, 1 microM)-mediated stimulation of I(Ca) via cGMP activation of the cGMP-stimulated phosphodiesterase (PDE2). Complete inhibition of the ISO (100 nM) response was observed at 1 mM SNP. 3. When SNP was applied locally, i.e. to one-half of the cell, and ISO to the whole cell, the response of I(Ca) to ISO was strongly antagonized in the cell half exposed to SNP (up to 100 % inhibition at 1 mM SNP) but a relatively small depression was observed in the other half of the cell (only 20 % inhibition at 1 mM SNP). 4. The NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (carboxy-PTIO, 1 mM) reversed the local effect of SNAP (3 microM) on FSK-stimulated I(Ca) when applied to the same side as the NO donor, but had no effect when applied to the other side of the cell. 5. A local application of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA, 30 microM), a selective inhibitor of PDE2, fully reversed the local effect of SNP (100 microM) or SNAP (10 microM) on I(Ca) but had no effect on the distant response. 6. When EHNA was applied on the distant side, with SNP (1 mM) and ISO (100 nM) applied locally, the distant effect of SNP was fully reversed. 7. Our results demonstrate that in frog ventricular myocytes stimulation of guanylyl cyclase by NO leads to a strong local depletion of cAMP near the L-type Ca(2+) channels due to activation of PDE2, but only to a modest reduction of cAMP in the rest of the cell. This may be explained by the existence of a tight microdomain between L-type Ca(2+) channels and PDE2.