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

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Abstract
Objective: Several studies have shown that myocardial ischemia leads to functional failure of endothelial cells (EC) whereby disturbance of Ca2+ homeostasis may play an important role. The mechanisms leading to Ca2+ disbalance in ischemic EC are not fully understood. The aim of this study was to test effects of different components of simulated ischemia (glucose deprivation, anoxia, low extracellular pH (pHo) and lactate) on Ca2+ homeostasis in EC. Methods: Cytosolic Ca2+ (Cai), cytosolic pH (pHi) and ATP content were measured in cultured rat coronary EC. Results: In normoxic cells 60 min glucose deprivation at pHo 7.4 had no effect on pHi. It only slightly increased Cai and decreased ATP content. Reduction of pHo to 6.5 under these conditions led to marked cytosolic acidosis and Cai overload, but had no effect on ATP content. Anoxia at pHo 6.5 had no additional effect on Cai overload, but significantly reduced cellular ATP. Addition of 20 mmol/l lactate to anoxia at pHo 6.5 accelerated Cai overload due to faster cytosolic acidification. Acidosis-induced Cai overload was prevented by inhibition of Ca2+ release channels of endoplasmic reticulum (ER) with 3 μmol/l ryanodine or by pre-emptying the ER with thapsigargin. Re-normalisation of pHo for 30 min led to recovery of pHi, but not of Cai. Conclusion: The ischemic factors leading to cytosolic acidosis (low pHo and lactate) cause Cai overload in endothelial cells, while anoxia and glucose deprivation play only a minor role. The ER is the main source for this Cai rise. Cai overload is not readily reversible.

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