Effects of calcium channel blockers on human erythrocyte ghost membranes

The effects of calcium channel blockers (CAB’s), verapamil, diltiazem and nicardipine, on erythrocyte ghost membranes have been studied. Using the fluorospectroscopic method, it was observed that the fluidity of the inner layer of ghost membranes was increased with an increase of drug concentrations but did not any changes in the fluidity of the outer layer. These drugs showed protective effect against hypotonic hemolysis of erythrocytes. Thus, the expansion of surface area in response to corpuscular volume of erythrocytes in the presence of CAB’s is seemed to play an important role in protecting hypotonic hemolysis of erythrocytes.     

The effect of calcium channel blockers and calmodulin inhibitors on the macrophage factor-stimulated synthesis of collagenase by rabbit chondrocytes

Macrophages and monocytes secrete a factor(s) which can stimulate the synthesis of collagenase in synovial cells and in chondrocytes. Incubation of rabbit chondrocytes with macrophage conditioned medium (MCM) and with the calcium channel blockers, nifedipine, verapamil or diltiazem (up to 200 microM) had no effect on collagenase synthesis. However, TMB-8 (8-[N,N-diethylamino]-octyl 3,4,5-trimethoxybenzoate hydrochloride), an inhibitor of internal calcium movement, did inhibit the process with an IC50 of approximately 130 microM. The calmodulin antagonists, trifluoperazine, chlorpromazine and calmidazolium (R-24571) were effective inhibitors of the process with IC50's of 40 microM, 18 microM and 3.5 microM, respectively. Collagenase activity itself was not affected by these agents. The data suggests that calmodulin and/or internal calcium movement may play a role in the macrophage factor-stimulated synthesis of collagenase in rabbit chondrocytes.     

Modulation of vascular and cardiac contractile protein regulatory mechanisms by calmodulin inhibitors and related compounds

The abilities of several calmodulin antagonists and other compounds belonging to different pharmacological classes to modulate Ca2+ X calmodulin mediated arterial myosin light chain phosphorylation and Ca2+-troponin C regulated cardiac myofibrillar ATPase activity have been quantitated in Triton X-100 purified preparations of bovine aortic actomyosin and canine ventricular myofibrils. At submaximal free Ca2+ concentrations, all calmodulin antagonists inhibited myosin phosphorylation; however, some (calmidazolium, trifluoperazine, chlorpromazine, pimozide) stimulated myofibrillar ATPase activity, some (compound 48/80, W-5) had no effect on activity, while others (W-7, haloperidol, mastoparan) inhibited ATPase activity. The relative order of potency for several agents in both preparations was the same, as IC50 values for inhibition of arterial myosin phosphorylation were: calmidazolium, 0.5 microM; trifluoperazine, 22 microM; perhexiline, 35 microM; and concentrations which stimulated cardiac myofibrillar ATPase activity by 50% were: calmidazolium, 9 microM; trifluoperazine, 45 microM; perhexiline, 90 microM. A common feature of stimulation of cardiac ATPase activity by these agents was a leftward shift in the pCa relationship, although different shape changes in the pCa curves were also apparent. Maximum ATPase activity was either not affected or inhibited (trifluoperazine). Several other agents belonging to diverse pharmacological classes also had differential effects on myosin phosphorylation and ATPase activity. These results show that structurally-distinct calmodulin antagonists and other compounds differentially affect cardiac myofibrillar ATPase activity. Moreover, several agents have been identified which inhibit arterial, and stimulate cardiac, contractile protein regulatory mechanisms. Thus, it may be possible to develop mechanistically novel cardiotonic/vasodilator agents, Ca2+ binding protein modulators, which function primarily by altering the Ca2+ sensitivity of contractile protein interactions.     

