Wortmannin inhibits the myofilament Ca2+ sensitization induced by endothelin-1

Endothelin-1 induces a positive inotropic effect due to a combination of an increase in Ca2+ transients and myofilament Ca2+ sensitivity in rabbit ventricular myocardium. We carried out the experiments to examine the potential contribution of myosin light chain kinase to the Ca2+ sensitization induced by endothelin-1 by use of wortmannin that inhibits myosin light chain kinase at high concentrations (IC50=200 nM). Wortmannin at 3 microM suppressed the basal force of contraction, but did not affect the positive inotropic effect mediated by beta-adrenoceptors. Wortmannin at 1 and 3 microM markedly inhibited the positive inotropic effect of endothelin-1, but did not affect the increase in Ca2+ transients elicited by endothelin-1. The present findings imply that the increase in myofilament Ca2+ sensitivity induced by endothelin-1 may be in part due to activation of myosin light chain kinase in rabbit ventricular myocardium.     

Ouabain increases myofibrillar Ca2+ sensitivity but does not influence the Ca2+ release in human skinned fibres

The present study investigated the impact of the Na(+) pump inhibitor ouabain (g-strophanthin) on Ca(2+) sensitivity and Ca(2+) release in human right auricular trabeculae (coronary bypass) and in skinned muscle fibres from left ventricular myocardium (cardiac transplantation, dilated cardiomyopathy). A time-dependent increase in force of contraction was observed in right auricular trabeculae in response to ouabain (100 nM) before the intracellular Ca(2+) transient (fura-2) increased (n=6). In triton X-skinned fibres (no sarcoplasmic reticulum) of human failing myocardium, ouabain (1-100 nM) concentration-dependently increased tension at a free extracellular Ca(2+) concentration of 1 microM and the Hill coefficient of the Ca(2+)-dependent tension development. Ouabain (1-100 nM) did not directly induce a Ca(2+) release out of the sarcoplasmic reticulum, nor did it alter the caffeine (10 mM) induced sarcoplasmic reticulum Ca(2+) release in saponin-skinned fibre preparations in which the sarcoplasmic reticulum had been Ca(2+)-loaded. In conclusion, ouabain increases myofibrillar Ca(2+) sensitivity possibly due to an increase in the cooperativity of the thick and thin myofilaments. This mechanism may additionally contribute to the positive inotropic effect of ouabain.     

The mechanism of action of maitotoxin in relation to Ca2+ movements in guinea-pig and rat cardiac muscles.

Maitotoxin (MTX), the most potent marine toxin known, produced a dose-dependent positive inotropic effect on guinea-pig isolated left atria and rat ventricle strips at concentrations of 0.1 ng to 4 ng ml-1. MTX (4 ng ml-1) also exhibited a positive chronotropic effect on guinea-pig right atria. The MTX-induced inotropic effect was almost abolished by Co2+ or verapamil, but was little affected by propranolol, reserpine or tetrodotoxin. The tissue Ca content of guinea-pig left atria was increased by MTX (2-30 ng ml-1) in a dose-dependent manner, and this increase was markedly inhibited by Co2+ or verapamil. Furthermore, on the rat isolated cardiac myocytes MTX (0.01-10 ng ml-1) caused an arrhythmogenic effect which was followed by their transformation into irreversibly rounded cells. The effects of MTX on the isolated cells were inhibited by verapamil or Ca2+-free solution. These results suggest that the excitatory effects of MTX on heart muscle are caused by a direct action on the cardiac muscle membrane mainly due to an increase in Ca2+ permeability.     

