Mechanism of Ca2+ overload in endothelial cells exposed to simulated ischemia

Objective: Several studies have shown that myocardial ischemia leads to fun ctional failure of endothelial cells (EC) whereby disturbance of Ca2+ homeo stasis may play an important role. The mechanisms leading to Ca2+ disbalanc e in ischemic EC are not fully understood. The aim of this study was to tes t effects of different components of simulated ischemia (glucose deprivatio n, anoxia, low extracellular pH (pH(i)) and lactate) on Ca2+ homeostasis in EC. Methods: Cytosolic Ca2+ (Ca-i), cytosolic pH (pH(i)) and ATP content w ere measured in cultured rat coronary EC. Results: In normoxic cells 60 min glucose deprivation at pHo 7.4 had no effect on pH(1). It only slightly in creased Cai and decreased ATP content. Reduction of pH, to 6.5 under these conditions led to marked cytosolic acidosis and Ca-i overload, but had no e ffect on ATP content. Anoxia at pH, 6.5 had no additional effect on Ca-i ov erload, but significantly reduced cellular ATP. Addition of 20 mmol/l lacta te to anoxia at pH(o) 6.5 accelerated Ca-i overload due to faster cytosolic acidification. Acidosis-induced Ca-i overload was prevented by inhibition of Ca2+ release channels of endoplasmic reticulum (ER) with 3 mu mol/l ryan odine or by pre-emptying the ER with thapsigargin. Re-normalisation of pH, for 30 min led to recovery of pi-Ii, but not of Ca-i Conclusion: The ischem ic factors leading to cytosolic acidosis (low pH, and lactate) cause Ca-i o verload in endothelial cells, while anoxia and glucose deprivation play onl y a minor role. The ER is the main source for this Ca-i rise. Ca-i overload is not readily reversible. (C) 2000 Elsevier Science B.V. All rights reser ved.