Is the efflux pump inhibitor Verapamil a potential booster for isoniazid against Mycobacterium tuberculosis?

The membrane-based efflux pump systems are recognized to have an important role in pathogenicity and drug resistance in Mycobacterium tuberculosis by the extrusion of toxic substrates and drugs from the inner bacillus. This study aimed to investigate the in vitro interaction of Verapamil (VP), an efflux pump inhibitor, with the classical first-line anti-tuberculosis drug isoniazid (INH) in resistant and susceptible M. tuberculosis clinical isolates. Seven multidrug-resistant (MDR), three INH monoresistant and four susceptible M. tuberculosis clinical isolates were tested for the INH and VP combination by modified Resazurin Microtiter Assay Plate (REMA). Fractional Inhibitory Concentration (FIC) and Modulation Factor (MF) were determined. The INH plus VP combination showed no significant change in the Minimum inhibitory concentration (MIC) values of INH (FIC≥ 0.5; MF=1 or 2).The use of VP in tuberculosis therapy should be managed carefully, considering the resistance caused by specific mutation in katG and inhA genes, in which the use of these EPIs may have no success. The use of EPIs as an adjunctive drug in the anti-tuberculosis therapy should be further investigated on a larger number of M. tuberculosis clinical isolates with different resistant profile.

The effects of verapamil and its combinations with glutamate and glycine on cardiodynamics, coronary flow and oxidative stress in isolated rat heart.

The role of N-methyl-D-aspartate receptor (NMDA-R) in heart is still unclear. For these ionotropic glutamate receptors is characteristic the necessity of both co-agonists, glutamate and glycine, for their activation, which primarily allows influx of calcium. The aim of the present study was to examine the effects of verapamil, as a calcium channel blocker, alone and its combination with glycine and/or glutamate on cardiac function, coronary flow, and oxidative stress in isolated rat heart or to examine the effects of potential activation of NMDA-R in isolated rat heart. The hearts of male Wistar albino rats were excised and perfused according to Langendorff technique, and cardiodynamic parameters and coronary flow were determined during the administration of verapamil and its combinations with glutamate and/or glycine. The oxidative stress biomarkers, including thiobarbituric acid-reactive substances, nitrites, superoxide anion radical, and hydrogen peroxide, were each determined spectrophotometrically from coronary venous effluent. The greatest decline in parameters of cardiac contractility and systolic pressure was in the group that was treated with verapamil only, while minimal changes were observed in group treated with all three tested substances. Also, the largest changes in coronary flow were in the group treated only with verapamil, and at least in the group that received all three tested substances, as well as the largest increase in oxidative stress parameters. Based on the obtained results, it can be concluded that NMDA-R activation allows sufficient influx of calcium to increase myocardial contractility and systolic pressure, as well as short-term increase of oxidative stress.

In Vitro Studies Indicate Intravenous Lipid Emulsion Acts as Lipid Sink in Verapamil Poisoning.

Intravenous lipid emulsion (ILE), a component of parenteral nutrition, consists of a fat emulsion of soy bean oil, egg phospholipids, and glycerin. Case reports suggest that ILE may reverse hypotension caused by acute poisoning with lipophilic drugs such as verapamil, but the mechanism remains unclear. The methods used are the following: (1) measurement of ILE concentration in serum samples from a patient with verapamil poisoning treated with ILE, (2) measurement of free verapamil concentrations in human serum mixed in vitro with increasing concentrations of ILE, and (3) measurement of murine ventricular cardiomyocyte L-type Ca(2+) currents, intracellular Ca(2+), and contractility in response to verapamil and/or ILE. Maximum patient serum ILE concentration after infusion of 1 L ILE over 1 h was approximately 1.6 vol%. In vitro GC/MS verapamil assays showed that addition of ILE (0.03-5.0 vol%) dose-dependently decreased the free verapamil concentration in human serum. In voltage-clamped myocytes, adding ILE to Tyrode's solution containing 5 µM verapamil recovered L-type Ca(2+) currents (ICa). Recovery was concentration dependent, with significant ICa recovery at ILE concentrations as low as 0.03 vol%. ILE had no effect on ICa in the absence of verapamil. In field-stimulated intact ventricular myocytes exposed to verapamil, adding ILE (0.5 %) resulted in a rapid and nearly complete recovery of myocyte contractility and intracellular Ca(2+). Our in vitro studies indicate that ILE acts as a lipid sink that rapidly reverses impaired cardiomyocyte contractility in the continued presence of verapamil.