Berbamine increases myocardial contractility via a Ca2+-independent mechanism

Berbamine (BM), a natural compound derived from Berberis vulgaris L, has been reported to inhibit cardiac contractile function at higher concentrations. Here, we report that BM had concentration-dependent biphasic effects on myocardial contraction in Langendorff-perfused rat hearts, that is, at lower concentrations (30-100 nM), it displayed positive inotropic and lusitropic effects, whereas at a higher concentration of 1 μM, it caused a negative inotropic effect after an initially weak increase. These effects were further confirmed in cardiomyocytes isolated from the left ventricles of rats. Moreover, the increased cell shortening by BM at concentrations from 0.1 to 100 nM was not associated with an alteration of intracellular Ca transients. Consistently, at 30 nM, BM shifted the cell shortening--Ca transient relationship curve induced by cumulative elevation of extracellular Ca concentration to the left. Furthermore, BM significantly increased membrane-bound but not filament-bound protein kinase C epsilon (PKCε) in the isolated hearts and cardiomyocytes. Such a translocation was inhibited by PKCε-specific inhibitor PKCε V1-2 concomitant with the abolishment of the BM-induced increase in contraction. These findings reveal the positive inotropic effect of BM in the myocardium and demonstrate that BM increases myocardial contractility by increasing myofilament Ca sensitivity via a PKCε-dependent signaling pathway.     

Muscarinic regulation of Ca2+ signaling in mammalian atrial and ventricular myocardium.

The differential regulation of the contractility of mammalian atrial and ventricular myocardium upon activation of muscarinic receptors can be ascribed, for the most part, to alterations in intracellular Ca2+ transients. However, alterations in myofibrillar sensitivity to Ca2+ ions also contribute to such regulation. In atrial muscle, the following actions are all associated with the corresponding alterations in the amplitude of Ca2+ transients in the same direction as those in the strength of the contractile force: (1) the direct inhibitory action on the basal force of contraction; (2) the increase (recovery) in force that is induced during the prolonged stimulation of muscarinic receptors; and (3) the rebound increase in force induced by washout of muscarinic receptor agonists. In addition, for a given decrease in force induced by muscarinic receptor stimulation in atrial muscle, the amplitude of Ca2+ transients is decreased to a smaller extent than the decrease in amplitude induced by reduction of extracellular Ca2+ concentration ([Ca2+]o), an indication that muscarinic receptor stimulation might increase myofibrillar sensitivity to Ca2+ ions simultaneously with the reduction in the amplitude of Ca2+ transients during induction of the direct inhibitory action. In mammalian ventricular myocardium, the direct inhibitory action of muscarinic receptor stimulation exhibits a wide range of species-dependent variation. A pronounced direct inhibitory action is induced in ferret papillary muscle, which is also associated with a definite increase in myofibrillar sensitivity to Ca2+ ions. By contrast, in the ventricular myocardium of other species including the rabbit and the dog, muscarinic receptor stimulation scarcely affects the baseline Ca2+ transients and the force, but it results in a pronounced decrease in Ca2+ transients and force when applied in the presence of beta-adrenoceptor stimulation, a phenomenon known as 'accentuated antagonism' or the 'indirect inhibitory action' of muscarinic receptor stimulation in mammalian ventricular myocardium. During induction of the indirect inhibitory action in mammalian ventricular myocardium, muscarinic receptor stimulation reverses all the effects induced by beta-adrenoceptor stimulation, including the increase in Ca2+ transients, the positive inotropic and lusitropic effects, and the decrease in myofibrillar sensitivity to Ca2+ ions. The relationship between the amplitude of Ca2+ transients and force is unaffected during induction of the indirect inhibitory action in rabbit and dog ventricular myocardium. The direct and indirect inhibitory actions of muscarinic receptor stimulation on Ca2+ transients have clearly different dependences on frequency: the former is more pronounced at a higher rate of stimulation, while the latter is more pronounced at a lower rate. The more complex interaction of muscarinic receptor and beta-adrenoceptor stimulation in mammalian atrial muscle and ferret ventricular muscle might be explained by the contribution of both the direct and the indirect regulatory mechanisms to the interaction.     

Amiloride derivatives as blockers of Na+/Ca2+ exchange: effects on mechanical and electrical function of guinea-pig myocardium.