Verapamil, diltiazem and nifedipine interactions with calmodulin stimulated (Ca2+ + Mg2+)-ATPase

The functional interactions of the three prototype Ca2+ antagonists, verapamil, diltiazem and nifedipine, were examined in relation to the calmodulin regulated plasma membrane Ca2+ pump ATPase. For this we used low ionic strength derived, calmodulin depleted, human red cell ghost membranes. Exogenously added calmodulin activated basal (Ca2+ + Mg2+)-ATPase in a concentration-dependent manner. Half-maximal activation by 6 nM calmodulin was antagonized by 10(-3) M verapamil and 10(-3) M diltiazem 25.1 and 12.1% respectively. The inhibition appeared to be specific for calmodulin activation since basal activity was not affected by these agents. Nifedipine had no effects on basal or calmodulin stimulated (Ca2+ + Mg2+)-ATPase activity. Unlike dihydropyridine modulation of verapamil and diltiazem binding at high affinity channel sites, nifedipine in this system did not alter the inhibitory responses of verapamil and diltiazem. The calmodulin directed antagonism of the two drugs was shown to be strictly additive over a full range of calmodulin concentrations and appeared to change predominantly the Vmax and, to a lesser degree, the affinity of calmodulin for the (Ca2+ + Mg2+)-ATPase. It is concluded that this model system provides evidence for additional functional discrepancies among the various classes of Ca2+ antagonists.     

Effects of nifedipine, verapamil, diltiazem and trifluoperazine on the antinociceptive activity of acetaminophen

The influence of the calcium channel blockers (CCBs) nifedipine, verapamil and diltiazem, and the calmodulin antagonist trifluoperazine on the antinociceptive activity of acetaminophen was studied in male albino mice. The nociceptive response was determined by the acetic acid writhing test. Nifedipine (50 or 20 mg/kg), verapamil (20 mg/kg), diltiazem (70 mg/kg) and trifluoperazine (3 mg/kg) were administered orally alone or 1 h before acetaminophen (100 mg/kg). Nifedipine (50 mg/kg), verapamil, diltiazem and trifluoperazine administered alone demonstrated significant antinociceptive effects compared to controls. Nifedipine, verapamil, diltiazem and trifluoperazine applied 1 h before acetaminophen potentiated its antinociceptive activity, which was strongest in mice injected with verapamil and nifedipine (20 mg/kg). It was established that 1 h after nifedipine (50 mg/kg) treatment, cytochrome P450 content, NADPH cytochrome c reductase and ethylmorphine-N-demethylase (EMND) activities were increased in the liver microsomes. Verapamil, diltiazem and trifluoperazine did not change the drug metabolizing enzymes studied. It is assumed that their effect on acetaminophen analgesia is not associated with the changes in acetaminophen oxidative metabolism in the liver.     

The in vitro hemolytic effect of diltiazem on erythrocytes exposed to varying osmolarity

The hemolytic effect of diltiazem (a calcium channel blocker) on red blood cells (RBCs) exposed to varying osmolarity was investigated. Previous work on the in vitro hemolytic effect of verapamil shed some light on the potential for some drugs to induce hemolysis at moderate-to-high doses and in pre-existing osmotic stress. The current experimental approach used a modified red cell hemolysis assay with concentrations of diltiazem ranging from 50–1500 μM compared to drug free controls. The time-course of hemolytic effects was also investigated. In conditions representing decreasing osmolarity (dilution from 140 to 0?mM NaCl) there was a significant increase in erythrocyte hemolysis that was also dependent on diltiazem concentration (ANOVA, p?<?0.05). The red cells also showed a significantly increased rate of hemolysis over 5?h with increasing concentration of diltiazem (ANOVA, p?<?0.05). Overall the data suggested that diltiazem can cause hemolysis of RBCs in a predictable time- and concentration-dependent manner, and that diltiazem increases the fragility of the erythrocytes further during hypotonic osmotic stress. The mechanism of diltiazem-dependent hemolysis could involve various ion transport pathways (i.e. Ca pump, Ca Channels) and subsequent effects on cell volume control or membrane fragility.     

Failure of Calcium Antagonistic Agents to Prevent Hepatotoxicity Induced by Diclofenac

Abstract Diclofenac (0.5–2 mM) dose- and time-dependently reduces the viability of isolated hepatocytes. This effect cannot be counteracted by the calcium channel blockers diltiazem (0.05–0.1 mM) and verapamil (0.05–0.5 mM), the calmodulin antagonist calmidazolium (0.01 mM) or Quin 2-AM (0.1 mM), an intracellular calcium chelating agent. On the contrary, verapamil even accentuates the toxic effects of diclofenac. It is concluded from these results, that diclofenac causes cell damage by other mechanisms than calcium overload.     

Characterization of the spontaneous motor activity of the isolated human pregnant myometrium.

