The calcium channel agonist Bay K 8644 promotes the growth of human liver cancer HepG2 cells in vitro: suppression with overexpressed regucalcin.

Hepatocellular carcinoma is one of the most prevalent malignant diseases and causes a third of cancer-related death. The consequences of altered calcium homeostasis in cancer cells may contribute to tumor progression. Regucalcin plays an inhibitory role in calcium signaling linked to transcription regulation. Regucalcin gene expression is downregulated in the tumor tissues of liver cancer patients, suggesting an involvement as a suppressor in hepatocarcinogenesis. We investigated whether Bay K 8644, an agonist of the L-type Ca2+ channel, promotes the growth of human liver cancer and if the effect of Bay K 8644 is suppressed by overexpressed regucalcin using the HepG2 cell model. The colony formation and growth of HepG2 cells were promoted by culturing with Bay K 8644 (0.1-10 nM). This effect was suppressed by inhibitors of signaling processes linked to cell proliferation, including PD98059 and wortmannin. Death of HepG2 cells was stimulated by Bay K 8644 with higher concentrations (25 and 100 nM). The effects of Bay K 8644 on cell growth and death were abolished by verapamil, an antagonist of calcium channel. Mechanistically, culturing with Bay K 8644 increased levels of mitogen-activated protein kinase (MAPK) and phospho-MAPK. Notably, overexpressed regucalcin suppressed Bay K 8644-promoted growth and death of HepG2 cells. Furthermore, overexpressed regucalcin prevented growth and increased death induced by thapsigargin, which induces the release of intracellular stored calcium. Thus, higher regucalcin expression suppresses calcium signaling linked to the growth of liver cancer cells, providing a novel strategy in treatment of hepatocellular carcinoma with delivery of the regucalcin gene.

Anti-arrhythmic effect of verapamil is accompanied by preservation of cx43 protein in rat heart.

The present study was to test the hypothesis that anti-arrhythmic properties of verapamil may be accompanied by preserving connexin43 (Cx43) protein via calcium influx inhibition. In an in vivo study, myocardial ischemic arrhythmia was induced by occlusion of the left anterior descending (LAD) coronary artery for 45 min in Sprague-Dawley rats. Verapamil, a calcium channel antagonist, was injected i.v. into a femoral vein prior to ischemia. Effects of verapamil on arrhythmias induced by Bay K8644 (a calcium channel agonist) were also determined. In an ex vivo study, the isolated heart underwent an initial 10 min of baseline normal perfusion and was subjected to high calcium perfusion in the absence or presence of verapamil. Cardiac arrhythmia was measured by electrocardiogram (ECG) and Cx43 protein was determined by immunohistochemistry and western blotting. Administration of verapamil prior to myocardial ischemia significantly reduced the incidence of ventricular arrhythmias and total arrhythmia scores, with the reductions in heat rate, mean arterial pressure and left ventricular systolic pressure. Verapamil also inhibited arrhythmias induced by Bay K8644 and high calcium perfusion. Effect of verapamil on ischemic arrhythmia scores was abolished by heptanol, a Cx43 protein uncoupler and Gap 26, a Cx43 channels inhibitor. Immunohistochemistry data showed that ischemia-induced redistribution and reduced immunostaining of Cx43 were prevented by verapamil. In addition, diminished expression of Cx43 protein determined by western blotting was observed following myocardial ischemia in vivo or following high calcium perfusion ex vivo and was preserved after verapamil administration. Our data suggest that verapamil may confer an anti-arrhythmic effect via calcium influx inhibition, inhibition of oxygen consumption and accompanied by preservation of Cx43 protein.

Investigation of mechanisms involved in (-)-borneol-induced vasorelaxant response on rat thoracic aorta.