Effect of verapamil, a calcium channel blocker, on the odontogenic activity of human dental pulp cells cultured with silicate-based materials.

INTRODUCTION: This study examines how calcium silicate cement extracts influence the behavior of human dental pulp cells (hDPCs) through calcium channels and active mitogen-activated protein kinase pathways, in particular extracellular signal-related kinase (ERK). METHODS: HDPCs are treated with various silicon concentrations both with and without verapamil, after which the cells' viability and odontogenic differentiation markers are determined by using PrestoBlue assay and Western blot, respectively. RESULTS: The silicon promoted cell proliferation and inhibited calcium channel blockers. It was also found that silicon increased ERK and p38 activity in a dose-dependent manner. Furthermore, it raised the expression and secretion of alkaline phosphatase, osteocalcin, dentin sialophosphoprotein, and dentin matrix protein-1. In addition, statistically significant differences (P < .05) have been found in the secretion of osteocalcin in ERK inhibitor + verapamil between the silicon concentrations; these varations are dose-dependent and indicate that ERK signaling is involved in the silicon-induced odontogenic differentiation of hDPCs. CONCLUSIONS: The current study shows that silicon ions released from calcium silicate substrates play a key role in odontoblastic differentiation of hDPCs through calcium channels and modulate ERK activation.

Modeling cyclosporine A inhibition of the distribution of a P-glycoprotein PET ligand, 11C-verapamil, into the maternal brain and fetal liver of the pregnant nonhuman primate: impact of tissue blood flow and site of inhibition.

UNLABELLED: Through PET imaging, our laboratory has studied the dynamic biodistribution of (11)C-verapamil, a P-gp substrate, in the nonhuman primate Macaca nemestrina. To gain detailed insight into the kinetics of verapamil transport across the blood-brain barrier (BBB) and the blood-placental barrier (BPB), we analyzed these dynamic biodistribution data by compartmental modeling. METHODS: Thirteen pregnant macaques (gestational age, 71-159 d; term, ∼172 d) underwent PET imaging with (11)C-verapamil before and during infusion (6, 12, or 24 mg/kg/h) of cyclosporine A (CsA, a P-glycoprotein [P-gp] inhibitor). Dynamic (11)C-verapamil brain or fetal liver (reporter of placental P-gp function) activity was assessed by a 1- or 2-tissue-compartment model. RESULTS: The 1-tissue-compartment model best explained the observed brain and fetal liver distribution of (11)C-radioactivity. When P-gp was completely inhibited, the brain and fetal liver distribution clearance (K1) approximated tissue blood flow (Q); that is, extraction ratio (K1/Q) was approximately 1, indicating that in the absence of P-gp function, the distribution of (11)C-verapamil radioactivity into these compartments is limited by blood flow. The potency of CsA to inhibit P-gp was tissue-independent (maternal BBB half-maximal inhibitory concentration [IC50], 5.67 ± 1.07 µM, vs. BPB IC50, 7.63 ± 3.16 µM). CONCLUSION: We propose that on deliberate or inadvertent P-gp inhibition, the upper boundary of increase in human brain (or fetal) distribution of lipophilic drugs such as verapamil will be limited by tissue blood flow. This finding provides a means to predict the magnitude of P-gp-based drug interactions at the BBB and BPB when only the baseline distribution of the drug (i.e., in the absence of P-gp inhibition) across these barriers is available through PET. Our data suggest that P-gp-based drug interactions at the human BBB and BPB can be clinically significant, particularly for those P-gp substrate drugs for which P-gp plays a significant role in excluding the drug from these privileged compartments.

Auto-inhibition of verapamil metabolism in rat hepatocytes of gel entrapment culture.

