Mechanisms of reoxygenation-induced calcium overload in cardiac myocytes: dependence on pHi.
This study investigated the selective effects of intracellular (pHi) or extracellular change in pH on reoxygenation-induced Ca2+ overload in simulated myocardial hypoxia. Experiments were performed in cultured cardiomyocytes isolated from the ventricle of neonatal ICR mouse. A model of chemical hypoxia with 2 mM sodium cyanide was developed to mimic the ATP depletion of hypoxia. This chemical hypoxia was "reoxygenated" and the dynamics in intracellular Ca2+ concentration ([Ca2+]i) and pHi were monitored using the fluorescent dyes fura-2 and 2', 7'-bis (2-carboxyethyl)-5(6)-carboxyfluorescein, respectively. During a 40-min chemical hypoxia, pHi progressively fell from 7.2 to 6.6. Reoxygenation with control solution caused rapid recovery of pHi and a marked increase in [Ca2+]i (1884 +/- 136 nM). Intracellular acidotic reoxygenation produced by lactate apparently prolonged the time course of pHi recovery and significantly suppressed reoxygenation-induced Ca2+ overload (1170 +/- 118 nM, P = 0.008). Extracellular acidotic reoxygenation with 2-(N-morpholino) ethanesulfonic acid (pK = 5.96) buffer somewhat suppressed the Ca2+ overload; however, the maximal value of [Ca2+]i was not reduced significantly compared with the control (1790 +/- 122 nM, P = 0.130). In addition, inhibition of Na(+)-H+ exchange by amiloride potentiated prolongation of intracellular acidosis during reoxygenation and resulted in a minimal increase in [Ca2+]i (985 +/- 102 nM, P = 0.004). These results suggest that reoxygenation-induced Ca2+ overload is closely correlated with intracellular pH in the initial phase of reoxygenation, and the protective effects of extracellular acidosis is principally mediated by intracellular acidification of reoxygenated cardiomyocytes.
Department of Surgery, Tottori University School of Medicine, Yonago, Japan.
N
N
Matsuda
T
T
Mori
H
H
Nakamura
M
M
Shigekawa
D000143:Acids / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D006863:Hydrogen-Ion Concentration; D000860:Hypoxia / Q000378:metabolism; D007773:Lactates / Q000494:pharmacology; D019344:Lactic Acid; D051379:Mice; D008813:Mice, Inbred ICR; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D010100:Oxygen / Q000494:pharmacology*
D000143:Acids; D007773:Lactates; D019344:Lactic Acid; D010100:Oxygen; D002118:Calcium