Entry Blocker Verapamil Research Articles

Influence of Calcium Entry Blockers and Calmodulin Inhibitors on 5-Hydroxytryptamine-, Potassium- and Calcium-induced Contractions in Human Umbilical Artery In-vitro

The effects of calcium (Ca2+) withdrawal, Ca2+ entry blockers and calmodulin inhibitors on the contractile responses to 5-hydroxytryptamine (5-HT), K+ and CaCl2 have been studied in human isolated umbilical artery. Following Ca2+ withdrawal from the medium (30 min), both 5-HT- and K(+)-induced contractions were virtually abolished. Ca2+ entry blockers (nifedipine, verapamil, diltiazem and cinnarizine) were significantly more effective in inhibiting 5-HT- than K(+)-induced contractions. In relation to calmodulin inhibitors, trifluoperazine inhibited all types of contractions, whereas W-7 (N-6-aminohexyl-5-chloro-1-naphthalene sulphonamide) only inhibited 5-HT- and CaCl2-induced contractions. All drugs inhibited CaCl2-induced contractions in a non-competitive manner. These findings indicate that the mechanisms by which these drugs exert their inhibitory effects on human umbilical artery are markedly different from those reported in other vascular beds.

Effects of nifedipine, verapamil and diltiazem on serum biochemical parameters and aortic composition of atherosclerotic chickens

Calcium appears to be involved in many of the cellular events, which are thought to be important in atherogenesis. In this study, we examine the effects of three calcium entry blockers (nifedipine, verapamil, and diltiazem at clinical and higher doses) on serum biochemical parameters and aortic calcium, cholesterol and triglyceride concentrations of atherosclerotic egg-fed chickens. All egg-fed chickens (treated and non-treated) showed an increase in serum total cholesterol, LDL-cholesterol and triglycerides without significant effect when calcium entry blockers were used. Increased HDL values were observed in clinical and high-dose nifedipine and clinical dose verapamil groups. The high-dose diltiazem group presented increased zinc values with respect to the clinical dose diltiazem and control groups. The sodium concentrations were significantly decreased in all the groups of animals treated with calcium entry blockers at high-doses and nifedipine at clinical doses. Measurements of aortic calcium concentration showed a significant decrease in the high-dose nifedipine and verapamil groups. Calcium channel blockers had a tendency to decrease total cholesterol in aortas. The values were statistically significant for the high-dose verapamil, and nifedipine groups. Only nifedipine showed a significant decrease for this parameter at clinical dosages. Triglyceride concentrations in aortas were significantly low in animals fed an atherogenic diet and treated with calcium channel blockers, without differences between drugs or dosages used in the experiment. In addition, the chicken atherosclerosis model has proved itself useful and very suitable for in vivo drug intervention studies.

Blockade of calcium entry in smooth muscle cells by the antidepressant imipramine

The present study was designed to evaluate the effects of antidepressants on smooth muscle contractile activity. In rat aortic rings, the antidepressants imipramine, mianserin and sertraline provoked concentration-dependent inhibitions of the mechanical responses evoked by K + (30 mM) depolarization. These myorelaxant effects were not modified by the presence of glibenclamide or 80 mM K + in the bathing medium. Moreover, the vasodilator properties of imipramine were not affected by atropine, phentolamine and pyrilamine. Radioisotopic experiments indicated that imipramine failed to enhance 86 Rb outflow from prelabelled and perifused aortic rings whilst counteracting the increase in 45 Ca outflow provoked by a rise in the extracellular K + concentration. Simultaneous measurements of contractile activity and fura-2 fluorescence revealed that, in aortic rings, imipramine reduced the mechanical and fluorimetric response to K + challenge. In A7r5 smooth muscle cells, whole cell recordings further demonstrated that imipramine inhibited the inward Ca 2+ current. Under different experimental conditions, the ionic and relaxation responses to the antidepressants were reminiscent of those mediated by the Ca 2+ entry blocker verapamil. Lastly, it should be pointed out that imipramine exhibited a myorelaxant effect of similar amplitude on rat aorta and on rat distal colon. All together, these findings suggest that the myorelaxant properties of imipramine, and probably also setraline and mianserin, could result from their capacity to inhibit the voltage-sensitive Ca 2+ channels.