Mechanism-based inactivation (MBI) of cytochrome P450 (CYP) 3A by verapamil and the resulting drug-drug interactions have been studied in vitro, but the inhibition of verapamil on its own metabolic clearance in clinic, namely auto-inhibition of verapamil metabolism, has never been reproduced in vitro. This paper aimed to evaluate the utility of gel entrapped rat hepatocytes in reflecting such metabolic auto-inhibition using hepatocyte monolayer as a control. Despite being a similar concentration- and time-dependent profile, auto-inhibition of verapamil metabolism showed apparent distinctions between the two culture models. Firstly, gel entrapped hepatocytes were more sensitive to such inhibition, which could be largely due to their higher CYP3A activity detected by the formation rates of 6ß-hydroxy testosterone and 1'-hydroxy midazolam. Furthermore, the inhibitory effect of ketoconazole and verapamil on CYP 3A activity as well as the reduction of verapamil intrinsic clearance (CL(int)) by ketoconazole was only observed in gel-entrapped hepatocytes. In this respect, the involvement of CYP3A in auto-inhibition of verapamil metabolism could be illustrated in gel-entrapped hepatocytes but not in hepatocyte monolayer. All of these results indicated that hepatocytes of gel entrapment reflected more of verapamil metabolic auto-inhibition than hepatocyte monolayer and could serve as a suitable system for investigating drug metabolism.

Functional expression of P-glycoprotein in primary cultures of human cytotrophoblasts and BeWo cells.

The objective of this study was to investigate the functional expression of the efflux transporter, P-glycoprotein (P-gp), in primary cultures of human cytotrophoblasts and BeWo cell monolayers. Uptake studies with primary cultures of human cytotrophoblasts or BeWo cells were conducted with calcein-AM and vinblastine (P-gp markers) or fluorescein (MRP marker) in the presence of specific P-gp or MRP inhibitors. Results showed that the accumulation of P-gp substrates calcein-AM and vinblastine by BeWo cells or primary cultures of human cytotrophoblasts was significantly enhanced in the presence of a typical P-gp inhibitor, cyclosporin-A, or other inhibitors such as quinidine, verapamil, and dipyridamole. MRP inhibitors had no effect on the accumulation of calcein or fluorescein by BeWo cells. Western blots confirmed the presence of multidrug resistant gene product 1 (MDR1) in both primary cultures of human cytotrophoblasts and BeWo cells. This study demonstrates functional P-gp in term human trophoblasts and further supports the use of primary cultures of human cytotrophoblasts and BeWo cells as in vitro models of the trophoblast to investigate mechanisms regulating drug distribution across the placenta.

Direct interaction between verapamil and doxorubicin causes the lack of reversal effect of verapamil on P-glycoprotein mediated resistance to doxorubicin in vitro using L1210/VCR cells.

Mouse leukemic cell subline L1210/VCR exerts expressive multidrug resistance (MDR) that is mediated by P-glycoprotein. Cells originally adapted to vincristine are also extremely resistant to doxorubicin. Resistance to both vincristine and doxorubicin is connected with depression of drug uptake. While resistance of L1210 cells to vincristine could be reversed by verapamil as chemosensitizer, resistance of cells to doxorubicin was insensitive to verapamil. Action of verapamil (well-known inhibitor of PGP activity) on multidrug resistance was often used as evidence that MDR is mediated by PGP. From this point it may be possible that the resistance of L1210/VCR cells to vincristine is mediated by PGP and the resistance to doxorubicin is mediated by other PGP-independent system. Another and more probable explanation of different effect of verapamil on resistance of L1210/VCR cells to vincristine and doxorubicin may be deduced from the following fact: Using UV spectroscopy we found that doxorubicin dissolved in water buffered medium interacts effectively with verapamil. This interaction may be responsible for the decrease of concentration of both drugs in free effective form and consequently for higher survival of cells. In contrast to doxorubicin vincristine does not give any interaction with verapamil that is measurable by UV spectroscopy and resistance of L1210/VCR cells to vincristine may be fully reversed by verapamil.

Neuron degeneration induced by verapamil and attenuated by EGb761.

Calcium channel blockers are used as neuroprotective agents, as glutamate antagonists. However, it has been found that calcium channel blockers may compromise neuronal survival after long-term exposure. To explore the mechanisms of the toxicity of calcium channel blockers on neurons, we studied the morphological characteristics and biochemical changes of cultured cortical neurons treated with verapamil, a calcium channel blocker. We now report that cerebral cortical cultures exposed to verapamil for 48 h undergo neuronal degeneration in both concentration-dependent and time-dependent fashion, possibly partially through the activation of apoptosis. On the other hand, it was found that Ginkgo biloba extract (EGb761) attenuated verapamil-induced neuronal injury, suggesting the possibility of using verapamil combined with EGb761 clinically. Furthermore, both B-50 immunoactivity (BIA) and the concentration of intracellular calcium in single neurons ([Ca2+]i) decreased after a 48-h exposure to verapamil, suggesting that the mechanisms of verapamil-induced degeneration may be associated with the disruption of intracellular calcium homeostasis and the inhibition of normal axonal elongation.

