Regulation of Ca2+-independent smooth muscle contraction by alternative staurosporine-sensitive kinase.

It is well known that inhibition of myosin phosphatase induces smooth muscle contraction in the absence of Ca2+. We characterized the kinase(s) which plays a role in Ca2+-independent, microcystin-LR-induced contraction in permeabilized smooth muscle of the rabbit portal vein. Assessments of various protein kinase inhibitors revealed this kinase(s) (1) was sensitive to staurosporine (1 microM), but resistant to other agents including wortmannin (10 microM), Y-27632 ((R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide+ ++, 100 microM). HA1077 (1-(5-isoquinolinylsulfonyl)-homopiperazine, 100 microM), H-7 (1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, 100 microM), and calphostin C (100 microM), and (2) induced phosphorylation of 20 kDa myosin light chain at serine-19. We concluded that other kinases exist which phosphorylate myosin light chain at serine-19 and induce Ca2+-independent smooth muscle contraction, distinct from Rho-associated kinase, myosin light chain kinase, and protein kinase C.     

Inhibitory effects of protein kinase inhibitors and cytoskeletal inhibitors on Ca2+-free contraction of rat uterus

In rat uterine smooth muscle, sustained Ca²⁺-free contraction was observed by oxytocin in Ca²⁺-free solution. This Ca²⁺-free contraction was effectively inhibited by protein kinase inhibitors and cytoskeletal inhibitors but myosin-light chain kinase (MLCK) inhibitors were not so effective. Simultaneous addition of a protein kinase inhibitor and a cytoskeletal inhibitor caused synergestic inhibition. These results suggest that the mechanism for Ca²⁺-free contraction involves some protein kinase and cytoskeletal elements rather than MLCK.     

Selective potentiation of extracellular Ca2+-dependent contraction by neuropeptide Y in rabbit mesenteric arteries

1. Neuropeptide Y (NPY) potentiated the contractile responses induced by electrical transmural stimulation, noradrenaline and KCl in the rabbit mesenteric artery. 2. In preparations treated with noradrenaline or KCl in Ca2+ free medium, NPY also potentiated the contractile response induced by resupplementation of Ca2+. 3. 3H-efflux from the arteries preincubated with [3H]-noradrenaline was not affected by NPY. 4. These results suggest that NPY selectively acts on the postsynaptic membrane and potentiates the contractions mediated through receptor-operated and voltage-dependent Ca channels.     

Effect of Ca2+ entry blockers on myosin light-chain kinase and protein kinase C

Thirteen Ca2+ entry blockers were compared with respect to their inhibitory effects on the activity of smooth muscle myosin light-chain kinase and smooth muscle protein kinase C, and the Ca2+-induced contraction of basilar artery rings. Comparison of the IC50 values obtained for these compounds, suggests that with the exception of ferdiline inhibition of the enzymes does not contribute to vascular smooth muscle relaxation.     

Phospholipid-sensitive Ca2+-dependent protein kinase inhibition by R-24571, A calmodulin antagonist

R-24571 (calmidazolium), a derivative of the antimycotic agent miconazole, inhibited phospholipid-sensitive Ca²⁺-dependent protein kinase (PL-Ca-PK), with an ic₅₀ (the concentration causing 50% inhibition) of 5.3 μM. It also inhibited the calmodulin/Ca²⁺-stimulated enzymes, with ic₅₀ values of 1.6 and 0.1 μM for myosin light chain kinase (MLCK) and phosphodiesterase respectively. Analysis of inhibition by R-24571 of PL-Ca-PK and MLCK revealed complex kinetics, suggesting that the agent interacted with the cofactors, the enzyme, and/or the cofactor-enzyme complexes. At saturating concentrations of the cofactors, R-24571 inhibited PL-Ca-PK and MLCK noncompetitively with their respective cofactors. Inhibition of MLCK by R-24571 was completely overcome by phosphatidylserine, indicating a strong hydrophobic interaction between R-24571 and the phospholipid in the presence of calmodulin. R-24571 also inhibited phosphorylation of various endogenous proteins in brain stimulated specifically by phosphatidylserine/Ca²⁺ or calmodulin/Ca²⁺. The present findings inducated that R-24571 has little specificity in inhibiting two types of Ca²⁺-dependent protein kinases sensitive to phospholipid or calmodulin.     