Verapamil, a phenylalkylamine Ca2+ channel blocker, inhibits ATP-sensitive K+ channels in insulin-secreting cells from rats.

Radioisotopic and electrophysiological techniques were used to assess the effects of verapamil, a phenylalkylamine Ca2+ channel blocker, on K+ permeability of insulin-secreting cells. Verapamil provoked a concentration-dependent inhibition of 86Rb (42K substitute) outflow from prelabelled and perifused rat pancreatic islets. This property appears to be inherent to the phenylalkylamine Ca2+ channel blockers since gallopamil, a methoxyderivative of verapamil, but not nifedipine, a 1,4-dihydropyridine Ca2+ channel blocker, inhibited 86Rb outflow. The experimental data further revealed that verapamil interacted with a Ca2+-independent, glucose- and glibenclamide-sensitive modality of 86Rb extrusion. Moreover, verapamil prevented the increase in 86Rb outflow brought about by BPDZ 44; a potent activator of the ATP-sensitive K+ channel. Single-channel current recordings by the patch clamp technique confirmed that verapamil elicited a dose-dependent inhibition of the ATP-dependent K+ channel. Lastly, under experimental conditions in which verapamil clearly inhibited the ATP-sensitive K+ channels, the drug did not affect 45Ca outflow, the cytosolic free Ca2+ concentration or insulin release. It is concluded that the Ca2+ entry blocker verapamil inhibits ATP-sensitive K+ channels in pancreatic beta cells. This effect was not associated with stimulation of insulin release.

Ca2+ channel blockers verapamil and nifedipine inhibit apoptosis induced by 25-hydroxycholesterol in human aortic smooth muscle cells

We have characterized the death of human aortic smooth muscle cells induced by 25-hydroxycholesterol, an oxidation product of cholesterol. Chromatin condensation characteristic of apoptosis was observed by enzymatic (TUNEL) staining of chromatin, and by electron microscopy. Fourteen percent of cells treated with 5 microg/ml of 25-hydroxycholesterol for 24 h displayed chromatin degradation as determined by positive TUNEL staining. Addition of TNF alpha (10 ng/ml) and IFN gamma (20 ng/ml) increased the proportion of TUNEL positive cells to 30%, whereas the cytokines alone were without effect. After 48 h, 40% of the cells treated with 5 microg/ml of 25-hydroxycholesterol were TUNEL positive, and 21% of the cells displayed chromatin condensation. Oligonucleosomal DNA fragmentation typical of apoptosis was demonstrated by agarose gel electrophoresis. Furthermore, activation of the ICE-like protease caspase 3 (CPP32) was observed in cells treated with 25-hydroxycholesterol. Addition of the Ca2+ entry blockers verapamil or nifedipine to the culture medium inhibited apoptosis by more than 70% and reduced cytotoxicity, while removal of Ca2+ from culture medium reduced apoptosis by 42%. Within a few minutes after addition, 25-hydroxycholesterol induced intracellular Ca2+ oscillations with a frequency of approximately 0.3-0.4 min(-1). Thus it appears that Ca2+ influx through plasma membrane channels is an important signal in oxysterol-induced apoptosis. Addition of TNF alpha and IFN gamma enhanced cytotoxicity and resulted in a higher proportion of apoptotic cells, suggesting that inflammatory cytokines can increase the cytotoxicity of lipid oxidation products.