The amiloride derivatives, 2',3'-benzobenzamil (BB), 3',4'-dichlorobenzamil (DCB), and 5-(N-4-chlorobenzyl)-2',4'-dimethylbenzamil (CBDB) are known as inhibitors of the Na+/Ca2+ exchange. This kind of drug action was recently suggested to be a new inotropic mechanism. In guinea-pig myocardium, we have studied the inotropic and the accompanying electrophysiological effects of the three compounds in order to assess their selectivity of action. In left atria and in papillary muscle, force of contraction increased with DCB and CBDB (atria only) at a high concentration (5 x 10(-5)-10(-4) mol/l) and after long exposure time, whereas BB produced a negative inotropic effect. In the isolated perfused Langendorff heart, the amiloride derivatives tested decreased spontaneous heart rate and force of contraction and prolonged the duration of contraction. In isolated cardiac myocytes, sodium current, calcium current and the delayed rectifier were reduced by concentrations of BB, DCB and CBDB similar to the IC50 values reported for the inhibition of the Na+/Ca2+ exchange. Our results demonstrate that the amiloride derivatives have multiple sites of action. It is concluded that more specific modulators of the Na+/Ca2+ exchange are required in order to define their contribution to the regulation of contractile activation of the heart.     

Diamide: positive inotropic effect in isolated atria and inhibition of Na+/Ca2+ exchange in cardiomyocytes

The influence of frequency of stimulation and external calcium on the positive inotropic response of guinea-pig left atria to diamide and the inhibitory action on Na+/Ca2+ exchange activity of rat cardiomyocytes by this oxidant of sulphhydryl groups have been investigated. Diamide (50-500 microM) induces a concentration-dependent positive inotropic effect which is more pronounced when atria are driven at 1.0 Hz rather than at 0.5 and 0.1 Hz, and are bathed in 2.72 mM rather than in 1.36 mM external calcium. A decrease in the positive inotropic effect at 35 degrees C with respect to 29 degrees C is also observed. In addition, diamide in positive inotropic concentrations (100-300 microM) significantly reduces Na+/Ca2+ exchange activity and cytoplasmic glutathione levels in adult rat cardiomyocytes. The thiol reducing agent dithiothreitol either reverses or prevents diamide effects both in isolated atria and cardiomyocytes, suggesting that the actions of diamide are correlated to its property to oxidize sulphhydryl groups to disulphides. In view of the functional importance of Na+/Ca2+ exchange in myocardial contractility, it is proposed that diamide may increase the heart force of contraction by an inhibition of the sarcolemmal Na+/Ca2+ exchange activity.     

Voltage-dependence of Ca2+ agonist effect of YC-170 on cardiac L-type Ca2+ channels.

1. We investigated the voltage-dependence of the agonist actions of YC-170, a dihydropyridine (DHP) derivative, on cardiac L-type Ca2+ channels in rabbit ventricular cells, using the patch clamp technique. The characteristics of YC-170 were compared with those of other DHP Ca2+ agonists (Bay K 8644, CGP 28392). Ca2+ channel activities were elicited by depolarizing pulses to 0 mV from a holding potential (HP) of either -80 mV or -40 mV. 2. YC-170 (10 microM) increased Ca2+ channel activities when HP was set at -80 mV. However, decreasing HP to -40 mV abolished the agonist action. The agonist effect of Bay K 8644 (1 microM) on Ca2+ channels was elicited to the same extent at either HP. CGP 28392 (10 microM) also increased Ca2+ channel activities at both HPs, but its agonist effect was significantly larger at an HP of -80 mV than at -40 mV. 3. All of the three DHP Ca2+agonists prolonged open times of Ca2+ channels, but the prolongation did not correspond to the voltage-dependence of Ca2+ agonist effects of the three DHPs. 4. YC-170 markedly reduced the closed time of the Ca2+ channel when the HP was at -80 mV, but prolonged it at HP of -40 mV. Bay K 8644 reduced closed times at an HP of -80 mV. At an HP of -40 mV, Bay K 8644 slightly reduced closed times. CGP 28392 reduced closed times at an HP of -80 mV and prolonged those at an HP of -40 mV. Thus the voltage-dependence of the agonist effects of these agents was in good agreement with the voltage-dependence of changes in closed times of Ca2+ channel. 5. Two mechanisms may be involved in the agonist action of YC-170; a prolongation of open times, and a reduction of closed times of Ca2+ channels, i.e. an increase in reopening. The former mechanism is not dependent on Hp and the latter mechanism is highly dependent on HP. Thus, the voltage-dependence of the agonist action may be attributed to the voltage-dependence of their enhancing effect on reopening of Ca2+ channels.     

