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In this study we have characterized the bumetanide-sensitive K+/Na+/Cl- cotransport in cultured rat cardiac myocytes. 1) It carries about 10% of the total K+ influx. 2) It is sensitive to furosemide (Ki0.5 = 10(-6)M) and bumetanide (Ki0.5 = 10(-7)M). 3) It is strongly dependent on the extracellular concentrations of Na+ and Cl-. 4) It carries out influx of both ions, K+ and Na+. A therapeutic concentration of ouabain (10(-7) M) stimulated the bumetanide-sensitive K+ influx (as measured by 86Rb+), in the cultured myocytes, with no effect on the bumetanide-resistant K+ influx, which was mediated mostly by the Na+/K+ pump. Stimulation of the bumetanide-sensitive Rb+ influx by a low ouabain concentration was strongly dependent on Na+ and Cl- in the extracellular medium. A low concentration of ouabain (10(-7) M) was found to increase the steady-state level of cytosolic Na+ by 15%. This increase was abolished by the addition of bumetanide or furosemide. These findings suggest that ouabain, at a low (10(-7) M) concentration, induced its positive inotropic effect in rat cardiac myocytes by increasing Na+ influx into the cells through the bumetanide-sensitive Na+/K+/Cl- cotransporter. In order to examine this hypothesis, we measured the effect of bumetanide on the increased amplitude of systolic cell motion induced by ouabain. Bumetanide or furosemide, added to cultured cardiac myocytes, inhibited the increased amplitude of systolic cell motion induced by ouabain. Neither bumetanide nor furosemide alone has any significant effect on the basal amplitude of systolic cell motion. We propose that stimulation of bumetanide-sensitive Na+ influx plays an essential role in the positive inotropic effect in rat cardiac myocytes induced by low concentration of ouabain.

The "lethal hit" induced by viral specific, sensitized, cytotoxic T lymphocytes (CTL) attacking virus-infected heart cells is important in the pathogenesis of viral myocarditis and reflects the key role of CTL in this immune response. The mechanisms involved are incompletely understood. Studies of the physiological changes induced in mengovirus-infected, cultured, neonatal, rat heart cells by CTL that had been previously sensitized by the same virus are presented. The CTL were obtained from spleens of mengovirus-infected, major histocompatibility complex (MHC) matched adult rats. Cell wall motion was measured by an optical method, action potentials with intracellular microelectrodes, and total exchangeable calcium content by 45Ca tracer measurements after loading the myocytes with 45Ca and then exposing them to CTL. After 50 min (mean time) of exposing mengovirus-infected myocytes to the CTL, the mechanical relaxation of the myocyte was slowed, with a subsequent slowing of beating rate and a reduced amplitude of contraction. Impaired relaxation progressed, and prolonged oscillatory contractions lasting up to several seconds appeared, with accompanying oscillations in the prolonged plateau phase of the action potentials. Arrest of the myocyte contractions appeared 98 min (mean time) after exposure to CTL. These changes in action potentials and contractions were reversible either by washout with the normal medium or by the addition of verapamil. The amount of total exchangeable calcium in the cultured myocytes, 1 h after exposure to CTL, was significantly increased. This increase was prevented by pretreatment with verapamil. (ABSTRACT TRUNCATED AT 250 WORDS)