Pravdić Danijel, Vladić Nikolina, Bosnjak Zeljko Josip
Department of Physiology, School of Medicine, University of Mostar, Mostar, Bosnia and Herzegovina.
Coll Antropol. 2009 Dec;33 Suppl 2(0 2):121-6.
We investigated the effects of different ischemia-mimetic factors on intracellular Ca2+ concentration ([Ca2+]i). Ventricular myocytes were isolated from adult Wistar rats, and [Ca2+]i was measured using fluorescent indicator fluo-4 AM by confocal microscopy. Intracellular pH was measured using c5-(and-6)-carboxy SNARF-1 AM, a dual emission pH-sensitive ionophore. Myocytes were exposed to hypoxia, extracellular acidosis (pH(o) 6.8), Na-lactate (10 mM), or to combination of those factors for 25 min. Monitoring of [Ca2+]i using fluo-4 AM fluorescent indicator revealed that [Ca2+]i accumulation increased immediately after exposing the cells to Na-lactate and extracellular acidosis, but not during cell exposure to moderate ischemia. Increase in [Ca2+]i during Na-lactate exposure decreased to control levels at the end of exposure period at extracellular pH 7.4, but not at pH 6.8. When combined, Na-lactate and acidosis had an additive effect on [Ca2+]i increase. After removal of solutions, [Ca2+]i continued to rise only when acidosis, hypoxia, and Na-lactate were applied together. Analysis of intracellular pH revealed that treatment of cells by Na-lactate and acidosis caused intracellular acidification, while short ischemia did not significantly change intracellular pH. Our experiments suggest that increase in [Ca2+]i during short hypoxia does not occur if pH(i) does not fall, while extracellular acidosis is required for sustained rise in [Ca2+]i induced by Na-lactate. Comparing to the effect of Na-lactate, extracellular acidosis induced slower [Ca2+]i elevation, accompanied with slower decrease in intracellular pH. These multiple effects of hypoxia, extracellular acidosis, and Na-lactate are likely to cause [Ca2+]i accumulation after the hypoxic stress.
我们研究了不同缺血模拟因素对细胞内钙离子浓度（[Ca2+]i）的影响。从成年Wistar大鼠分离出心室肌细胞，使用荧光指示剂fluo-4 AM通过共聚焦显微镜测量[Ca2+]i。使用c5-（和-6）-羧基SNARF-1 AM（一种双发射pH敏感离子载体）测量细胞内pH。将肌细胞暴露于缺氧、细胞外酸中毒（pH(o) 6.8）、乳酸钠（10 mM）或这些因素的组合中25分钟。使用fluo-4 AM荧光指示剂监测[Ca2+]i发现，将细胞暴露于乳酸钠和细胞外酸中毒后，[Ca2+]i积累立即增加，但在细胞暴露于中度缺血期间未增加。在细胞外pH 7.4时，乳酸钠暴露期间[Ca2+]i的增加在暴露期结束时降至对照水平，但在pH 6.8时未降至对照水平。当乳酸钠和酸中毒联合使用时，对[Ca2+]i增加具有相加作用。去除溶液后，仅当酸中毒、缺氧和乳酸钠一起应用时，[Ca2+]i才继续升高。细胞内pH分析显示，乳酸钠和酸中毒处理细胞导致细胞内酸化，而短暂缺血并未显著改变细胞内pH。我们的实验表明，如果细胞内pH不下降，短暂缺氧期间[Ca2+]i不会增加，而细胞外酸中毒是乳酸钠诱导的[Ca2+]i持续升高所必需的。与乳酸钠的作用相比，细胞外酸中毒诱导的[Ca2+]i升高较慢，同时细胞内pH下降也较慢。缺氧、细胞外酸中毒和乳酸钠的这些多种作用可能导致缺氧应激后[Ca2+]i积累。