Ca(2+)-dependent inhibition of Ca(2+)-independent contraction in uterine smooth muscle.

Uterine smooth muscle of the rat shows Ca(2+)-independent contraction in response to oxytocin in Ca(2+)-free medium. Micromolar Ca2+ inhibits this contraction. We now tested whether Ca2+ itself is the cause of this inhibition. The ratio of fura-2 fluorescence, the indicator of the intracellular level of Ca2+, was increased in parallel with the degree of inhibition by Ca2+. When inhibition was elicited by Ca2+, EGTA released the inhibition. Comparison of the dose-response curve for oxytocin in Ca(2+)-free solution and that in the medium with 1 microM Ca2+ showed that the inhibition by Ca2+ is non-competitive. EGTA chelation of the intracellular Ca2+ by loading of EGTA as its acetoxymethylester resulted in diminution of inhibition by Ca2+. EGTA suppressed the Ca(2+)-induced contraction but did not affect Ca(2+)-independent contraction. It is concluded that the inhibition is induced by intracellular Ca2+ itself.     

Origin of ATP for Ca2+-induced contraction in the guinea-pig femoral artery

Previously, we have described differences between the rat proximal colon and femoral artery with respect to the role of ATP newly synthesized by creatine kinase. In the present study the role of newly synthesized ATP was studied in the guinea-pig femoral artery to examine species differences. In the -toxin-permeabilized preparation of the guinea-pig femoral artery, the rapid Ca²⁺-induced contraction was suppressed when creatine kinase activity was inhibited. The contraction was restored completely by treatment with NaN₃, an inhibitor of ecto-ATPase, the enzyme that breaks down exogenous ATP. Thus, ATP newly synthesized by creatine kinase may have no role in contraction of the guinea-pig femoral artery. This is in marked contrast to the rat femoral artery, in which Ca²⁺-induced contractions are almost completely inhibited by inhibition of creatine kinase activity but only partly restored by NaN₃. To characterize the difference between the guinea-pig and rat tissue, the origin of ATP required for contraction was determined in intact preparations. Monoiodoacetic acid, an inhibitor of glycolysis, inhibited the high K⁺-induced contraction in the guinea-pig femoral artery more potently than in the rat tissue. In contrast, an inhibitor of mitochondrial respiration, carbonylcyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), inhibited contraction in femoral arteries from rats, but not from guinea-pigs. These results suggest that contraction in the rat femoral artery is dependent largely on oxidative phosphorylation, while contraction in the guinea-pig tissue is dependent only on glycolysis. Because oxidative phosphorylation generates ATP and phosphocreatine, while glycolysis generates only ATP, the strong dependence of the contraction of the rat femoral artery on the oxidative phosphorylation is consistent with its dependence on ATP newly synthesized by creatine kinase from ADP and phosphocreatine, as previously shown. Thus, it is proposed that ATP, newly synthesized by creatine kinase, in addition to ATP generated by oxidative phosphorylation, is utilized for contraction in the rat femoral artery, while glycolysis produces sufficient ATP for contraction in the guinea-pig femoral artery.     

Stimulation of myosin light-chain kinase by Cd2+ and Pb2+

 The effect of Cd²⁺ on myosin light chain kinase (MLCK) reported in the literature is controversial, apparently because the level of Ca²⁺ contaminating the reaction mixture could not be accurately controlled by the addition of a metal chelator when Cd²⁺ was also present. In the present study, we have reduced the contaminating Ca²⁺ to a trace level that did not interfere with the enzyme activity; thus the use of a metal chelator was not necessary. We showed that Cd²⁺, or Pb²⁺ had a biphasic effect on MLCK isolated from chicken gizzard: stimulation at low and inhibition at high concentrations. (The stimulatory effect of on the enzyme activity isolated from chicken gizzard: stimulation at low and inhibition at high concentrations). The stimulatory effect of Cd²⁺ or Pb²⁺ on MLCK activity was not seen in the absence of calmodulin, and was abolished by trifluoperazine, a calmodulin antagonist, indicating that the heavy metals exert their activation via calmodulin. The inhibition of the enzyme activity by Cd²⁺ or Pb²⁺ at higher concentrations was also seen with the calmodulin-independent catalytic fragment of MLCK, suggesting that the inhibition is probably through their binding to sulfhydryl groups that are essential for catalytic activity. Pb²⁺ was more effective than Cd²⁺ in stimulating the enzyme activity, but less potent in inhibition. The extent of stimulation by heavy metals most likely resulted from a combination of the biphasic effects. Dithiothreitol and N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylenediamine selectively chelated Cd²⁺ and Pb²⁺ over Ca²⁺, and reversed their stimulatory or inhibitory effect on MLCK. Thus, they are useful agents to discriminate between the action of Ca²⁺ from that of these heavy metal cations. Cd²⁺ and Pb²⁺ not only activited MLCK in vitro, but also triggered smooth muscle contraction in an isolated rabbit aortic strip. Received: 18 April 1994 / Accepted: 5 July 1994     