The monoterpene (-)-borneol is present in essential oils of several medicinal plants. The aim of this study was to evaluate (-)-borneol effects on rat thoracic aorta artery rings. The cumulative addition of (-)-borneol (10(-9) -3 × 10(-4) M) on a phenylephrine-induced pre-contraction (10(-6) M) promoted a vasorelaxant effect in a concentration-dependent manner and independent of vascular endothelium. A similar effect was obtained on KCl-induced pre-contractions (80 mM). (-)-Borneol (10(-5) -3 × 10(-4 ) M) inhibited contractions induced by cumulative addition of CaCl2 (10(-6) -3 × 10(-2) M) in depolarizing medium without Ca(2+) in a concentration-dependent manner. On S-(-) Bay K 8644-induced pre-contractions (10(-7) M), (-)-borneol did not induce significant changes compared with KCl-induced pre-contractions. In a Ca(2+) -free medium, (-)-borneol (10(-5) , 10(-4) or 10(-3) M) interfered in calcium mobilization from phenylephrine (10(-6) M)- or caffeine (20 mM)-sensitive intracellular stores. The involvement of K(+) channels was evaluated by tetraethylammonium (3 mM), 4-aminopyridine (1 mM) and glibenclamide (10(-5) M) pre-treatment, and (-)-borneol-induced vasorelaxation was markedly attenuated. Thus, this vasorelaxant effect can probably be attributed to calcium influx blockade through voltage-operated calcium channels (CaV L), calcium mobilization from intracellular stores and potassium channels activation.

Cellular basis for the electrocardiographic and arrhythmic manifestations of Timothy syndrome: effects of ranolazine.

BACKGROUND: Timothy syndrome is a multisystem disorder associated with QT interval prolongation and ventricular cardiac arrhythmias. The syndrome has been linked to mutations in Ca(V)1.2 resulting in gain of function of the L-type calcium current (I(Ca,L)). Ranolazine is an antianginal agent shown to exert an antiarrhythmic effect in experimental models of long QT syndrome. OBJECTIVE: The purpose of this study was to develop and characterize an experimental model of Timothy syndrome by using BayK8644 to mimic the gain of function of I(Ca,L) and to examine the effects of ranolazine. METHODS: Action potentials from epicardial and M regions and a pseudo-electrocardiogram (ECG) were simultaneously recorded from coronary-perfused left ventricular wedge preparations, before and after addition of BayK8644 (1 microM). RESULTS: BayK8644 preferentially prolonged action potential duration of the M cell, leading to prolongation of the QT interval and an increase in transmural dispersion of repolarization (from 44.3 +/- 7 ms to 86.5 +/- 25 ms). Stimulation at cycle lengths of 250-500 ms led to ST-T wave alternans due to alternation of the plateau voltage of the M cell action potential as well as development of delayed afterdepolarizations in epicardial and M cell action potentials. Ventricular extrasystoles and tachycardia (monomorphic, bidirectional, or torsades de pointes) developed spontaneously or after rapid pacing. Peak and late I(Na) were unaffected by BayK8644. Clinically relevant concentrations of ranolazine (10 microM) suppressed all actions of BayK8644. CONCLUSION: A left ventricular wedge model of long QT syndrome created by augmentation of I(Ca,L) recapitulates the ECG and arrhythmic manifestations of Timothy syndrome, which can be suppressed by ranolazine.

Isolated perfused and paced guinea pig heart to test for drug-induced changes of the QT interval.

INTRODUCTION: One of the biomarkers for assessing the risk of a cardiac adverse event is drug-induced prolongation of the QT interval. A model is needed for evaluating the potential liability of test compounds on QT interval in vitro. Since QT intervals can be generated from paced or spontaneously beating hearts, data so generated can also be used for validating QT(c) correction equations. METHODS: Isolated guinea pig hearts were perfused in Locke's solution according to the Langendorff method. QT intervals were routinely measured from Lead II ECG waveforms. RESULTS: Compounds known to inhibit HERG channel, such as dofetilide, prolonged the QT interval in this model. (+/-)Bay K8644, a calcium channel activator, prolonged the QT interval, while verapamil, a calcium channel blocker, shortened it. Procainamide, a sodium channel blocker, also prolonged the QT interval. Many of the compounds, which prolonged the QT interval, also prolonged PR interval, suggesting dual inhibition of the Ikr channel, the rapid component of delayed rectifier potassium channel, and the calcium channel. The QT/RR intervals exhibited a curvilinear relationship, which could be corrected into nearly straight horizontal lines by using correction equations derived from linear, parabolic, and hyperbolic models. However, these correction equations yielded different results on the QT prolongation produced by sotalol, which also slowed down the heart rate. With the data set obtained in this investigation, correction equations derived from linear and parabolic models worked better than the equations derived from the hyperbolic model. The exponential model did not fit at all. CONCLUSION: QT intervals obtained under paced conditions provide the most direct and reliable QT information for a drug. The isolated perfused and paced guinea pig heart is a convenient model for studying the effect of compounds on QT interval in vitro.