The motor activity of myometrial strips from pregnant human uterus was characterized in vitro by the use of inhibitory compounds acting on the smooth muscle contractility at different levels. Spontaneous contractions were not inhibited by tetrodotoxin or a series of membrane receptor antagonists, like anticholinergics, antihistaminics, alpha-adrenergic blocking agents, antiserotoninergics and opioid receptor antagonists, thus excluding neuronal involvements or a local release of endogenous mediators active on the respective membrane receptors. The ineffectiveness of indomethacin (10(-5) M) minimizes a role for excitatory prostaglandins. Isoprenaline and selective beta 2-adrenergic stimulants, like salbutamol and hexoprenaline (up to 10(-5) M), failed to affect the amplitude of spontaneous contractions. Conversely the adenylate cyclase activator, forskolin, had a concentration-dependent inhibitory effect. Ca2(+)-free medium, trifluoperazine and all the calcium channel blockers examined produced a concentration-dependent inhibition of the spontaneous contractions in the following order of sensitivity: nifedipine much greater than verapamil much greater than diltiazem greater than trifluoperazine. The inhibitory effect of nifedipine was not overcome by excess calcium concentration in the bathing medium, but was completely restored by addition of the calcium agonist Bay K 8644 10(-7) M. From these data it can be concluded that the spontaneous activity of pregnant human myometrium in vitro is independent of neural or humoral mechanisms. The inhibitory effect of Ca(+)-free medium and the efficacy of calcium channel blockers support the view that calcium influx is an important step in initiating the contractile activity of uterine smooth muscle.     

Effects of Ca2+ and calmodulin antagonists on the oxygen uptake induced by acetylcholine or substance P in rat submandibular gland slices

Effects of Ca2+ and calmodulin antagonists on the oxygen uptake induced by acetylcholine (ACh) or substance P (SP) were investigated in rat submandibular gland slices. The oxygen uptake induced by ACh or SP was significantly inhibited by removing Ca2+ from the medium and the slices. The oxygen uptake by ACh in the Ca2+-deficient slices was almost completely recovered by the addition of 3.0 and 5.0 mM Ca2+, whereas that by SP was not recovered by the addition of 3.0 mM Ca2+, but recovered by 5.0 mM Ca2+. Ca2+ antagonists, diltiazem, verapamil and La3+, significantly inhibited the ACh-induced oxygen uptake. On the other hand, the SP-induced oxygen uptake was inhibited by diltiazem and La3+, but not by verapamil. Calmodulin antagonists, trifluoperazine, chlorpromazine and W-7, had no inhibitory effects on the ACh-induced oxygen uptake. The SP-induced oxygen uptake was not affected by trifluoperazine, chlorpromazine and low concentrations of W-7, but was inhibited by high concentrations of W-7. These results suggest that the ACh- or SP-induced oxygen uptake is dependent on the presence and permeability of Ca2+ with a subtle difference between the ACh and the SP mechanisms and that the oxygen uptake is independent of calmodulin.     

The role of a Ca2+/calmodulin dependent plasma membrane Ca2+ channel during concanavalin A activation of MC9 mast cells

The effects of Con A on free cytoplasmic calcium concentrations in the cloned murine mast cell, MC9, have been measured using the fluorescent calcium indicator quin 2. Con A causes a rapid, small yet sustained rise in free cytosolic calcium (up to 245 nM) followed closely by increased 45calcium uptake and more slowly by histamine release. The increases in 45calcium uptake and histamine release require extracellular calcium. However, the Ca2+ influx blockers, nifedipine and verapamil inhibit these responses only at concentrations significantly higher than those used in smooth muscle to oppose potential-dependent events, and diltiazem is inactive. These observations suggest that, in these mast cells, other types of channels control Ca2+ entry. In contrast, the intracellular Ca2+ blocker, TMB-8, inhibits both the Con A-induced histamine release and the Ca2+ changes. The calmodulin antagonists calmidazolium, trifluoperazine and W-7 are also highly effective inhibitors of both the Ca2+ changes and histamine release in direct proportion to their potency against calmodulin-dependent phosphodiesterase, implicating calmodulin in the regulation of stimulus-secretion in MC9 cells. These data imply that histamine release follows increases in intracellular Ca2+ concentration. Free intracellular Ca2+ results from rapid release from internal stores and is followed by a slower but more sustained influx of extracellular Ca2+.     