Augmented sphingosylphosphorylcholine-induced Ca2+-sensitization of mesenteric artery contraction in spontaneously hypertensive rat

Sphingosylphosphorylcholine (SPC) is a vasoconstricting lysosphingolipid, and the RhoA/Rho-kinase pathway plays an important role in SPC-induced contraction. Since RhoA/Rho-kinase-mediated signaling is involved in the generation and/or maintenance of hypertension, we compared the effect of SPC on the contractility of endothelium-denuded small mesenteric arteries in spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY). Fura-2 Ca²⁺ signals, contractile responses, and phosphorylation of 20-kDa myosin light chains (MLC₂₀) were measured. Ten μM SPC induced a gradual and sustained vasoconstriction, which was greater in arteries of the SHR (82.5±4.3%, n=9) than in those of the WKY (26.7±4.5%, n=10). In Ca²⁺-free media, SPC gradually increased vascular tone in the SHR, but caused little vasoconstriction in the WKY. In the SHR and WKY, SPC evoked a greater vasoconstriction than did high K⁺depolarization at a given Ca²⁺ ratio, and the Ca²⁺ ratio–tension curve induced by SPC was significantly shifted to the left compared with that induced by high K⁺ depolarization. However, the magnitude of shift to the left was greater in the SHR than in the WKY. The Rho-kinase inhibitor Y-27632 significantly inhibited SPC-induced contractions, but neither the protein kinase C inhibitor calphostin-C nor PD98059, which inhibits activation of some mitogen-activated protein kinases, had any effect on the SHR or the WKY. SPC significantly increased the phosphorylation of MLC₂₀ in both the SHR and the WKY, and Y-27632 inhibited the SPC-induced increase in MLC₂₀ phosphorylation in the SHR. Our results suggest that SPC induces greater vascular tone in the SHR than in the WKY. Furthermore, our results indicate that activation of the Rho-kinase pathway plays an important role in the SPC-induced Ca²⁺ sensitization in the SHR.     

Inhibition of erythrocyte Ca2(+)-pump by Ca2+ antagonists

Inside-out vesicularized membrane fragments from human erythrocytes were prepared to study the effects of various Ca2+ channel entry blockers of plasma membrane Ca2+ transport and (Ca2+ + Mg2+)-ATPase activity concomitantly. Verapamil and diltiazem (0.01 to 5 mM) inhibited both (Ca2+ + Mg2+)-ATPase activity and initial rates of 45Ca2+ net uptake analogously. In general, the parameter affected most by these drugs, using either Ca2+ transport or (Ca2+ + Mg2+)-5'-adenosine-triphospho-hydrolase (EC 3.6.1.3) ([Ca2+ + Mg2+]-ATPase) measurements, was the stimulation by calmodulin. However, the specificity and selectivity of inhibition appeared to be highly concentration and membrane preparation dependent. Verapamil and diltiazem inhibited the calmodulin-Ca2+ transport concentration-effect relationship by changing its apparent affinity as well as the maximal velocity of the process. In a "white ghost" membrane preparation, bepridil inhibited calmodulin activation with a high degree of selectivity as opposed to its effects on calmodulin activation in the vesicular preparation. Nifedipine failed to exhibit any specificity and modestly inhibited basal and calmodulin-activated inside-out vesicular Ca2+ transport and (Ca2+ + Mg2+)-ATPase alike. Our results suggest that verapamil, diltiazem and bepridil (0.01 to 0.3 mM), but not nifedipine (1 nM to 0.01 mM), in relatively high concentrations can antagonize the calmodulin-stimulated Ca2(+)-pump, i.e. the ATPase as well as the transport process. The inhibitors differed with regard to potency, selectivity, and the type of inhibition they produced.     