Endothelin dissociates muscle tension from cytosolic Ca2+ in vascular smooth muscle of rat carotid artery

Endothelin induced rapid increase followed by a decrease in cytosolic Ca2+ [( Ca2+]i) and a slow increase in muscle tension in the vascular smooth muscle strip of rat carotid artery. Thus, the endothelin-induced contraction was smaller, and it became gradually greater than high K-induced contraction at a given [Ca2+]i. In Ca2+-free solution, endothelin induced a transient increase in [Ca2+]i and a sustained contraction. These results suggest that endothelin-induced contraction is due to the increase in [Ca2+]i, the time-dependent change in Ca2+-sensitivity of contractile elements, and the mechanism which is independent of the increment in [Ca2+]i.     

Pharmacological dissection of Ca2+ channels in the rat aorta by Ca2+ entry modulators.

Ca2+ agonists, Bay K 8644 and CGP 28392, induced concentration-dependent contractions in segments of rat aorta partially depolarized with 15 mmol/l K+. The responses caused by Bay K 8644 were greater than those elicited by CGP 28392. The actions of these Ca2+ agonists were blocked by nifedipine. CGP 28392 and mainly Bay K 8644 increased the contractions induced by different K+ concentrations but did not alter those caused by noradrenaline (NA) and higher K+ concentrations. Bay K 8644 enhanced and nifedipine reduced the contractions elicited by Ca2+ addition in a Ca2(+)-free solution containing K+ (25, 50 or 75 mmol/l). This enhancement was blocked by nifedipine. Nifedipine and verapamil reduced more effectively the responses induced by K+ than those caused by NA; they did not modify contractions evoked by Ca2+ ionophore A23187. They also reduced the increases in K+ contractions elicited by CGP 28392 and Bay K 8644. Bay K 8644 increased 45Ca2+ uptake induced by K+ and did not affect that caused by NA. Nifedipine blocked this increase as well as 45Ca2+ uptake elicited by NA and K+. Basal 45Ca2+ efflux was not modified by verapamil and Bay K 8644. NA and K+ significantly increased this efflux in normal solution, but not in Ca2(+)-free medium. Bay K 8644 enhanced 45Ca2+ efflux induced by K+ in normal medium, whereas it did not affect those elicited by NA and K+ in a solution without Ca2+. This Ca2+ agonist had scant ability to induce Ca2+ efflux in a Ca2(+)-free medium. These results indicate that in this vascular preparation: (1) Ca2+ agonists (Bay K 8644 particularly) have the ability to produce Ca2+ influx (and subsequent contraction) and Ca2+ efflux by activation of voltage-operated Ca2+ channels (VOCs), only when they are preactivated with K+; (2) VOCs and receptor-operated Ca2+ channels (ROCs) appear to constitute two different populations, and (3) Ca2+ antagonists possess, in comparison with Ca2+ agonists, a small range of selectivity to block only VOCs; the latter drugs are better to pharmacologically discriminate between VOCs and ROCs in the rat aorta.     