Self-biting induced by activation of L-type calcium channels in mice: dopaminergic influences.

The L-type calcium channel activator +/-Bay K 8644 induces repetitive self-biting and self-injurious behavior in young mice. Since dopaminergic systems have been implicated in prior studies of these behaviors in both humans and animals, the present experiments were designed to test whether drugs influencing the dopaminergic systems could modify the behavioral responses to +/-Bay K 8644. The ability of +/-Bay K 8644 to provoke self-biting and self-injurious behavior was increased by amphetamine and GBR 12909, drugs that augment synaptic dopaminergic concentrations by blocking the reuptake and/or stimulating the release of dopamine. Conversely, self-biting and self-injurious behavior were decreased by tetrabenazine or reserpine, two drugs that deplete vesicular stores of dopamine. These results suggest that dopaminergic systems may play a role in the ability of +/-Bay K 8644 to provoke self-biting and self-injurious behavior.

Calcium channel activation and self-biting in mice.

The L type calcium channel agonist (+/-)Bay K 8644 has been reported to cause characteristic motor abnormalities in adult mice. The current study shows that administration of this drug can also cause the unusual phenomenon of self-injurious biting, particularly when given to young mice. Self-biting is provoked by injecting small quantities of (+/-)Bay K 8644 directly into the lateral ventricle of the brain, suggesting a central effect of the drug. Similar behaviors can be provoked by administration of another L type calcium channel agonist, FPL 64176. The self-biting provoked by (+/-)Bay K 8644 can be inhibited by pretreating the mice with dihydropyridine L type calcium channel antagonists such as nifedipine, nimodipine, or nitrendipine. However, self-biting is not inhibited by nondihydropyridine antagonists including diltiazem, flunarizine, or verapamil. The known actions of (+/-)Bay K 8644 as an L type calcium channel agonist, the reproduction of similar behavior with another L type calcium channel agonist, and the protection afforded by certain L type calcium channel antagonists implicate calcium channels in the mediation of the self-biting behavior. This phenomenon provides a model for studying the neurobiology of this unusual behavior.

Canine left ventricular hypertrophy predisposes to ventricular tachycardia induction by phase 2 early afterdepolarizations after administration of BAY K 8644.

OBJECTIVES: The purpose of this study was to test the hypothesis that the longer duration of ventricular action potentials in hypertrophied hearts predisposes to the development of early after-depolarizations and triggered ventricular tachyarrhythmias. BACKGROUND: For unknown reasons, the incidence of sudden death is greater in patients with myocardial hypertrophy. METHODS: We measured left ventricular monophasic action potentials in normal dogs and dogs with left ventricular hypertrophy before and after administration of the calcium agonist BAY K 8644 and the potassium channel blocker cesium. RESULTS: We demonstrated longer action potential durations in dogs with than in those without left ventricular hypertrophy. Also, BAY K 8644 produced phase 2 early afterdepolarizations and ventricular tachyarrhythmias more frequently in the dogs with than in those without left ventricular hypertrophy. Phenylephrine, an alpha agonist, further increased the action potential duration in hypertrophied hearts and the propensity to develop early afterdepolarizations and ventricular tachyarrhythmia after administration of BAY K 8644. Control and hypertrophied hearts developed early afterdepolarizations and ventricular tachyarrhythmia equally when exposed to cesium. CONCLUSIONS: Although in vitro studies have shown that fibers of hypertrophied ventricular myocardium can develop triggered activity as a result of both early and late afterdepolarizations, the present study is the first to show in vivo that the hypertrophied ventricular myocardium compared with the normal ventricle is predisposed to develop phase 2 early afterdepolarizations that appear to trigger ventricular tachyarrhythmia. It is possible that such a mechanism contributes to the development of ventricular tachyarrhythmia and sudden cardiac death in patients with left ventricular hypertrophy. If this is shown to be true, specific pharmacologic interventions can be suggested.