Effects of ion channel blockers on rapid postmortem changes in extracellular dopamine and serotonin levels in the rat nucleus accumbens

In the present study, we used in vivo brain microdialysis to examine the effects of ion channel blockers tetrodotoxin (TTX), EGTA-free Ca2+ and verapamil on rapid postmortem changes in extracellular levels of doapmine (DA), serotonin (5-HT) and their metabolites dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindoleacetic acid (5-HIAA) in the ACC of freely moving rats. Extracellular ACC DA levels decreased following the perfusion of the three ion channel blockers in freely moving rats, and then, at death by cervical dislocation, maximum respective 220-, 60- and 90-fold increases were observed in the extracellular output of DA in animals treated with EGTA, verapamil and TTX, respectively. Also, ACC 5-HT decreased following perfusion with the three blockers in the freely moving rats, and then maximum increases of 80-, 30- and 45-fold in the extracellular output of 5-HT were observed at death in animals treated with EGTA, verapamil and TTX, respectively, compared to the baseline. Cervical dislocation-induced rapid postmortem changes were inhibited markedly by perfusion with CSF containing the Ca2+ entry blocker verapamil. These observations suggested that rapid postmortem changes in ACC DA and 5-HT release were associated with the action of calcium ion channels and/or voltage gated channels in the CNS.

Inhibition of calcium entry preserves contractility of arterial smooth muscle in culture.

The addition of the growth stimulator fetal calf serum (FCS, 10%) to rings of rat tail artery causes an increase in [Ca2+]i, accompanied by contraction. This response was inhibited by the calcium entry blocker verapamil (1 microM). To investigate the effect of Ca2+ entry blockade on growth and contractility, rings of rat tail artery were cultured for 4 days in medium with or without FCS and then mounted for tension registration and stimulated with noradrenaline (NA) or high-K+ solution. In cultured rings growth was quantitated by [3H]-thymidine incorporation and increase in protein contents. FCS in the medium stimulated DNA synthesis by about 2-fold and increased protein contents by about 70%. The growth-stimulated cultured rings developed less force than freshly prepared rings (2.2 +/- 0.3 vs. 8.3 +/- 1.0 mN/mm). The addition of 1 microM verapamil to the medium during culture increased maximal NA-evoked force to 5.0 +/- 0.4 mN/mm but had no effect on the increases in DNA synthesis and protein contents. Force developed by growth-arrested rings, cultured in the absence of FCS, was not different from that of freshly prepared rings (7.2 +/- 0.6 mM/mm). Verapamil did not affect maximal force in these rings. Similar responses were seen when contraction was elicited by high-K+ solution. We conclude that verapamil, present during culture, preserves contractility of arterial smooth muscle, and that this effect is not parallel to inhibition of growth.

Effect of verapamil on intimal thickening and vascular reactivity in the collared carotid artery of the rabbit.

1. Intimal thickening is a common site for atherosclerosis. Therefore, we investigated whether the calcium entry blocker verapamil (10 mg kg-1 body weight day-1, s.c.) can retard intimal thickening and changes in vascular reactivity induced by a non-occlusive, silicone collar positioned around the left carotid artery of rabbits. The contralateral carotid artery was sham-operated and served as a control. 2. Verapamil and placebo (saline 0.1 ml kg-1, day-1, s.c.) treatments were initiated 7 days before placing the collar and lasted 3 weeks. Thereafter, segments were cut from collared and sham-treated arteries for histology and isometric tension recording. 3. The intima/media (I/M ratio increased after 14 days of collar treatment, but intimal thickening was not inhibited by verapamil (I/M ratio placebo 0.31 +/- 0.07, verapamil 0.32 +/- 0.09). 4. The collar decreased the capacity to develop force, as indicated by the response to a supramaximal concentration of KCl, decreased the sensitivity (pD2) to acetylcholine (ACh) and phenylephrine (Phe), but increased the sensitivity to 5-hydroxytryamine (5-HT). 5. Although verapamil did not affect intimal thickening, it normalized the hypersensitivity to 5-HT in collared arteries. 6. The contraction to the supramaximal concentration of KCl was not affected by verapamil. Verapamil decreased the Emax of ACh, but this was only seen in collar-treated arteries. Verapamil also decreased the sensitivity to ACh and Phe, in both sham- and collar-treated arteries. 7. We conclude that verapamil, without preventing thickening of the intima, can modify collar-induced changes in vascular reactivity.