Ca2+ entry blockers, force staircase and the onset of the positive inotropic action of cardiotonic steroids in isolated cardiac muscle

1. Effects of alterations of Ca2+ fluxes on the force staircase phenomenon and on the positive inotropic action of strophanthidin or ouabain were examined in left atrial muscle preparations isolated from rabbit or rat heart. 2. The organic Ca2+ entry blockers converted the positive force staircase observed in rabbit heart to the negative staircase; however, Ni2+, Co2+ or a reduction of the extracellular Ca2+ concentration which reduces Ca2+ influx via the Na+/Ca2+ exchange mechanism as well as via the Ca2+ channels, failed to convert the force staircase. 3. All of these interventions or ryanodine, which inhibits Ca2+ release from the sarcoplasmic reticulum, delayed the onset of the positive inotropic effect of strophanthidin or ouabain without reducing the peak inotropic effect. 4. These results indicate that either Ca2+ antagonists reduce the Na+ influx in addition to reducing Ca2+ influx, or Ca2+ influx per se and not the Na+ influx is important for the staircase phenomenon, and that intracellular Ca2+ accumulation plays an important role in the early phase of the positive inotropic effect of the cardiotonic steroids.     

Interaction of three structurally distinct Ca2+ channel activators with single L-type Ca2+ channels

The dihydropyridine S(-)-Bay K 8644 (Bay K), the benzoylpyrrole FPL 64176 (FPL) and the benzodiazocine CGP 48506 (CGP) are structurally unrelated L-type Ca2+ channels agonists. The aim of our study was to investigate whether these three drugs interact with different binding sites and thereby modulate the behaviour of L-type Ca2+ channels in a qualitatively different manner. Single-channel recordings were performed on CHO cells stably expressing the alpha1C-b subunit of the L-type Ca2+ channel. Mean open time and open probability were determined sweep by sweep and the effects of CGP (10(-4) M), Bay K (10(-6) M) and FPL (10(-6) M) were compared. All three compounds increased mean open time and open probability when applied alone. However, the gating pattern changes induced by each drug were qualitatively and quantitatively different. We also applied binary mixtures and analysed the resulting sweeps with respect to their gating pattern. The application of mixtures did result in a gating pattern not seen with any of the single drugs. The mixture of CGP and FPL led to a prolonged mean open time compared with each single drug. The mixture of Bay K and FPL exhibited an open probability lower than with each single drug. The mixture of CGP and Bay K increased the mean open time per sweep like Bay K, but the number of openings was similar to the level seen with CGP alone. These results cannot be explained by assuming alternative binding of the drugs to a single binding site. We therefore conclude that Bay K, CGP and FPL bind to different but interacting sites on the L-type Ca2+ channel.     

Effects of the calcium channel facilitator, CGP 28,392, on different modes of contraction in smooth muscle of rabbit and rat aortae and guinea-pig taenia caeci.

Effects of a Ca2+ channel facilitator, CGP 28,392, on smooth muscle contractions were examined in order to delineate characteristics of Ca2+ channels in rabbit and rat aortae and guinea-pig taenia caeci. Application of increasing concentrations of KCl induced contractile responses in these smooth muscles and CGP 28,392 shifted the concentration-response curve for KCl to the left. The maximum response was also increased in rat aorta and guinea-pig taenia. CGP 28,392 also shifted the concentration-response curves for noradrenaline in rat aorta and for histamine in taenia to the left and increased the maximum response in rat aorta. However, the corresponding curve for noradrenaline in rabbit aorta was not affected by CGP 28,392. The sustained contractions induced by KCl were inhibited by cumulative application of verapamil in these smooth muscles. Pretreatment of the muscle with CGP 28,392 decreased the inhibitory effect of verapamil. The noradrenaline-induced contraction in rat aorta and the histamine-induced contraction in taenia were also inhibited by verapamil, and CGP 28,392 antagonized the effect of verapamil. The noradrenaline-induced contraction in rabbit aorta was only slightly inhibited by verapamil, and CGP 28,392 did not modify the effect of verapamil. In these smooth muscles, cumulative application of Ca2+ to the Ca2+-depleted, KCl-treated muscle induced contraction, and the concentration-response curve for Ca2+ was shifted to the left by CGP 28,392 and to the right by verapamil. The concentration-response curves for Ca2+ in Ca2+-depleted, noradrenaline-treated rabbit and rat aortae and in Ca2+-depleted, histamine-treated taenia were also shifted to the left by CGP 28,392 and to the right by verapamil.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of extracellular Ca2+ and related ions on the cardiac action of milrinone