Amrinone as an antidote in experimental verapamil overdose.

OBJECTIVE: To evaluate the effect of amrinone as a treatment for the hemodynamic effects of verapamil overdose in a canine model. METHODS: This nonblind interventional study was performed in an established canine model of verapamil toxicity, without concurrent control animals. Pentobarbital-anesthetized and instrumented dogs (n = 8) were maintained and observed for 60 minutes or until death. The animals were overdosed with verapamil, 15 mg/ kg IV, over 30 minutes. Hemodynamic parameters, including cardiac index (CI), heart rate (HR), and mean arterial pressure (MAP), were monitored. Completion of the verapamil infusion represented the defined point of toxicity; at that point, all the animals received an amrinone bolus of 2 mg/kg IV over 2 minutes followed by an amrinone drip at 10 micrograms/kg/min. The hemodynamic values at the defined point of toxicity were compared with those obtained postinitiation of the amrinone infusion. RESULTS: Two animals died before the 60-minute observation period elapsed. Baseline CI was 5.6 L/min/m2. Following verapamil-induced toxicity, mean CI was 2.2 L/min/m2. After administration of amrinone, a significant (p < 0.05) increase in CI was observed at 30 minutes (CI = 3.6 L/min/m2), 45 minutes (CI = 4.2 L/ min/m2), and 60 minutes (CI = 4.2 L/min/m2). There was no statistically significant difference noted for MAP or HR compared with "point of toxicity" values. CONCLUSION: Amrinone appears to reverse the depressed cardiac index associated with verapamil overdose in a canine model while having no significant effect on the hypotension or bradycardia.

Membrane properties of complex spike firing neurons of the mouse dorsal cochlear nucleus in vitro.

Intracellular recordings were made from complex spike firing neurons of the mouse dorsal cochlear nucleus (DCN) in vitro. The whole cochlear nucleus was dissected out and maintained submerged in rapidly flowing artificial cerebrospinal fluid (CSF). Recordings were made with current clamp techniques in the presence or absence of ion channel blocking drugs tetrodotoxin (TTX, 1 microM), tetraethylammonium (TEA, 20 mM), 4-aminopyridine (4-AP, 5 mM) or verapamil (50, 100, 150, 250 microM). The cells showed both spontaneous firing and responses to injections of depolarising current consisting of a mixture of a tall single action potential and complexes of 2 to 3 smaller wider action potentials superimposed on a plateau depolarisation. The membrane properties were: resting membrane potential -68.8 +/- 8.5 mV, cell resistance 54.1 +/- 26.5 M omega, time constant 9.6 +/- 5.4 ms and capacitance 0.25 +/- 0.5 nF; the first three variables had bimodel distributions. The current/voltage (I/V) relationship at membrane below resting was non-linear. Previously published histological evidence from the mouse DCN has shown that both cartwheel cells and Purkinje-like neurons are present. Both DCN cartwheel cells and cerebellar Purkinje cells are known to fire both tall single action potentials and complexes of smaller wider action potentials. It is therefore possible that the recordings shown here were made from these neuron types. TTX (1 microM) abolished both the tall single and the complexes of smaller action potentials, suggesting that the tall single action potentials are sodium dependent and possibly that a TTX sensitive sodium channel is responsible for the plateau as is suggested for Purkinje cells in the cerebellum. Verapamil (100 microM) abolished only the complex action potentials and the plateau leaving the tall narrow action potentials intact, which is consistent with the smaller complexes being calcium dependent. Higher concentrations abolished all spiking activity. TEA and 4-AP used separately both caused marked depolarisation to around -20 mV, suggesting that there is a large potassium current active at and near resting.

Studies on the interactions between the angiotensin converting enzyme inhibitor enalaprilat and calcium antagonists in rats.

