Cellular mechanisms underlying calcium-proton interactions in cultured chick ventricular cells.
1. Cytosolic free Ca2+ concentration ([Ca2+]i) in cardiac muscle cells is influenced by many factors including intracellular pH. Intracellular alkalinization has been shown to reduce, whereas acidification has been shown to augment [Ca2+]i. We examined the cellular mechanisms underlying Ca2+-H+ interactions using cultured chick embryo ventricular cells. 2. Cells were loaded with fura-2 or BCECF (2,7-biscarboxyethyl-5(6)-carboxy-fluorescein) and changes in time-averaged [Ca2+]i or pHi were monitored continuously using a dual-wavelength spectrofluorometer. 3. Exposure of cells to 20 mM-NH4Cl (intracellular alkalinization) produced a rapid decrease in [Ca2+]i; subsequent wash-out of NH4Cl (intracellular acidification) resulted in an increase in [Ca2+]i to levels above control. Intracellular acidification produced by elevated CO2 content also resulted in an increase in [Ca2+]i. The Na+-H+ exchange inhibitor ethylisopropylamiloride (10 microM) inhibited completely the rise but not the fall in [Ca2+]i in response to manipulation of pHi with NH4Cl. 4. In the presence of caffeine (20 mM), NH4Cl produced a decrease in [Ca2+]i similar to that observed in the absence of caffeine, but subsequent removal of NH4Cl resulted in an increase in [Ca2+]i that was distinctly smaller than that observed in the absence of caffeine. Ryanodine (10 microM) had no significant influence on NH4Cl-induced changes in [Ca2+]i. 5. Following treatment with the mitochondrial inhibitors sodium cyanide (5 mM), CCCP (carbonyl cyanide m-chlorophenyl hydrazone, 10 microM) or rotenone (10 microM), the NH4Cl-induced decrease in [Ca2+]i was markedly diminished, but wash-out of NH4Cl resulted in increases in [Ca2+]i similar to those observed in control cells. 6. Inhibition of glycolysis with 20 mM-2-deoxyglucose did not significantly alter the changes in [Ca2+]i induced by NH4Cl addition or its wash-out, but 2-deoxyglucose plus cyanide abolished the decrease in [Ca2+]i produced by intracellular alkalinization and nearly completely blocked the increase in [Ca2+]i produced by acidification. 7. In all experiments, the increase in [Ca2+]i during wash-out of NH4Cl was inhibited by ethylisopropylamiloride.(ABSTRACT TRUNCATED AT 400 WORDS)
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Cardiovascular Division, Brigham and Women's Hospital, Boston, MA.
Smith
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D000643:Ammonium Chloride; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D002642:Chick Embryo; D006352:Heart Ventricles; D006863:Hydrogen-Ion Concentration; D009206:Myocardium / Q000378:metabolism*; D011522:Protons
D011522:Protons; D000643:Ammonium Chloride; D002118:Calcium