Verapamil and flunarizine protect the isolated perfused rat liver against warm ischemia and reperfusion injury.

Using the model of the isolated perfused rat liver, we investigated the influence of the two pharmacologically different calcium channel blockers, verapamil and flunarizine, on changes of ion homeostasis, liver weights, pH deviations and enzyme activities during warm ischemia (37 degrees C) and reperfusion. The LDH and GLDH activities were determined and the calcium, potassium, and sodium concentrations were measured in the effluent. Warm ischemia (180 min) caused an increased enzyme release, a high influx of calcium and sodium into the liver and a massive potassium efflux current. Normoxic reperfusion led to a further increase in hepatic enzyme release and although the loss of potassium ceased, the calcium influx into the liver continued. By the end of reperfusion the liver weight had increased significantly (P < 0.01) in the control group. The two calcium entry blockers were added to the perfusate in various concentrations. Both substances protected the liver against warm ischemia and normoxic reperfusion damage, but they did not inhibit calcium inflow. However, the potassium efflux was significantly reduced by all concentration tasted (P < 0.001). After reperfusion the liver weights were significantly lower in the treated groups (P < 0.001) than in control animals. Thus, the calcium entry blockers verapamil and flunarizine protect liver cells against damage caused by warm ischemia and reperfusion. Furthermore, they prevent the disruption of intracellular potassium homeostasis, which seems to be related to improved volume regulation of liver cells.

Effect of atrial natriuretic peptide and calcium antagonists on platelet-activating factor-induced contraction and intracellular calcium mobilization in rat mesangial cells.

We studied the effect of platelet-activating factor (PAF-acether) on rat mesangial cells (MC) and the intracellular mechanisms for PAF-acether-mediated MC activation. Contraction was measured as changes in planar cell surface area (PCSA), and intracellular free calcium concentration ([Ca2+]i) was measured as changes in the signal of the fluorescent indicator fura-2. PAF-acether induced a time- and dose-dependent reduction in PCSA and a dose-dependent increase in [Ca2+]i. Both phenomena were abolished by preincubation of the cells with the PAF-acether-binding blockers L652,731 and BN 52021. TMB-8, an inhibitor of intracellular calcium mobilization, partially inhibited both the reduction in PCSA and the increase in [Ca2+]i, whereas the Ca2+ entry blocker verapamil completely inhibited the reduction in PCSA but only partially blocked the increase in [Ca2+]i. Atrial natriuretic peptide (ANP) produced a dose-dependent inhibition of PAF-acether-induced cell contraction, but its effect was not statistically related to changes in [Ca2+]i. These results show that PAF-acether-induced MC contraction is associated with transient increases in [Ca2+]i, but the relaxing effects of ANP and verapamil may require additional physiologic interactions that involve mechanisms other than the [Ca2+]i transient.

Protective Effect of Verapamil on Shock Wave Induced Renal Tubular Dysfunction

In a prospective randomized study, the effects of the calcium entry blocker verapamil on shock wave induced tubular impairment were examined. A total of 24 patients with renal pelvis or caliceal stones undergoing anesthesia-free extracorporeal shock wave lithotripsy (ESWL**Dornier Medical Systems, Inc., Marietta, Georgia.) without auxiliary measures was randomly assigned to the verapamil group (12) or the control group (12). Four doses of verapamil (80mg. each) were given orally starting the night before ESWL. Controls received no medication. To assess renal tubular function the urinary excretion of α1-microglobulin, N-acetyl-β5-glucosaminidase and Tamm-Horsfall protein were determined before, immediately, and 12 and 24hours after ESWL. After ESWL there was an increase in urinary α1-microglobulin and N-acetyl-β5-glucosaminidase, which was significantly higher in the control than in the verapamil group. Tamm-Horsfall protein, a glycoprotein synthesized by the distal tubules, decreased significantly less in the verapamil group compared to the controls. Our results indicate that verapamil exhibits a protective effect on shock wave induced tubular damage. The underlying mechanisms are not elucidated yet, and direct actions on tubular cells and interference with renal hemodynamics are to be discussed.