OBJECTIVE: To investigate the hypotheses that the synergistic hypotensive interaction between angiotensin converting enzyme (ACE) inhibitors and calcium antagonists is mediated via alpha1-adrenoceptor blockade and that in the presence of ACE inhibitors the alpha1-adrenoceptor potency of clinically used calcium antagonists may be sufficiently enhanced to add to the hypotensive effect. METHODS: The interactions between the ACE inhibitor enalaprilat and the calcium antagonists diltiazem, cinnarizine, felodipine and verapamil were studied in anesthetized rats for effects on blood pressure and in isolated perfused rat tail arteries for effects at alpha1-adrenoceptors. RESULTS: It was found that in isolated tail arteries enalaprilat had no effect on the weak alpha1-adrenoceptor antagonist actions of diltiazem, cinnarizine and felodipine. Similarly, enalaprilat did not affect the hypotensive responses to these calcium antagonists. However, enalaprilat was found to potentiate the alpha1-adrenoceptor antagonist action of verapamil, both in vitro and in vivo, as well as the hypotensive action of verapamil. CONCLUSIONS: These results support the above hypothesis in the case of verapamil only; for calcium antagonists in general the hypothesis was not supported. The results are consistent with a growing body of evidence showing that the distinction between calcium antagonists and alpha1-adrenoceptor antagonists is becoming less sharply defined.

[Changes in the chrono-inotropic properties of the myocardium and oxygen consumption as affected by strophanthin-K, isoptin and their combination]. / Izmeneniia khronoinotropnykh svoistv miokarda i potrebleniia im kisloroda pod vliianiem strofantina-K, izoptina i ikh kombinatsii.

In the experiments on isolated frog atria isometrically contracting, the author studied myocardial contractility and oxygen consumption under the action of strophanthin K, isoptin, and their combination. Strophanthin was demonstrated to exert two-phase effects on the inotropic function of an isolated myocardial stripe, which depended on the time of cardiotonic action. The calcium antagonist isoptin in combination with strophanthin K reduced myocardial oxygen uptake, potentiated the negative chronotropic effect of the cardiac glycoside, decreased the cardiostimulant action of the latter, slowing down its development.

Protection of reperfused ischemic pig myocardium by nexopamil, a new combined Ca2+ and serotonin antagonist.

We investigated the ability of a newly developed calcium and serotonin (5-HT2) antagonist, nexopamil, to protect the heart from ischemia- and reperfusion-induced myocardial injury. Anesthetized open-chest minipigs were subjected to 1 h left anterior descending coronary artery (LAD) occlusion and 3-h reperfusion. Thirty minutes before occlusion, one group of pigs (n = 7) received nexopamil (0.1 mg/kg intravenously, i.v.) and another group (n = 9) received vehicle. Nexopamil reduced infarct size (IS: tetrazolium stain) from 47 +/- 4% (vehicle) to 21 +/- 7% of the ischemic area (p < 0.05). In nexopamil-treated pigs, this was paralleled by reduced release of creatine kinase (CK) into coronary venous blood. In addition, nexopamil prevented reperfusion-associated myocardial contracture. Nexopamil decreased left ventricular peak pressure (LVPP) and pressure rate index (PRI) immediately before coronary occlusion by 11 and 18%, respectively. Coadministration of methoxamine (2 mg/kg, n = 6) with nexopamil increased LVPP and PRI to values of vehicle-treated pigs but did not prevent reduction in infarct size or CK activity in plasma. During reperfusion, neutrophil granulocytes showed increased formation of reactive oxygen metabolites (chemiluminescence) after stimulation with zymosan. Neutrophil counts in coronary venous blood were significantly reduced at 3 h reperfusion. Both changes were attenuated in nexopamil-treated pigs. Coronary occlusion resulted in increased platelet reactivity in coronary venous blood (collagen-induced aggregation) that was prevented by nexopamil. Nexopamil significantly increased the transcardiac (coronary venous-arterial) concentration gradients of 6-oxo-prostaglandin F1 alpha (PGF1 alpha) without changing thromboxane (B2 (TBX2) concentrations, indicating a selective increase in cardiocoronary PGI2 formation. Nexopamil reduces myocardial injury in reperfused ischemic myocardium. Besides calcium channel blocking activity, inhibition of ischemia-induced neutrophil activation and enhanced endogenous PGI2 formation may be factors contributing to the beneficial effects of nexopamil.

Avanços terapêuticos em cardiologia: parte 1 - Antagonistas do cálcio: estado atual em cardiologia / Therapeutic advances in cardiology: part 1 - Calcium antagonists: up-to-date state in cardiology

O cálcio representa a base celular para a excitaçäo e contraçäo da musculatura cardiovascular. Os bloqueadores do cálcio têm estruturas moleculares diferentes, com efeitos comuns, e repercussöes cardiovasculares com potências desiguais. Aumentam o fluxo coronário, diminuem a pós-carga, o consumo de oxigênio e o espasmo coronário. Têm múltiplas indicaçöes clínicas, representando um marco na estratégia terapêutica em cardiologia

Cromakalim (BRL 34915) counteracts the epileptiform activity elicited by diltiazem and verapamil in rats.

