Masayuki Endou

Inhibitory effect mediated by α1-adrenoceptors on transient outward current in isolated rat ventricular cells

In order to clarify the underlying ionic mechanism(s) by whichα1-adrenoceptor stimulation prolongs the action potential duration (APD), single rat ventricular cells were voltage-clamped under a Na+-free condition using patch pipettes. Depolarizing pulses from a holding potential of -77 mV induced a 4-aminopyridine-sensitive transient outward current (Ito). Phenylephrine, in the presence of theβ-blocker propranolol (1μM), inhibited Ito in a concentration-dependent fashion and the maximum inhibition of Ito (42.5±10.0%, n=5) was produced by 30μM phenylephrine. The inhibitory effect of phenylephrine on Ito was almost abolished by 1μM. prazosin, a selectiveα1-blocker, indicating that the Ito inhibition is mediated byα1-adrenoceptors. On the other hand, phenylephrine had little influence on the Ca2+ current in the presence of 4-aminopyridine. In isolated rat papillary muscles, both theα1-adrenoceptor-mediated APD prolongation and positive inotropic response were markedly attenuated by pretreatment with 1.5 mM 4-aminopyridine. These results suggest that the inhibition of Ito is a primary cause of the prolongation of APD produced byα1-adrenoceptor stimulation and that the Ito inhibition may be causally related to the positive inotropic effect mediated byα1-adrenoceptors.

Electrophysiologic mechanisms responsible for inotropic responses to ketamine in guinea pig and rat myocardium

Inotropic and electrophysiologic effects of ketamine were investigated in cardiac preparations isolated from guinea pigs and rats. Ketamine produced a concentration-dependent negative inotropic effect in electrically driven guinea pig papillary muscles, an effect that was accompanied by a decrease in action potential duration at the 0-mV level (APD0). In contrast, ketamine produced a concentration-dependent positive inotropic effect in rat left atria in the presence of 10(-6) M propranolol. The increase in force of contraction was accompanied by an increase in APD0. Experiments using patch clamp techniques revealed that ketamine reduced the transsarcolemmal Ca2+ current (ICa) as well as the inward rectifier K+ current and delayed outward K+ current in guinea pig single ventricular cells. These results indicate that the shortening of APD0 observed in guinea pig papillary muscles might result from the suppression of ICa. In rat single ventricular cells ketamine reduced the Ca(2+)-insensitive transient outward current (Ito) and did not enhance ICa, suggesting that the ketamine-induced prolongation of APD0 observed in rat left atria is due to a decrease in Ito rather than an increase in ICa. Treatment of rat left atria with the specific Ca(2+)-insensitive Ito inhibitor 4-aminopyridine (2 mM) produced a positive inotropic effect and prolongation of APD0, and these effects were equivalent to those caused by the highest concentration of ketamine. In the presence of 4-aminopyridine, ketamine failed to induce a positive inotropic effect and instead caused a negative inotropic one. In conclusion, the negative and positive inotropic effects of ketamine may result from the suppression of ICa and Ito, respectively. The inhibitory action on these membrane currents may at least in part explain the species and tissue differences in inotropic responses to ketamine.

Inability of endothelin to increase Ca2+ current in guinea-pig heart cells.

Effects of endothelin, a novel vasoconstrictor peptide derived from vascular endothelial cells, on cardiac contractility and membrane currents, were examined in guinea‐pig cardiac preparations. Endothelin (3–1000 nm) produced a positive inotropic effect in papillary muscles in a concentration‐dependent manner. In whole‐cell voltage clamp recording, endothelin (250 nm) decreased the amplitude of Ca2+ current (ICa, 25.0 ± 6.6%) in ventricular myocytes. The endothelin‐induced decrease in ICa was abolished by pretreatment with ryanodine (1 μm). These results suggest that endothelin does not activate cardiac sarcolemmal Ca2+ channels. The enhancement of the sarcoplasmic reticulum function may play an important role in the positive inotropic effect of endothelin.