Inhibitory effects of protein kinase inhibitors and cytoskeletal inhibitors on Ca2(+)-free contraction of rat uterus

In rat uterine smooth muscle, sustained Ca2(+)-free contraction was observed by oxytocin in Ca2(+)-free solution. This Ca2(+)-free contraction was effectively inhibited by protein kinase inhibitors and cytoskeletal inhibitors but myosin-light chain kinase (MLCK) inhibitors were not so effective. Simultaneous addition of a protein kinase inhibitor and a cytoskeletal inhibitor caused synergistic inhibition. These results suggest that the mechanism for Ca2(+)-free contraction involves some protein kinase and cytoskeletal elements rather than MLCK.     

二氢杨梅素通过增加细胞内钙而收缩离体犬颈动脉环

目的观察二氢杨梅素(Dihydromyricetin)对离体犬颈动脉环的作用以及相关机制。方法应用等张收缩法记录犬离体颈动脉环张力,应用激光共聚焦显微镜测定急性分离犬颈动脉平滑肌细胞胞质内游离Ca2+浓度([Ca2+]i)。结果在内皮完整和去内皮血管上,二氢杨梅素均引起浓度(1~300μmol.L-1)依赖性的血管张力增加;无钙液中,二氢杨梅素引起的张力增加作用明显降低,同有钙液相比,统计学上差异有显著性(P<0.05);电压依赖性钙通道阻断剂维拉帕米不能阻断其收缩作用。激光共聚焦检测细胞内钙的结果表明,二氢杨梅素浓度依赖性(100、300μmol.L-1)增加了静息状态平滑肌细胞胞质内钙浓度。结论二氢杨梅素对血管平滑肌具有剂量依赖性的收缩作用,这一作用为非内皮依赖性,与外钙内流致胞质内钙升高有关。     

Role of Ca2+-sensitive protein kinase C in phenylephrine enhancement of Ca2+ sensitivity in rat tail artery

We investigated the role of protein kinase C (PKC) isoforms on changes in sensitivity of contractile mechanisms to intracellular Ca(2+) (force /[Ca(2+)]i) by phenylephrine (0.1-100 microM) in rat tail arterial helical strips using simultaneous measurements of force and [Ca(2+)]i. Force/[Ca(2+)]Ii induced by phenylephrine was greater than that induced by 80 mM K+. Force/[Ca(2+)]i induced by phenylephrine in physiologic saline solution or low Ca(2+) solution was dependent on the agonist concentration. Removal of Ca(2+) completely abolished the phenylephrine-induced contraction. The PKC inhibitors staurosporine and calphostin C inhibited the increase in force/[Ca(2+)]i induced by phenylephrine to a much greater extent than that induced by 80 mM K+. LY379196, a specific PKCbeta inhibitor, did not inhibit the increase of calcium sensitivity due to phenylephrine. The classic PKC isoforms, alpha, betaI, and II not gamma were demonstrated in the artery by immunohistochemistry. These results suggest that in rat tail arterial smooth muscle, PKCalpha, and not beta or gamma, mediates the increase of changes in sensitivity of contractile mechanisms to intracellular Ca(2+) to high dose of alpha1 receptor stimulation (phenylephrine 100 microM) on nonphysiologic conditions.     

Effects of isosorbide dinitrate and diltiazem on Ca2+ flux and contraction in artery

We studied the effects of isosorbide dinitrate and diltiazem on histamine-stimulated 45Ca fluxes and contractions of isolated porcine coronary artery. Isosorbide dinitrate was slightly more potent as an inhibitor of intracellular compared to extracellular calcium-dependent contraction. Isosorbide dinitrate inhibited histamine-stimulated calcium efflux and intracellular calcium-dependent contraction over similar concentration ranges. Isosorbide dinitrate partially inhibited histamine-stimulated calcium influx, but this effect was significant only at high concentration and correlated weakly with inhibition of contraction that was dependent on extracellular calcium. Diltiazem more potently inhibited extracellular vs. intracellular calcium-dependent contraction. Diltiazem partially inhibited histamine-stimulated calcium efflux and intracellular calcium-dependent contraction to similar extents (55-60%) and produced similar concentration-response relationships for inhibition of histamine-stimulated calcium influx and extracellular calcium-dependent contraction. The data suggest that alterations of cellular calcium metabolism are major mechanisms of vascular smooth muscle relaxation by isosorbide dinitrate and diltiazem, but that the specific alterations differ for the two drugs. Isosorbide dinitrate may inhibit contraction primarily by enhancing intracellular calcium sequestration, but possibly also by inhibiting agonist-stimulated calcium influx at high isosorbide dinitrate concentrations. Diltiazem primarily inhibits stimulated calcium influx, but may also inhibit intracellular calcium release.     