An Analysis of Control of the Ventricle of the Mollusc Mercenaria Mercenaria: II. Ionic Mechanisms Involved in Excitation By 5-Hydroxytryptamine

ABSTRACT This study focuses on the ionic mechanisms involved in serotonergic control of the ventricle from the mollusc MercenariaMercenaria. The effects of calcium (Ca2+), sodium (Na+), potassium (K+), magnesium (Mg2+) and chloride (Cl−) ions on the action of 5-hydroxytryptamine (5-HT) were tested using a sucrose-gap technique. 5-HT increased the amplitude and frequency of the cardiac action potentials (APs) and coupled systolic force in a range of concentrations from the threshold, at 10−10 mol l−1, to 10−6 mol l−1. Low, physiological doses of 5-HT increased the rate of rise and amplitude of the fast rising phase of the AP, and hastened the process of repolarization. Doses of 5-HT higher than 10−5mol l−1caused systolic arrest. The action of 5-HT was highly dependent on the presence of physiological levels of extracellular Ca2+. It had a maximal effect on systolic activity in a calcium chloride concentration range of 9–18mmol l−1. The activity of 5-HT was blocked by treatment with Ca2+-free saline, with inorganic Ca2+ blockers (lanthanum or cobalt) or with an organic Ca2+ entry blocker (verapamil). The effects of 5-HT were potentiated by treatment with barium ions (Ba2+), by a dihydropyridine-sensitive Ca2+ agonist, Bay K 8644, or by a vertebrate Ca2+ entry blocker, diltiazem.Removal of extracellular Na+ or treatment with a Na+ ionophore, monensin, did not significantly affect excitation by 10−6 mol l−15-HT; nor did the removal of Cl− or Mg2+. Unlike Ca2+, these three ions probably did not have a critical role during the excitatory action of 5-HT. The excitatory action of 5-HT was not significantly altered by treatment with K+-free saline. When the membrane was depolarized by high-K+ salines, however, 5-HT was unable to elicit any APs or systolic contractions, suggesting that its mechanism may involve voltage-sensitive channels.

Inhibition of Na +/Ca 2+ overload with R 56 865 protects against cardiac arrhythmias elicited by ouabain in vivo in guinea-pigs

Several studies have suggested a central role for Na +/Ca 2+ in the pathogenesis of ouabain-induced cardiac arrhythmias. To test this hypothesis, the effects on ouabain-induced arrhythmias of i.v. pretreatment with R 56 865, a Na + and Ca 2+ overload inhibitor, were compared with those of lidocaine, verapamil and tetrodotoxin in anesthetized guinea-pigs. Cardiac arrhythmias were induced by i.v. infusion of ouabain (10 μg/kg per min). All nine guinea-pigs pretreated with saline developed ventricular premature beats at an ouabain dose of 159 ± 9 μg/kg (mean ± S.E.M.), ventricular tachycardia at a dose of 190 ± 10 μg/kg, ventricular fibrillation at a dose of 253 ± 18 μg/kg and died at a dose of 269 ± 16 μg/kg; none of the animals developed heart block or asytole. Pretreatment with R 56 865 (1.25 mg/kg, n = 6) significantly increased the ouabain doses required to induce ventricular premature beats, ventricular tachycardia, ventricular fibrillation and death relative to those for the saline group. Pretreatment with a Ca 2+ entry blocker verapamil (0.32 mg/kg, n = 6) also significantly increased the ouabain doses required to provoke ventricular arrhythmias and death; this medication was associated with second or third degree heart block during ouabain infusion in four out of six animals. Pretreatment with lidocaine ((10 mg/kg, n = 6) caused a significant increase in the dose of ouabain needed to initiate cardiac arrhythmias and to cause death. Pretreatment with a selective Na + channel blocker tetrodotoxin (4 μg/kg, n = 6) also significantly increased the ouabain does required to provoke ventricular premature beats, ventricular tachycardia, ventricular fibrillation, and death. The present results suggest that intracellular Na + and Ca 2+ loading plays an important role in the ouabain-induced cardiac arrhythmias and demonstrate the capacity of R 56 865 to reduce the ventricular arrhythmias and death caused by ouabain toxicity in vivo.