The effects of phenothiazines and other calmodulin antagonists on the sarcoplasmic and endoplasmic reticulum Ca(2+) pumps

The effects of a number of phenothiazines and other calmodulin antagonists on the Ca(2+)-ATPase activity of sarcoplasmic reticulum (SR) and endoplasmic reticulum (ER) were investigated. The drugs used in this study were trifluoperazine, calmidazolium, fluphenazine, chlorpromazine, W-7, and calmodulin-binding peptide. Our results showed that calmidazolium and calmodulin-binding peptide were the most potent inhibitors of skeletal muscle SR Ca(2+)-ATPase activity (isoform SERCA 1) (IC(50) values of 0.5 and 7 microM, respectively), while W-7 was the least potent inhibitor (IC(50), 125 microM). All of the antagonists had little effect on the cerebellar ER Ca(2+)-ATPase activity (isoform SERCA 2b), except for trifluoperazine, which had a biphasic effect, causing stimulation at low concentrations and inhibition at higher concentrations. Our results suggest that the effects of these calmodulin antagonists are independent of calmodulin and that they inhibit the Ca(2+)-ATPase in an isoform-specific manner. It was found that these antagonists inhibit the skeletal muscle isoform of the Ca(2+) pump by altering the Ca(2+) affinity and the associated Ca(2+)-binding steps, as well as possibly stabilising the E1 conformational state of the enzyme.     

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.     

Comparison of negative inotropic potency, reversibility, and effects on calcium influx of six calcium channel antagonists in cultured myocardial cells.

The negative inotropic effects of calcium channel antagonists on the myocardium were used as a standard for the definition and determination of potency of this group of drugs. The effects of six calcium channel antagonists (verapamil, methoxyverapamil (D600), nifedipine, lidoflazine, perhexiline and diltiazem) were compared on cultured chick embryo ventricular cells. Drug concentrations producing 50% inhibition of contractile amplitude, derived from linearized concentration-response curves, varied from 2.8 X 10(-8)M for nifedipine to 8.3 X 10(-7)M for perhexiline. Equipotent negative inotropic concentrations of verapamil, D600, perhexiline, diltiazem and lidoflazine produced a similar inhibitory effect on 45Ca uptake into cultured cells. Nifedipine produced no significant inhibition of 45Ca uptake. The time required for recovery of contractility after cessation of drug superfusion varied in the order lidoflazine greater than perhexiline greater than D600 greater than verapamil greater than nifedipine greater than diltiazem. This relative order accords closely with the reported in vivo half-lives of these drugs. It is concluded that while some inhibition of 45Ca2+ uptake into cardiac cells can be demonstrated with five of the six calcium channel blockers studied, the relationship between the degree of inhibition of calcium influx and negative inotropic effects may not be uniform for all calcium channel antagonists.     

DILTIAZEM AND VERAPAMIL LOWER BLOOD PRESSURE IN THE UNANAESTHETIZED RAT THROUGH CNS MECHANISMS INVOLVING ENDOGENOUS OPIOIDS

1. To evaluate and compare the effects of the calcium channel blockers, diltiazem and verapamil, on CNS modulation of blood pressure, unanaesthetized and unrestrained rats with catheters previously inserted into the lateral cerebral ventricle and femoral artery received intracerebroventricular (i.c.v.) administration of diltiazem or verapamil, 10 or 50 micrograms/kg, or their diluent. 2. Diltiazem, at both 10 and 50 micrograms/kg i.c.v., produced significant (P less than 0.05) decreases in systolic and diastolic blood pressure and heart rate. Verapamil, at 50 micrograms/kg but not at 10 micrograms/kg i.c.v., produced a significant (P less than 0.05) decrease in blood pressure, while both doses significantly (P less than 0.05) decreased heart rate. 3. To examine the endogenous opioid systems as potential modulators of the effects of these calcium antagonists, the mu opioid antagonist naloxone, 20 micrograms/kg, was administered i.c.v. either before or after each calcium antagonist. Naloxone reversed and prevented the reduction in blood pressure produced by both agents. The decrease in heart rate produced by verapamil but not diltiazem was reversed by naloxone. 4. The results suggest that: (1) calcium channels in neuron membranes in the CNS play a role in blood pressure regulation; (2) at least part of the blood pressure reduction produced by calcium blockers may be effected in the CNS; and (3) central opioid mechanisms modulate part of the action of the calcium antagonists verapamil and diltiazem on blood pressure.     