The biphasic single dose, dose-response curve of milrinone was sensitive to [Ca2+]0. At concentrations of 1.8 nM Ca2+ or less this biphasic response is observed but at [Ca2+]0 of 4.5 mM or more the dose response curve becomes monotonic. The inotropic response to milrinone in contrast to norepinephrine is highly sensitive to the extracellular [Ca2+]0. At low [Ca2+]0 of 0.15 mM milrinone could produce a negative inotropic effect. The positive inotropic effect of milrinone was proportional to [Ca2+]0 up to 2.7 mM. With [Ca2+]0 above 3.6 mM and low [Na+]0, the inotropic response to milrinone was reduced. These effects were due to increased [Ca2+]i and not due to the increase in contractile force produced by Ca2+. The positive inotropic effect of milrinone in contrast to norepinephrine is increased with an increase in [K+]0 possibly due to the depolarization produced by K+. The positive inotropic response to 10 micrograms of milrinone when [Ka+]0 = 4 mM was not significantly changed by Ca2+ channel blocking agents. In depolarized tissue (20 mM K+) the electropharmacological and contractile effects of milrinone are blocked by verapamil and ruthenium red. This suggests that under these conditions different mechanisms of Ca2+ channel activation are operative. Substitution of Sr2+ for Ca2+ increased contractile force and prolonged time to peak tension and relaxation time. Milrinone decreased time to peak tension but had no detectable effect on relaxation time. The results are discussed and it is suggested that milrinone acts on Ca2+ channels in the sarcolemma and intracellularly by increasing cyclic AMP which activates Ca2+ release and uptake from the sarcoplasmic reticulum.     

Ca2+ entry activated by emptying of intracellular Ca2+ stores in ileal smooth muscle of the rat.

1. The effects of depletion of intracellular Ca2+ stores on muscle tension and the intracellular Ca2+ concentration ([Ca2+])i were studied in fura-2 loaded longitudinal smooth muscle cells of the rat ileum. 2. After exposure to a Ca(2+)-free solution, application of Ca2+ caused a small contraction and a rise in [Ca2+]i, both of which were potentiated when the muscle was challenged with carbachol or caffeine before the addition of Ca2+. 3. Cyclopiazonic acid (CPA), a specific inhibitor of sarcoplasmic reticulum Ca(2+)-ATPase, dose-dependently decreased tension development and the rises in [Ca2+]i induced by carbachol and caffeine in the Ca(2+)-free solution, but conversely increased the Ca(2+)-induced responses even in the presence of the voltage-dependent Ca2+ channel blockers, methoxyverapamil and nifedipine. 4. The contraction and rise in [Ca2+]i evoked by Ca2+ gradually declined with time after removal of CPA, while the reverse was the case for the responses to carbachol and caffeine. 5. The Ca(2+)-induced contraction and rise in [Ca2+]i in the presence of CPA were inhibited by the replacement of Na+ with K+ or Cs+, and by the addition of Cd2+, Ba2+, Ni2+ or La3+. 6. The influx of Mn2+ was much greater in extent in the presence of CPA than in its absence. 7. These results suggest that the emptying of intracellular Ca2+ stores may activate Ca2+ influx not associated with voltage-dependent Ca2+ channels in the rat ileal smooth muscle.     