Effect of verapamil on hepatic ischemia/reperfusion injury

Alterations in cellular calcium homeostasis are a critical factor in the pathogenesis of hepatic ischemic damage and may mediate oxygen free radical injury during the reperfusion period. We investigated the effect of the calcium channel blocker verapamil on hepatic ischemia/reperfusion injury in normal rats and rats sensitized to oxidative injury by chemical depletion of the endogenous antioxidant glutathione. Forty-five minutes of complete hepatic ischemia followed by reperfusion caused an increase in the serum glutamic pyruvic transaminase (SGPT) level and a decline in the endogenous hepatic glutathione level but no increase in hepatic lipid peroxidation products. Chemical depletion of hepatic glutathione with diethylmaleate did not induce hepatocellular injury but augmented hepatic ischemia/reperfusion-induced SGPT release and promoted lipid peroxidation. Pretreatment with the calcium entry blocker verapamil protected against the ischemia/reperfusion-induced drop in hepatic glutathione but did not reduce SGPT release in normal rats. In rats sensitized to oxidative injury by chemical depletion of endogenous glutathione, the calcium channel blocker verapamil protected against ischemia/reperfusion-induced lipid peroxidation and reduced the release of SGPT. These findings indicate that the rat liver is protected against oxidative injury after short periods of total ischemia by its rich supply of endogenous glutathione. A beneficial effect of verapamil occurs only in rats sensitized to oxidative injury, suggesting that the calcium channel blocker protects against oxygen radical attack.

Idiopathic Long QT Syndrome Exacerbated by Beta‐Adrenergic Blockade and Responsive to Left Cardiac Sympathetic Denervation: Implications Regarding Electrophysiologic Substrate and Adrenergic Modulation

Electrophysiologic Substrate of Long QT Syndrome. Introduction: The mechanisms of T wave abnormalities and arrhythmias in patients affected by the long QT syndrome (LQTS) are unclear, as are the reasons why a high‐risk subgroup (20%) continues to have syncope during therapy with beta blockers but is protected after left cardiac sympathetic denervation (LCSD). Afterdepolarizations, both early (EAD) or delayed (DAD), have been implicated as likely electrophysiologic substrates for the ECG abnormalities and arrhythmias in LQTS. To test this hypothesis, we analyzed nine Hotter recordings of a LQTS patient typical of the high‐risk subgroup. Methods and Results: We applied to the ECG rules derived from previous in vitro and in vivo studies, which relate the coupling intervals of the dysrhythmic beats to the preceding cycle lengths, to discriminate among arrhythmia mechanisms. In the absence of therapy, the patient's ECG showed peaked T waves, with a notched second component accentuated by sinus pauses. The amplitude of the notches increased directly with the preceding RR interval (r = 0.58, P &lt; 0.05), as it is observed with EAD. Therapy with the beta blocker propranolol did not modify the notches, which maintained their amplitude and their relationship with the preceding RR (r = 0.62, P &lt; 0.05). Moreover, during beta‐blocker therapy, ventricular premature beats, couplets, and runs of ventricular tachycardia (VT) occurred. The coupling intervals of ventricular premature beats and couplets were not influenced by the preceding RR intervals. By contrast, the coupling interval of the first beat of VT decreased as the preceding RR shortened, as it occurs with DAD‐induced triggered activity. After LCSD, T wave notch amplitude was reduced (P &lt; 0.05) and was no longer influenced by the preceding RR (r = 0.31, P = 0.22). The same was true when the calcium entry blocker verapamil was administered. Conclusion: In this patient, T wave abnormalities and some ventricular arrhythmias had the behavior observed in vitro for EAD and the consequent triggered activity. However, the cycle length dependence of VT manifested the behavior observed in vitro for DAD and the related triggered activity. The fact that these phenomena were accentuated by beta blockade and disappeared after LCSD suggests that the alpha‐adrenergic receptor‐effector system may be a modulator of T wave abnormalities and arrhythmias in some patients unresponsive to beta blockade and protected by LCSD. (J Cardiovasc Electrophysiol, Vol. 3, pp. 295–305, August 1992)