The effect of calmodulin antagonists on the activation of RAW-264 macrophage-like cells for tumor cell killing

The effects of several calmodulin antagonists on the activation of RAW-264 macrophage-like cells for tumor cell killing were investigated. At concentrations ranging from 5 X 10(-8) to 5 X 10(-7) M, calmidazolium, trifluoperazine, chlorprothixene, chlorpromazine and W-13 inhibited the development of cytolytic activity, evoked in RAW-264 by treatment with lymphokine and lipopolysaccharide, in a dose-dependent manner. Since the order of the potency of these drugs against the activation of RAW-264 cells was much the same as their ability to inhibit calmodulin-dependent phosphodiestherase activity: calmidazolium greater than trifluoperazine greater than chlorprothixene greater than chlorpromazine greater than W-13, and because W-12, a nonactive analog of W-13, failed to inhibit the process of activation, we believe that the development of cytolytic activity in RAW-264 cells may be dependent on calmodulin. At micromolar concentrations, calmodulin antagonists (except calmidazolium) enhanced the process of activation. The enhancement of cytolytic activity was neither the result of the toxicity of these drugs nor related to their effects on intracellular calcium. It was entirely dependent on the presence of stimulants but occurred independently from the stage of macrophage activation, and most likely was due to the nonspecific interference of these agents with calmodulin-independent processes.     

Action of calcium antagonists on multidrug resistant cells. Specific cytotoxicity independent of increased cancer drug accumulation

Previous studies have shown that calcium channel blockers can overcome, at least partially, multidrug resistance (MDR). This study was undertaken to attempt to determine the mechanisms whereby these agents bring about this effect. Their influence on the uptake and retention of several cancer drugs and on the toxic actions of these compounds was assessed employing MDR cell lines from several species. The wild-type drug sensitive parent cells proved to be more susceptible than the multidrug resistant variants to the effects of calcium channel blockers on cancer drug accumulation. This was shown for verapamil, nifedipine and the calmodulin inhibitor trifluoperazine acting on human, mouse and Chinese hamster ovary (CHO) cell lines. The enhancement of drug accumulation by calcium antagonists was similar to that caused by non-ionic detergents. Furthermore, verapamil was unable to alter 45Ca2+ accumulation in sensitive or resistant cells, suggesting that these agents act in a calcium-independent manner. Verapamil accumulation in multidrug resistant cells was reduced compared to sensitive cells. In spite of this reduced accumulation, however, verapamil alone was much more toxic to multidrug resistant cells than to the sensitive cells. This suggests that calcium channel blockers are specifically toxic to MDR cells by virtue of an interaction with the MDR cell surface distinct from that involved in promoting cellular accumulation.     

Calmodulin-stimulated plasma membrane (Ca2+ + Mg2+)-ATPase: inhibition by calcium channel entry blockers

Calcium channel entry blockers representing different structural classes were studied for their effects on human erythrocyte basal and calmodulin-stimulated (Ca2+ + Mg2+)-ATPase. Effects on the activity of (Mg2+)-ATPase and (Na+ + K+)-ATPase were also assessed. Of the four Ca2+ entry blockers tested, only verapamil and diltiazem specifically inhibited the calmodulin-stimulated (Ca2+ + Mg2+)-ATPase activity, the basal enzyme activity being unaltered by these drugs. Other membrane-associated ATPases were not affected. Calmodulin concentration effect curves showed the inhibition by verapamil (10(-3) M) and diltiazem (10(-3) M) to be non-competitive. This concentration inhibited the calmodulin-dependent increment (5.1 nM calmodulin) of the ATPase activity by 35 and 36% respectively. Similarly, both drugs inhibited the Ca2+-activation process of calmodulin-stimulated activity in a non-competitive manner, decreasing Vmax by 23 and 17% respectively. Basal (Ca2+ + Mg2+)-ATPase activity was not affected by verapamil or diltiazem at any calcium concentration. In contrast, cinnarizine non-specifically inhibited all four membrane ATPases including calmodulin-stimulated (Ca2+ + Mg2+)-ATPase activity at concentrations above 3 X 10(-6) M. Nifedipine was without effect on any of the four membrane ATPases. From this we conclude that certain calcium channel entry blockers can inhibit calmodulin-regulated plasma membrane Ca2+-pump ATPase. Therefore, this identifies an additional functional low affinity receptor in the plasma membrane for some of the calcium channel entry blockers.     