Dissociation of phosphoinositide hydrolysis and positive inotropic effect of histamine mediated by H1-receptors in guinea-pig left atria

SummaryThe present study was undertaken to determine whether the phosphoinositide hydrolysis is responsible for the positive inotropic effect of histamine in guinea-pig left atria. Histamine induced hydrolysis of phosphoinositides and a positive inotropic effect in a concentration-dependent manner. These effects were antagonized by chlorpheniramine (0.1 μmol/l) but not by cimetidine (10 μmol/l). At a concentration of 1 μmol/l histamine produced a dual-component positive inotropic response composed of an initial increasing phase and a second and late developing, greater positive inotropic phase. Histamine (10 μmol/l) caused a gradual increase in the formation of [3H]inositol trisphosphate (IP3) and a significant increase in the [3H]IP3 level was detected 10 min after the stimulation. Thus, the increase in IP3 did not precede the increase in force of contraction. The phospholipase C inhibitors 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (100 μmol/l) and neomycin (100 μmol/l) significantly reduced the histamine-induced [3H]inositol monophosphate accumulation. However, pretreatment with the phospholipase C inhibitors did not affect the positive inotropic effect of histamine, either in its extent or in its pattern. The phorbol esters 12-O-tetradecanoylphorbol-13-acetate (TPA) (100 nmol/l) and phorbol-12,13-dibutyrate (PDBu) (100 nmol/l) also significantly inhibited the phosphoinositide hydrolysis induced by histamine. The inhibitory effect of the phorbol esters on the phosphoinositide response was completely abolished in the presence of 10 μmol/l 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), a protein kinase C inhibitor. TPA significantly attenuated the positive inotropic effect of histamine without changing the dual-component pattern, whereas PDBu merged two distinct components of the histamine inotropic response into one and potentiated the early part of the positive inotropic effect. However, neither of the changes which the phorbol esters produced in the positive inotropic response to histamine was blocked by H-7. In addition, H-7 itself failed to modify the positive inotropic effect of histamine. These results indicate that histamine induces hydrolysis of phosphoinositides in guinea-pig left atria that is mediated by H1-receptors, but this biochemical event does not appear to contribute to the H1-receptor-mediated positive inotropic action.

Nonstereospecific actions of ketamine isomers on the force of contraction, spontaneous beating rate, and Ca2+ current in the guinea pig heart.

Ketamine possesses stereospecific actions of anesthesia with the S(+)-isomer being three to four times as potent an anesthetic as the R(-)-isomer. We investigated the mechanical and electrophysiologic effects of ketamine isomers in guinea pig cardiac preparations. Both isomers decreased the contractile force of electrically driven papillary muscles and the spontaneously beating rate of the right atria in a concentration-dependent manner. There were no significant differences between the S(+)- and R(-)-isomers for these measured variables. Consistent with the results from mechanical experiments, electrophysiologic experiments using whole cell voltage clamp techniques revealed that both isomers suppressed identically the transsarcolemmal Ca2+ current (I (Ca)), which plays a role in the generation of the force of contraction and the spontaneous firing of sinoatrial node cells. In conclusion, the optical isomers of ketamine have equipotent cardiodepressant effects in the guinea pig. Inasmuch as the S(+)-isomer is the more potent anesthetic, it could offer significant clinical advantage over the R(-)-isomer or the racemate, in terms of impairment of cardiac functions, if the present results could be extrapolated to the clinical setting. (Anesth Analg 1996;83:75-80)

Vascular effects of carvedilol, a new beta-adrenoceptor antagonist with vasodilating properties, in isolated canine coronary artery.

The pharmacologic properties of carvedilol, a beta-adrenoceptor antagonist with vasodilating activity, were investigated in isolated canine coronary artery ring preparations. Carvedilol competitively antagonized the relaxations caused by isoproterenol in 40 mM K+-depolarized preparations and its pA, value was 9.70 +/- 0.08. At concentrations greater than or equal to 3 x 10(-6) M, carvedilol significantly inhibited the contractile response to high [K+]o. Compared with the inhibitory effect on the KCl-induced contraction, the drug was less effective in suppressing the contraction induced by prostaglandin F2 alpha (PGF2 alpha). In addition, carvedilol (10(-7) to 3 x 10(-5) M) suppressed the contraction produced by Bay K 8644, a Ca2+ channel agonist. The concentration-response curve for Bay K 8644 was shifted downward by carvedilol in a concentration-dependent manner. The drug also produced a concentration-dependent inhibitory effect on the 4-aminopyridine-induced rhythmic contractions in a similar fashion to the Ca2+ channel antagonists. Carvedilol was ineffective in suppressing the contractions induced by PGF2 alpha in Ca2+-free solution and by A-23187. The results suggest that carvedilol exerts a vasodilating action possibly by inhibiting Ca2+ influx through potential-operated Ca2+ channels, although the concentrations required for producing the vasodilation are much higher than that for the beta-adrenoceptor antagonism in canine coronary artery.