Interaction of prostaglandins with adenosine diphosphate induced increase in cytosolic free calcium in human platelets.

The interaction of prostaglandins with changes in cytosolic Ca2+ concentration ([Ca2+]) and aggregation of human platelets induced by adenosine diphosphate (ADP) were investigated. Cytosolic [Ca2+] was measured with the fluorescent dye Quin2. Addition of ADP (0.25-2.5 mumol l-1) to platelet suspensions produced a dose dependent increase in cytosolic [Ca2+] from a basal level of 51 +/- 1 nmol l-1 to maximum levels exceeding 1 mumol l-1 and induced platelet aggregation. Chelation of extracellular calcium with 100 mumol l-1 EGTA markedly reduced the increase in cytosolic [Ca2+] induced by 0.25 mumol l-1 ADP, while pretreatment with the calcium entry blocker verapamil was without effect. Stimulation of cyclic AMP with prostaglandins (PGD2, PGE1, PGE2, PGI2, but not PGF2 alpha) and forskolin, or incubation with dibutyryl-cAMP, inhibited the rise in cytosolic [Ca2+] and platelet aggregation following ADP. We conclude that prostaglandins inhibit the increase in cytosolic [Ca2+] and aggregation of human platelets induced by ADP, probably by stimulation of cyclic AMP generation, thereby opposing the mechanism by which ADP increases cytosolic [Ca2+] and subsequently induces platelet aggregation.

Does verapamil act as an immunomodulatory drug in vivo?

Based on in vitro data, previous investigators have hypothesized that Ca2+ entry blockers might affect lymphocyte activation, proliferation and effector function. We have tested this hypothesis by comparing the in vitro and in vivo effects of the Ca2+ entry blocker verapamil on human lymphocytes. In vitro high concentrations of verapamil (100 gmM) inhibited mitogen-stimulated Ca2+ influx, inositol phosphate generation, and proliferation; similar effects were observed with diltiazem and nifedipine. In vivo treatment of healthy volunteers with verapamil (2–4 times 240 mg per day for 7 days) did not affect the number of circulating lymphocytes or their subset distribution. Moreover, we did not observe any effect of in vivo treatment with verapamil on mitogen-stimulated lymphocyte proliferation or expression of interleukin-2 receptors in vitro. We conclude that the inhibitory effects of verapamil on lymphocyte activation in vitro are unlikely to be of therapeutic relevance and may not be related to the Ca2+ entry blocking effects of this drug.

Angiotensin II-induced increase in slowly exchanging 45Ca2+ in relation to contractile responses of rat and guinea-pig aorta