Effects of calcium channel blockers and a calmodulin antagonist on contractions of guinea pig airways

Both antigen- and calcium ionophore A23187-induced airways contractions are dependent on increased concentrations of free intracellular calcium. Antigen, but not A23187, contracted airways in the absence of extracellular calcium, suggesting that antigen-induced airways contraction was partly dependent on the mobilization of intracellular calcium. A series of calcium entry blockers and a calmodulin antagonist were examined on airways contraction induced by antigen, A23187, histamine, and LTD4. Verapamil (10(-5)-3 X 10(-4) M) and nitrendipine (3 X 10(-5)-2.8 X 10(-4) M) dose-dependently inhibited antigen-induced contraction of trachea but only partly inhibited antigen-induced contraction of parenchyma and A23187-induced contractions of both trachea and parenchyma. Lanthanum chloride (up to 10(-4) M) and flunarizine (up to 2.1 X 10(-5) M) were relative ineffective. High concentrations of trifluoperazine (1-3 X 10(-4) M) were necessary to inhibit antigen- and A23187-induced airways contractions. Only verapamil and trifluoperazine were effective inhibitors of the responses to histamine and LTD4. In light of the relative ineffectiveness of the above calcium antagonists on the LTD4 dose-response curves, it is suggested that airways contraction is dependent on mobilization of intracellular calcium and that the action of nitrendipine and verapamil as inhibitors of antigen- and A23187-induced contractions is at the point of blocking mediator (i.e. LTC4 and LTD4) release. Verapamil and trifluoperazine also appear to affect contractions. However, the high concentrations of calcium channel and calmodulin antagonists to inhibit antigen- and A23187-induced contractions may limit their usefulness as anti-allergic, anti-asthmatic agents.     

Blockade by calmodulin inhibitors of Ca2+ channels in smooth muscle from rat vas deferens.

1. Effects of three compounds which are used as calmodulin inhibitors (trifluoperazine, W-7 and calmidazolium) on Ca2+ channels were investigated in smooth muscle from rat vas deferens. 2. All three calmodulin inhibitors relaxed the smooth muscle precontracted by a high concentration of KCl (63.7 mM). The order of potency for the relaxation was trifluoperazine > W-7 > calmidazolium. 3. In binding studies using a microsomal fraction of vas deferens, all these calmodulin inhibitors displaced specific [3H]-nimodipine binding. Trifluoperazine and W-7 inhibited the binding at concentrations that relaxed the smooth muscle whereas calmidazolium inhibited at concentrations much lower than those necessary for muscle relaxation. 4. Ba2+ current flowing through voltage-gated Ca2+ channels was measured under whole-cell voltage-clamp conditions in isolated smooth muscle cells. The Ba2+ current was suppressed by the three calmodulin inhibitors in the concentration-range where inhibition of [3H]-nimodipine binding was observed. Neither voltage-dependence nor the inactivation time course of Ba2+ current were affected by these compounds. 5. The results suggest that the calmodulin inhibitors directly block Ca2+ channels in the smooth muscle cells. The channel inhibition by trifluoperazine and W-7, but perhaps not that by calmidazolium, may be responsible for the muscle relaxation observed with these compounds.     

Dihydropyridine receptors in the pregnant human uterus in vitro

The stimulatory effect of the dihydropyridine derivative, Bay K 8644, on the isolated pregnant human uterus, and its interactions with the calcium channel blockers, nifedipine, verapamil and diltiazem and with the calmodulin inhibitor trifluoperazine were investigated. In uterine preparations showing spontaneous activity, Bay K 8644 (1 nmol/l-1 mumol/l) produced an increase in the frequency of contractions without effects on their amplitude. However, strong phasic contractions were induced in quiescent preparations. The stimulatory action of Bay K 8644 proved to be insensitive to calcium withdrawal, but was completely prevented in the presence of 1 mmol/l EGTA. Bay K 8644 shifted the inhibitory concentration-response curve of verapamil and nifedipine to the right, leaving the diltiazem- and trifluoperazine-induced effect virtually unchanged. Schild plot analysis revealed a competitive interaction of Bay K 8644 with nifedipine, while the interaction with verapamil was of the nonlinear type. These data demonstrated that the dihydropyridine derivative Bay K 8644 possesses calcium agonistic properties also in the isolated human uterus. Furthermore, the competitive interaction with nifedipine showed the existence of specific dihydropyridine receptors closely associated with the calcium channel.