Sodium fluoride produces a K+ efflux by increasing intracellular Ca2+ through Na+-Ca2+ exchange

Acute fluoride intoxication increases intracellular calcium (Cai), manifested by increased twitch tension in cardiac muscle, and by potassium efflux (mediated by Ca2+-dependent K+ channels) in fluoridated erythrocytes. Fluoride, like isoproterenol, stimulates adenylate cyclase, and could increase Cai via the effects of cAMP on Ca2+ channels. However, while the inotropic effects of fluoride mimicked isoproterenol in rat atria, their effects on the time course of isometric contraction were quite different. In addition, acetylcholine negated isoproterenol's effect on twitch tension but did not modulate the effects of fluoride. Further, the Ca2+ channel antagonist verapamil had no effect on fluoride-stimulated K+ efflux from erythrocytes. Fluoride also inhibits Na+-K+ ATPase, and increases intracellular Na+, so could increase Cai via Na+-Ca2+ exchange. Lanthanum, which blocks Na+-Ca2+ exchange, blocks fluoride-induced K+ efflux in erythrocytes. We conclude that the effects of fluoride on adenylate cyclase are not important in intact tissue, and that inhibition of Na+-K+ ATPase and subsequent Na2+-Ca2+ exchange may be the mechanism of increased Cai in acute fluoride toxicity.     

Ca2+ buffering action of sarcoplasmic reticulum on Bay k 8644-induced Ca2+ influx in rat femoral arterial smooth muscle

We examined the Ca2+ buffering action of sarcoplasmic reticulum during the stimulation of arterial smooth muscle with Bay k 8644 [methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyr idine-5-carboxylate]. The effects of Bay k 8644 on tension and cellular Ca2+ level were first determined in endothelium-denuded strips of rat femoral artery. The Ca2+ buffering action was examined by using cyclopiazonic acid and thapsigargin to inhibit Ca2+-ATPase of sarcoplasmic reticulum and ryanodine to deplete Ca2+ stored in sarcoplasmic reticulum. The addition of Bay k 8644 (0.3-300 nM) to the resting strips almost failed to cause a contraction. When the strips were preincubated with 10 microM cyclopiazonic acid, Bay k 8644 induced a concentration-dependent contraction that is antagonized by nifedipine. The maximum contraction induced by Bay k 8644 in the presence of cyclopiazonic acid was comparable to the maximum contraction induced by 65.9 mM K+-depolarization and the ED50 value for Bay k 8644 was around 5 nM. Similar results were obtained when the strips were preincubated with 30 nM thapsigargin or 10 microM ryanodine. Bay k 8644 also induced a strong contraction when the extracellular K+ concentration was elevated. During the stimulation with 100 nM Bay k 8644, the Ca2+ influx was increased. We conclude that in rat femoral arterial smooth muscle, (1) the Ca2+ influx induced by Bay k 8644 is completely buffered by Ca2+ uptake into the sarcoplasmic reticulum, and (2) this sarcoplasmic reticulum can buffer a large amount of Ca2+ that induces a maximum contraction.     

Ca(2+)-sensitizing effect is involved in the positive inotropic effect of troglitazone

1. Troglitazone, an insulin sensitizing agent, has a direct positive inotropic effect. However, the mechanism of this effect remains unclear. Thus, we examined the inotropic effect of troglitazone while focusing on intracellular Ca2+ handling. 2. Troglitazone significantly increased peak isovolumic left ventricular pressure (LVP(max)), peak rate of rise of LVP (dP/dt(max)), peak rate of fall of LVP (dP/dt(min)) in isolated rat hearts perfused at a constant coronary flow and heart rate. This inotropic effect of troglitazone was not inhibited by pretreatment with carbachol (muscarine receptor agonist), H89 (protein kinase A inhibitor), U73122 (phospholipase C inhibitor), H7 (protein kinase C inhibitor), verapamil (L-type Ca2+ channel antagonist), thapsigargin (Ca(2+)-adenosine triphosphatase inhibitor) or ryanodine (ryanodine receptor opener). 3. Radioimmunoassay showed that the cyclic adenosine monophosphate concentration in the left ventricle was not increased by troglitazone. 4. Whole-cell patch clamp analysis revealed that troglitazone had no effect on inward Ca2+ currents in cardiomyocytes. 5. In fura-2 loaded perfused rat hearts, troglitazone exerted its positive inotropic effect without increasing Ca2+ concentration. 6. These results suggest that neither the inward Ca2+ currents nor Ca2+ handling in the sarcoplasmic reticulum was involved in the inotropic effect of troglitazone. Furthermore, troglitazone exerted its positive inotropic effect without affecting the intracellular concentration of Ca2+. 7. In conclusion, the positive inotropic effect of troglitazone is mediated by a sensitization of Ca2+.     