To gain more information about sources of activator Ca2+ involved in the contraction of rat and guinea-pig aorta evoked by angiotensin II and their sensitivity to Ca2+ entry blockers, measurement of slowly exchanging 45Ca2+ was established. A more physiological procedure was used, replacing La(3+)- and EGTA-containing solutions by a normal Ca(2+)-containing buffer. It was demonstrated that the angiotensin II-induced increase in slowly exchanging 45Ca2+ in rat aorta was incompletely (by approximately 60%-70%) inhibited by the organic Ca2+ entry blockers nifedipine, verapamil and diltiazem and by other Ca2+ entry blocking compounds like CoCl2 and chlorpromazine. 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8) was able to inhibit the angiotensin II-induced increase in 45Ca2+ content completely, but this may be an intracellular storage effects. By contrast, the organic Ca2+ entry blockers completely inhibited that part of the angiotensin II-induced contraction of rat aorta which was dependent upon extracellular Ca2+. In guinea-pig aorta, the increase in 45Ca2+ content elicited by angiotensin II could be completely suppressed by all compounds under study. The results of these experiments correlated well with data from the functional experiments in guinea-pig aorta. In both preparations the release of Ca2+ from a rapidly as well as a slowly exchanging intracellular pool appears to contribute to the contractile response elicited by angiotensin II.

Increased toxicity of anthracycline antibiotics induced by calcium entry blockers in cultured cardiomyocytes

Calcium channel blocking drugs have been reported to reduce survival rate of laboratory animals treated with cardiotoxic antitumor anthracyclines. In order to elucidate the mechanisms of this drug interaction, cell toxicity of the anthracyclines, doxorubicin and daunorubicin, was evaluated in primary cultures of cardiac myocytes isolated from neonatal rats. Low concentrations of extracellular calcium ([Ca 2+] 0) and addition of calcium entry blockers (nifedipine or flunarizine) potentiated myocardial toxicity of anthracyclines as assessed by the release of lactate dehydrogenase from the cells. Accumulation of anthracyclines in the cardiomyocytes was increased by calcium entry blockers (nifedipine, flunarizine, and verapamil) and by low [Ca 2+] 0; efflux of [ 3H]daunorubicin from myocardial cells was inhibited by nifedipine. At a dose that exerts only modest calcium channel activity, R-verapamil failed to affect doxorubicin accumulation in cardiomyocytes, whereas the calcium channel activator, (±)-Bay K-8644, reduced the retention of anthracyclines; the calcium channel activity is thus required in order to increase the accumulation of anthracyclines in myocardial cells. Calcium channel blockers are also known to increase intracellular retention and toxicity of chemotherapeutic drugs in multidrug resistant tumor cells by inhibiting the efflux of cytotoxic agents from cells; however, the ability of the interacting drugs to inhibit the efflux of chemotherapeutic agents from tumor cells is not dependent on the calcium channel blocking activity. Therefore, the mechanism(s) by which calcium channel blocking drugs increase the accumulation of anthracyclines in resistant tumor cells and myocardial cells may be different. In accordance with previous investigations, the present in vitro study confirmed that anthracycline-induced cardiotoxicity may be potentiated by calcium channel blocking drugs. This indicates that, in the association of antineoplastic drugs with agents that reverse multidrug resistance, the potential exists for enhanced damage of normal cells and tissues; further studies are needed to evaluate the relevance of this adverse interaction.

Combined negative inotropic effects of calcium entry blockers and isoflurane on canine isolated heart muscles.

The combined negative inotropic effects of isoflurane and calcium entry blockers (verapamil, diltiazem, nifedipine, nicardipine) were studied utilizing isolated heart preparations of ventricular muscles from dogs. All of these calcium entry blockers exerted dose-dependent decreases in maximal velocity of shortening (Vmax), maximal developed isometric force (Fm), and the maximal first derivative of Fm (maximal dF/dt). Dose-dependent decreases of these variables of muscle mechanics were augmented in isoflurane-depressed myocardium. At equimolar concentrations, direct myocardial depression was demonstrated in the following order of severity: nifedipine > diltiazem = verapamil > nicardipine. Percent depressions of Vmax, Fm and maximal dF/dt were significantly greater in muscles when calcium entry blockers were combined with 1MAC isoflurane than in muscles of calcium entry blockers alone. These data suggest that the negative inotropic effects of verapamil, diltiazem, nifedipine, and nicardipine were potentiated by isoflurane.