Activation of a Ca2+-dependent K+ current by the oncogenic receptor protein tyrosine kinase v-Fms in mouse fibroblasts

We investigated the effects of the receptor-coupled protein tyrosine kinase (RTK) v-Fms on the membrane current properties of NIH3T3 mouse fibroblasts. We found that v-Fms, the oncogenic variant of the macrophage colony-stimulating factor receptor c-Fms, activates a K⁺ current that is absent in control cells. The activation of the K⁺ current was Ca²⁺-dependent, voltage-independent, and was completely blocked by the K⁺ channel blockers charybdotoxin, margatoxin and iberiotoxin with IC₅₀ values of 3nM, 18 nM and 76nM, respectively. To identify signalling components that mediate the activation of this K⁺ current, NIH3T3 cells that express different mutants of the wildtype v-Fms receptor were examined. Mutation of the binding site for the Ras-GTPase-activating protein led to a complete abolishment of the K⁺ current. A reduction of 76% and 63%, respectively, was observed upon mutation of either of the two binding sites for the growth factor receptor binding protein 2. Mutation of the ATP binding lobe, which disrupts the protein tyrosine kinase activity of v-Fms, led to a 55% reduction of the K⁺ current. Treatment of wild-type v-Fms cells with Clostridium sordellii lethal toxin or a farnesyl protein transferase inhibitor, both known to inhibit the biological function of Ras, reduced the K⁺ current amplitude to 17% and 6% of the control value, respectively. This is the first report showing that an oncogenic RTK can modulate K⁺ channel activity. Our results indicate that this effect is dependent on the binding of certain Ras-regulating proteins to the v-Fms receptor and is not abolished by disruption of its intrinsic protein tyrosine kinase activity. Furthermore, our data suggest that Ras plays a key role for K⁺ channel activation by the oncogenic RTK v-Fms. Received: 19 November 1997 / Accepted: 21 January 1998     

Mechanism of asynchronous Ca2+ waves underlying agonist-induced contraction in the rat basilar artery

Background and purpose:

Uridine 5''-triphosphate (UTP) is a potent vasoconstrictor of cerebral arteries and induces Ca²⁺ waves in vascular smooth muscle cells (VSMCs). This study aimed to determine the mechanisms underlying UTP-induced Ca²⁺ waves in VSMCs of the rat basilar artery.

Experimental approach:

Isometric force and intracellular Ca²⁺ ([Ca²⁺]_i) were measured in endothelium-denuded rat basilar artery using wire myography and confocal microscopy respectively.

Key results:

Uridine 5''-triphosphate (0.1–1000 µmol·L⁻¹) concentration-dependently induced tonic contraction (pEC₅₀ = 4.34 ± 0.13), associated with sustained repetitive oscillations in [Ca²⁺]_i propagating along the length of the VSMCs as asynchronized Ca²⁺ waves. Inhibition of Ca²⁺ reuptake in sarcoplasmic reticulum (SR) by cyclopiazonic acid abolished the Ca²⁺ waves and resulted in a dramatic drop in tonic contraction. Nifedipine reduced the frequency of Ca²⁺ waves by 40% and tonic contraction by 52%, and the nifedipine-insensitive component was abolished by SKF-96365, an inhibitor of receptor- and store-operated channels, and KB-R7943, an inhibitor of reverse-mode Na⁺/Ca²⁺ exchange. Ongoing Ca²⁺ waves and tonic contraction were also abolished after blockade of inositol-1,4,5-triphosphate-sensitive receptors by 2-aminoethoxydiphenylborate, but not by high concentrations of ryanodine or tetracaine. However, depletion of ryanodine-sensitive SR Ca²⁺ stores prior to UTP stimulation prevented Ca²⁺ waves.