Effects of amiloride in guinea-pig and rat left atrial contraction as affected by frequency of stimulation and [Ca2+]0-[Na+]0 ratio: role of Na+/Ca2+ exchange.

1. The effect of amiloride (0.5 mM) on guinea-pig and rat left atria driven at various rates of stimulation and different [Ca2+]0-[Na+]0 ratios has been studied. 2. Amiloride elicited a positive inotropic response in guinea-pig left atria driven at 0.1 Hz, 0.5 Hz and 1 Hz when [Ca2+]0 was 3.6 mM, 1.8 mM and 0.9 mM respectively but not when [Ca2+]0 was 2.7 mM at 0.1 Hz, 0.9 mM at 0.5 Hz and 0.45 mM at 1 Hz. 3. A positive inotropic response was obtained in guinea-pig left atria driven at 0.1 Hz and 1 Hz when [Ca2+]0-[Na+]0(2) was increased respectively from 8 x 10(-5) to 16 x 10(-5) and from 2 x 10(-5) to 8 x 10(-5). The positive inotropic effect was evident only when the ratio was increased by increasing [Ca2+]0 and not by decreasing [Na+]0. 4. In the presence of amiloride, the force of contraction of guinea-pig left atria decreased instead of increasing, when the rate of stimulation was lowered from 1 Hz to 0.01 Hz. Amiloride inhibited the post-rest potentiation. 5. In rat left atria amiloride was devoid of any effect in all the above-mentioned experimental conditions. 6. It is suggested that the pattern of cardiac actions of amiloride can be explained by the inhibition of the Na+/Ca2+ exchange system.     

Effects of endothelin on cytosolic Ca2+ level and mechanical activity in rat uterine smooth muscle.

The effects of endothelin (ET) on cytosolic Ca2+ level ([Ca2+]i) and mechanical activity were examined in isolated rat uterine smooth muscle. ET-1, ET-2, ET-3 and sarafotoxin S6b (STX) induced rhythmic contractions superimposed on an increased muscle tone. The concentration needed to induce a half-maximum contraction (EC50) was 1.6-3.3 nM for ET-1, ET-2 and STX and higher than 200 nM for ET-3, suggesting that the ET(A) receptor is responsible for these contractions. The sensitivity to ET-1 of uterus at day 20 of gestation was higher than that of non-pregnant rat uterus. Contraction induced by ET-1 followed an increase in [Ca2+]i. The relation between [Ca2+]i and muscle tension, an an indicator of Ca2+ sensitivity of contractile elements, in the presence of ET-1 was identical to that in the presence of high K+ in non-pregnant and pregnant rat uteri. The ET-1-induced increases in [Ca2+]i and muscle tension were strongly inhibited by verapamil in non-pregnant rat uterus. In pregnant rat uterus, however, verapamil only partially inhibited the increases. The verapamil-insensitive portions of [Ca2+]i and contraction were inhibited by EGTA. In the absence of external Ca2+, ET changed neither [Ca2+]i nor muscle tension. These results suggest that ET-1 acts on ET(A) receptors, increase [Ca2+]i and induces contraction without changing Ca2+ sensitivity of contractile elements. The increase in [Ca2+]i seemed to be mediated by opening of L-type Ca2+ channels in non-pregnant rat uterus and also of non-L-type Ca2+ channels in pregnant rat uterus, but not by Ca2+ release from intracellular storage sites.