Reoxygenation-induced Ca2+ rise is mediated via Ca2+ influx and Ca2+ release from the endoplasmic reticulum in cardiac endothelial cells.
OBJECTIVE
Conditions of ischemia-reperfusion disturb the homoeostasis of cytosolic Ca2+ in cardiac microvascular endothelial cells (CMEC), leading to numerous malfunctions of the endothelium. Reperfusion specifically aggravates the Ca2+ overload developed during sustained ischemia. The aim of this study was to identify the origin of the reperfusion-induced part of the Ca2+ overload. Our hypotheses were that this is either due to a Na+-dependent process, e.g. involving the Na+/H+ exchanger (NHE) and/or the Na+/Ca2+ exchanger (NCX), or a process involving the endoplasmic reticulum (ER) and store-operated channels (SOC).


RESULTS
Cultured CMEC from rats were exposed to conditions of simulated ischemia (hypoxia, pH 6.4) and reperfusion (reoxygenation, pH 7.4). Cytosolic Ca2+ ([Ca2+]i) and cytosolic Na+ ([Na+]i) concentrations and cytosolic pH (pHi) were measured with the use of fluorescent indicators. Removal of Ca2+ from the extracellular media during reoxygenation prevented the [Ca2+]i rise. Neither the activation of the NHE nor of the NCX in reoxygenated CMEC caused a change in this [Ca2+]i rise. Complete or partial removal of Na+ from the external media also had no effect on the [Ca2+]i rise. In contrast, specific inhibition of the inositol trisphosphate (InsP3) receptor by xestospongin C (3 micromol/l), of phospholipase (PLC) by U73122 (1 micromol/l), or of SOC by the inhibitors gadolinium (10 micromol/l) or 2-APB (50 micromol/l) lowered or abolished the reoxygenation-induced [Ca2+]i rise.


CONCLUSIONS
In CMEC exposed to reperfusion conditions, the enhanced Ca2+ overload is due to Ca2+ influx. The influx is not mediated by a Na+-dependent mechanism, but rather is due to activation of the InsP3 receptor of the ER and activation of SOC.
Peters
Saskia C
SC
Institute of Physiology, Justus-Liebig University Giessen, Aulweg 129, 35392 Giessen, Germany.
Piper
H Michael
HM
D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D015687:Cell Hypoxia; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D042783:Endothelial Cells / Q000378:metabolism; D006146:Guanidines / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007473:Ion Channels / Q000378:metabolism*; D008297:Male; D008833:Microcirculation; D015428:Myocardial Reperfusion Injury / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism*; D010042:Ouabain / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D012964:Sodium / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism; D013450:Sulfones / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology
D006146:Guanidines; D007473:Ion Channels; D017923:Sodium-Hydrogen Exchangers; D013450:Sulfones; D010042:Ouabain; D019284:Thapsigargin; C093373:cariporide; D012964:Sodium; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000254:Sodium-Potassium-Exchanging ATPase; D002118:Calcium