1. The effects of BRL 34915 (cromakalim), a potassium channel opener, have been tested on the epileptiform activity elicited by high dose/concentrations of some calcium antagonists in in vivo (diltiazem) and in vitro (diltiazem and verapamil) experiments in rats. 2. Diltiazem (150-300 mg kg-1, i.p.) induced behavioural and electroencephalographic (EEG) seizures that were completely prevented by cromakalim (10 nmol/10 microliters, i.c.v.). Whereas, pentobarbitone (5-10 mg kg-1, i.p.) only prevented the behavioural component of the seizures. 3. In hippocampal slices, verapamil (1.5-2.0 mM) produced, within 30-60 min of perfusion, a CA1 epileptiform bursting in 80% of the experiments. This epileptiform activity was prevented by the cromakalim concentration (50 microM) that did not affect the control CA1 synaptic transmission per se. Pentobarbitone also prevented verapamil-induced epileptiform bursting only at the concentration (100 microM) that also reduced control CA1 synaptic transmission. 4. Diltiazem (1.5 mM) produced a biphasic excitatory-depressant effect within 60 min of perfusion. A CA1 epileptiform bursting appeared in 100% of the experiments within 30 min of perfusion. These excitatory effects were followed by a depression phase, characterized by a reduction of the magnitude of CA1 excitatory postsynaptic potentials (e.p.s.ps) and population spike. 5. The diltiazem-induced epileptiform bursting was prevented by cromakalim at a concentration (50 microM) that did not affect the control CA1 synaptic transmission per se. Pentobarbitone also prevented the diltiazem-induced epileptiform bursting only at a concentration (100 microM) that also reduced the control CA1 synaptic transmission. Both cromakalim (50 microM) and pentobarbitone (100 microM) failed to affect the depressant effects of diltiazem on CA1 hippocampal area. On the contrary, high (3.3mM) calcium solutions prevented both the excitatory and the depressant effects of 1.5 mm diltiazem within 60 min.6. These data indicate an involvement of potassium currents in the epileptiform activity elicited by high doses of diltiazem and verapamil.

Verapamil upregulates sensitivity of human colon and breast cancer cells to LAK-cytotoxicity in vitro.

Pretreatment of human colon cancer LoVo-H cells and human breast cancer ZR-75 1A cells with low doses of verapamil, a Ca2+ channel blocker, for 48 h has a slight growth stimulatory effect and substantially increases cell sensitivity to lymphokine-activated killer (LAK) mediated cytotoxicity in the standard 51Cr release assay. The role of intracellular Ca2+ levels in determining verapamil effect is demonstrated by cytochemical evidence of intracellular Ca2+ lowering in verapamil-treated cells and by the reversal by the Ca2+ ionophore A-23187 of verapamil-induced sensitivity to LAK-mediated cytotoxicity.

Pharmacological properties of contraction caused by sodium removal in muscle strips isolated from canine coronary artery.

Contractions produced by Na+ removal were studied in muscle strips isolated from canine coronary artery. In the presence of 20 mM K+ and 0.5 mM Ca2+, rapid contractions were observed repeatedly on complete replacement of NaCl with sucrose. This contraction in the absence of Na+ (0-Na) was not affected by phentolamine but was strongly inhibited by verapamil. Ouabain slowly potentiated the O-Na contraction and markedly reduced the inhibition due to verapamil. The O-Na contraction was dependent on external Ca2+ both with and without ouabain. Bepridil had effects very similar to those of verapamil. Amiloride and excess Mg2+ reduced the O-Na contraction and the degree of their inhibition was similar after ouabain treatment. The decrease in verapamil susceptibility could suggest that the O-Na contraction has verapamil-sensitive and -insensitive components. The former is probably due to Ca2+ influx through voltage-dependent channels and the latter to Ca2+ influx through an Na(+)-Ca2+ exchange process. Ouabain is considered to increase the contribution of Na(+)-Ca2+ exchange to the O-Na contraction. Mg2+ may inhibit both verapamil-sensitive and -insensitive pathways. Amiloride probably exerts its inhibitory effect on the contractile machinery.