The effect of KB-R7943, an inhibitor of the plasmalemmal Na + -Ca 2+ exchanger, on mitochondrial Ca 2+ transporters was examined with membrane-permeabilized cardiomyocyte-derived H9c2 cells expressing the fluorescent Ca 2+ indicator, yellow cameleon 3.1, in the mitochondria. KB-R7943, as well as ruthenium red, inhibited the rise in mitochondrial Ca 2+ on increasing the extramitochondrial Ca 2+ concentration from 0 nM to 300 nM. CGP37157, but not KB-R7943, inhibited the decline in mitochondrial Ca 2+ on return to Ca 2+ free extramitochondrial solution. These results indicated that KB-R7943 has inhibitory effects on the mitochondrial Ca 2+ uniporter, but not on the mitochondrial Na + -Ca 2+ exchanger.
Intracellular Ca2+-mediated mechanisms for pacemaker depolarization were studied in sinus node tissue preparations from mice and guinea pigs. Microelectrode recordings revealed that the sinus node of the mouse, which had a higher beating rate, had a steeper slope of the pacemaker depolarization than that of the guinea pig. BAPTA and ryanodine, agents that interfere with intracellular Ca2+, significantly decreased the slope of the pacemaker depolarization in both species. In contrast, SEA0400, a specific inhibitor of the Na+-Ca2+ exchanger (NCX), as well as change to low Na+ extracellular solution, significantly decreased the slope in the mouse, but not in the guinea pig. Niflumic acid, a blocker of the Ca2+ activated Cl− channel, decreased the slope in both species. Confocal microscopy revealed the presence of spontaneous Ca2+ oscillations during the interval between Ca2+ transients; such phenomenon was more pronounced in the mouse than in the guinea pig. Thus, although intracellular Ca2+-mediated mechanisms were involved in the pacemaker depolarization of the sinus node in both species, the NCX current was involved in the mouse but not in the guinea pig.
The effect of blocking the persistent component of the sodium channel current (late I Na ) on the automatic activity of the isolated guinea pig pulmonary vein myocardium was examined. NCC-3902 blocked late I Na , but did not affect other major ion channel currents stably expressed in cell lines. In isolated pulmonary vein cardiomyocytes, NCC-3902 blocked the late I Na induced by a ramp depolarizing voltage clamp pulse similar to that of the pacemaker depolarization observed in the pulmonary vein myocardium. In isolated pulmonary vein tissue, NCC-3902 decreased the frequency of automatic firing of the myocardium through a reduction of the pacemaker depolarization slope. In isolated pulmonary vein cardiomyocytes, NCC-3902 significantly reduced the firing frequency of Ca 2 transients, but had no effect on Ca 2 sparks. NCC-3902 affected neither the spontaneous beating rate of the right atrium nor the contractile force of the ventricular myocardium. Selective blockers of late I Na like NCC-3902, which inhibit the automatic activity of the pulmonary vein myocardium, appear to be promising as drugs for the pharmacological treatment of atrial fibrillation.
We developed a method to evaluate the activity of the Na + /Ca 2+ exchanger (NCX) and sarco-endoplasmic reticulum Ca 2+ -ATPase (SERCA) with fluorescence microscopy in mouse ventricular cardiomyocytes. In non-beating ventricular cardiomyocytes, -adrenoceptor stimulation by phenylephrine caused a decrease in the cytoplasmic Ca 2+ concentration, which was inhibited by SEA0400, an NCX inhibitor, but not cyclopiazonic acid, a SERCA inhibitor. β-Adrenoceptor stimulation by isoprenaline caused a decrease in the cytoplasmic Ca 2+ concentration, which was inhibited by cyclopiazonic acid but not SEA0400. Ellagic acid, a phenolic phytochemical, also decreased the basal Ca 2+ concentration, which was inhibited by cyclopiazonic acid, but not SEA0400. Thus, this method using fluorescent microscopy and specific inhibitors would be useful for the evaluation of pharmacological agents acting on NCX and SERCA.
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