Conclusions and implications:

Uridine 5''-triphosphate-induced Ca²⁺ waves may underlie tonic contraction and appear to be produced by repetitive cycles of regenerative Ca²⁺ release from the SR through inositol-1,4,5-triphosphate-sensitive receptors. Maintenance of Ca²⁺ waves requires SR Ca²⁺ reuptake from Ca²⁺ entry across the plasma membrane via L-type Ca²⁺ channels, receptor- and store-operated channels, and reverse-mode Na⁺/Ca²⁺ exchange.     

Inhibition of alpha-receptor-induced Ca2+ release and Ca2+ influx by Mn2+ and La3+

The influence of Mn2+ and La3+ on alpha-receptor-stimulated Ca2+ movements was examined in arterial smooth muscle of the rabbit aorta. Both cations cause an inhibition of phenylephrine (PE) contractile response which exhibits a different pattern at low and high cation concentrations. At 0.1-1.0 mM inhibition by Mn2+ and La3+ was predominately due to a reduction in Ca2+ influx reflected as inhibition of the slow phase of contraction and reduction in PE-stimulated 45Ca uptake. PE log dose-response curves were shifted to the right in a non-parallel manner by 1 mM Mn2+ such that responses to lower PE concentrations were more inhibited. However, in the presence of 10 mM Mn2+ PE responses are equally inhibited at all PE levels. At 10 mM both Mn2+ and La3+ also inhibited PE-stimulated Ca2+ release resulting in a reduction in both the rapid phase of contraction and in the magnitude of PE stimulation of 45Ca efflux. The effects of Nm2+ (1 or 10 mM) on contraction and 45Ca efflux were rapidly reversible, while the effect of La3+ was not. Inhibition of Ca2+ release by 10 mM Mn2+ and La3+ was not caused by displacement of releasable Ca2+, but appeared to reflect their occupation of a superficially located receptor modulating site. The inhibition of Ca2+ influx by lower concentrations of Mn2+ may illustrate the functional consequence of configurational changes in the alpha 2-form of the receptor which have been recently described at lower concentrations of divalent cations.     

Morinda citrifolia Inhibits Both Cytosolic Ca2+-dependent Phospholipase A2 and Secretory Ca2+-dependent Phospholipase A2

This study investigated the effects of the methanol extracts of Morinda citrifolia containing numerous anthraquinone and iridoid on phospholipase A₂ (PLA₂) isozyme. PLA₂ activity was measured using various PLA₂ substrates, including 10-pyrene phosphatidylcholine, 1-palmitoyl-2-[¹⁴C]arachidonyl phosphatidylcholine ([¹⁴C]AA-PC), and [³H]arachidonic acid (AA). The methanol extracts suppressed melittin-induced [₃H]AA release in a concentration-dependent manner in RAW 264.7 cells, and inhibited cPLA₂/sPLA₂-induced hydrolysis of [¹⁴C]AA-PC in a concentration- and time-dependent manner. A Dixon plot showed that the inhibition by methanol extracts on cPLA₂ and sPLA₂ appeared to be competitive with inhibition constants (K_i) of 3.7µg/ml and 12.6µg/ml, respectively. These data suggest that methanol extracts of Morinda citrifolia inhibits both Ca²⁺-dependent PLA₂ such as, cPLA₂ and sPLA₂. Therefore, Morinda citrifolia may possess anti-inflammatory activity secondary to Ca²⁺-dependent PLA₂ inhibition.     

Inhibition in L-type Ca2+ channel by stimulation of protein kinase C in isolated guinea pig ventricular cardiomyocytes.

1. Electrophysiological effects of phorbol esters on the L-type Ca2+ current (ICa(L)) in isolated single ventricular cells from guinea pig hearts were investigated. 2. In whole-cell voltage-clamped myocytes, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) at 10(-7) M inhibited ICa(L). An antagonist of protein kinase C (PK-C), H-7, at 10(-5) M did not modify the TPA-induced inhibition. The time-course of inactivation process for ICa(L) was greatly slowed. 3. In cell-attached patch-clamp experiments, TPA (10(-7) M) also markedly decreased the opening of L-type Ca2+ channels. The conductance was unaffected. 4. Even H-7 (10(-5) M) alone inhibited the opening of the channels. Addition of TPA (10(-7)-10(-8) M) caused further decrease in the opening. 5. On the other hand, 4-alpha-phorbol-12,13-didecanoate (not a PK-C activator) had no effect on the Ca2+ channels. 6. These results indicate that the PK-C activation induced by TPA greatly depresses the opening of L-type Ca2+ channels in ventricular cell membranes.