Identification and characterization of histamine H1- and H2-receptors in guinea-pig left atrial membranes by [3H]-mepyramine and [3H]-tiotidine binding.

1 Histamine receptors in the membranes prepared from guinea‐pig left atria were characterized with [3H]‐mepyramine and [3H]‐tiotidine binding. 2 The binding of the H1‐antagonist, [3H]‐mepyramine, was saturable and of high affinity with a maximum binding capacity of 307 ± 27 fmol mg−1 protein (n = 14) and with an equilibrium dissociation constant (KD) of 1.5 ± 0.2 nm (n = 14). The binding was rapid and readily reversible. 3 The competition curve for [3H]‐mepyramine binding by histamine was biphasic and revealed high and low affinity states of binding. The addition of 5′‐guanylylimidodiphosphate (GppNHp) (100 μm) converted this heterogeneous binding into homogeneous binding of low affinity. 4 The competition curves of H1‐antagonists with [3H]‐mepyramine had Hill coefficients not significantly different from unity, consistent with competition with [3H]‐mepyramine at a single site. GppNHp did not shift the competition curves. 5 Dissociation constants for H1‐antagonists determined from inhibition of [3H]‐mepyramine binding correlated well with the constants derived from inhibition of the positive inotropic response of guinea‐pig left atria to histamine. 6 The H2‐antagonist, [3H]‐tiotidine, labelled an apparently homogeneous population of recognition sites with a maximum binding capacity of 41 ± 8 fmol mg−1 protein (n = 6) and a KD of 10.8 ± 1.2 nm (n = 6). 7 Although histamine competed for [3H]‐tiotidine binding in a concentration‐dependent manner, the curve was monophasic and was not shifted by GppNHp. 8 It is concluded that both H1 and H2‐receptors exist in guinea‐pig left atria. H1‐receptors probably couple to intracellular effector(s) through a guanine nucleotide‐dependent transducing mechanism. On the other hand, H2‐receptors seem unlikely to be linked to guanine nucleotide regulatory proteins in guinea‐pig left atria, which may explain the failure of histamine to cause an increase in cyclic AMP in spite of the presence of H2‐receptors.

Characterization of histamine receptors modulating inotropic and biochemical activities in rabbit left atria

The experiments were performed to identify histamine H1- and H2-receptors in rabbit left atrium and to characterize the pharmacological properties mediated by the respective subtypes of histamine receptors. High-affinity saturable binding to the left atrial membranes was obtained for [3H]mepyramine, yielding a maximum binding capacity (Bmax) of 96 fmol/mg of protein and an equilibrium dissociation constant (KD) of 3.8 nM and also for [3H]tiotidine, yielding a Bmax of 126 fmol/mg of protein and a KD of 14.7 nM. In isolated left atrium, histamine produced a concentration-dependent positive inotropic effect, an effect which was competitively antagonized by cimetidine but not altered by chlorpheniramine. Schild analysis showed that the pA2 value for cimetidine was 6.55 and the slope was not significantly different from unity. An excellent correlation was found between the increase in force of contraction and cyclic AMP in the presence of histamine, suggesting that the positive inotropic effect of histamine in rabbit left atrium is dependent on an increased level of intracellular cyclic AMP through stimulation of histamine H2-receptors. Histamine also produced concentration-dependent stimulation of phosphoinositide hydrolysis as measured by [3H]inositol monophosphate accumulation. The phosphoinositide response to histamine was blocked by chlorpheniramine and mepyramine but not by cimetidine. The data indicate that histamine H1-receptors, in addition to histamine H2-receptors, are present in the rabbit left atrium. Although this tissue lacks an inotropic response to histamine H1-receptor stimulation, the histamine H1-receptors interact with histamine to mediate the stimulation of phosphoinositide hydrolysis.

Interaction of edrophonium with muscarinic acetylcholine M2 and M3 receptors.

Background It has been reported that edrophonium can antagonize the negative chronotropic effect of carbachol. This study was undertaken to evaluate in detail the interaction of edrophonium with muscarinic M2 and M3 receptors. Methods A functional study was conducted to evaluate the effects of edrophonium on the concentration–response curves for the negative chronotropic effect and the bronchoconstricting effect of carbachol in spontaneously beating right atria and tracheas of guinea pigs. An electrophysiologic study was conducted to compare the effects of edrophonium on carbachol-, guanosine triphosphate (GTP)&ggr; S-, and adenosine-induced outward K+ currents in guinea pig atrial cells by whole cell voltage clamp technique. A radioligand binding study was conducted to examine the effects of edrophonium on specific [3H]N-methyl-scopolamine (NMS) binding to guinea pig atrial (M2) and submandibular gland (M3) membrane preparations, and on atropine-induced dissociation of [3H]NMS. Results Edrophonium shifted rightward the concentration–response curves for the negative chronotropic and bronchoconstricting effects of carbachol in a competitive manner. The pA2 values for cardiac and tracheal muscarinic receptors were 4.61 and 4.03, respectively. Edrophonium abolished the carbachol-induced outward current without affecting the GTP&ggr; S- and adenosine-induced currents in the atrial cells. Edrophonium inhibited [3H]NMS binding to M2 and M3 receptors in a concentration-dependent manner. The pseudo-Hill coefficient values and apparent dissociation constants of edrophonium for M2 and M3 receptors were 1.02 and 1.07 and 21 and 34 &mgr;m, respectively. Edrophonium also changed dissociation constant values of [3H]NMS without affecting its maximum binding capacities. Conclusion Edrophonium binds to muscarinic M2 and M3 receptors nonselectively, and acts as a competitive antagonist.

Electrophysiologic and anticholinergic effects of pirmenol enantiomers in guinea-pig myocardium

SummarySince it has been reported that several class I drugs stereoselectively block sodium channels, potassium channels and muscarinic receptors in cardiac tissues, electrophysiologic and anticholinergic effects of enantiomers of pirmenol, a class I antiarrhythmic drug, were examined. Both (+) and (−) pirmenol depressed the maximum upstroke velocity (Vmax) of the action potential in a concentration-dependent manner in guinea-pig papillary muscles driven at 1.0 Hz, and there was no significant difference in the potency of the class I effect between the enantiomers. The onset rates of use-dependent block (UDB) of (Vmax) at 2.0 Hz for 10 µmol/l (+) and (−) pirmenol were 0.30±0.03 and 0.29±0.01 per action potential, and the recovery time constants from UDB for (+) and (−) pirmenol were 27.0±2.7 and 27.7±1.9 s, respectively, indicating no difference in the binding and unbinding kinetics to the sodium channel between the enantiomers. Both (+) pirmenol and (−) pirmenol prolonged action potential duration (APD) at low concentrations (1–10 µmol/l) and shortened it at high concentrations (30–100 µmol/l) Again, there was little difference with respect to the effects on APD between the enantiomers. However, in the isolated guinea-pig left atria (−) pirmenol more potently antagonized the negative inotropic effect of carbachol than (+) pirmenol, and the pA2 values for (+) and (−) pirmenol were 6.41 and 6.71, respectively. The functional study was supported by the radioligand binding experiments using [3H]N-methyl scopolamine ([3H]NMS) in guinea-pig left atrial membranes. Specific ([3H]NMS) binding was competitively displaced by the enantiomers, and the apparent dissociation constant for (+) pirmenol (1.95±0.23 µmol/l) was significantly greater than that for (−) pirmenol (0.90±0.18 µmol/l). These results suggest that pirmenol enantiomers stereoselectively interact with cardiac muscarinic receptors but not so with cardiac sodium and potassium channels.