pHe, [Ca2+]e, and cell death during metabolic inhibition: role of phospholipase A2 and sarcolemmal phospholipids

Abstract

This study measures cellular lactate dehydrogenase (LDH) release during metabolic inhibition as a monitor of sarcolemmal integrity as affected by variation of external pH (pHe) and Ca2+ concentration ([Ca2+]e). The sigmoidal relationship between pHe and LDH release and pHe and net Ca2+ uptake was essentially identical with the 50% maximal value occurring at pH 7.0 for both. This suggests that a process(es) sensitive to both pHe and [Ca2+]eplays a role in cell lysis during the course of metabolic inhibition. Variation of pHe during metabolic inhibition did not alter the decline in cellular ATP, nor did it affect changes in sarcolemmal phospholipid topology. Intracellular pH followed changes of pHe with a few minutes lag. Cell lysis increased in a graded manner as pHe and [Ca2+]ewere increased, but pHe was the sole determinant of lysis, i.e., [Ca2+]elevel had no effect, at the lowest (6.2) and the highest (8.0) pHe levels. pHe variation did not affect the release of radiolabeled arachidonic acid, nor did inhibitors of phospholipase A2(PLA2) affect cell lysis at varying pHe. Therefore, cellular PLA2 activation could not be implicated for a role in cell lysis in the present model of metabolic inhibition. Alternatively, we propose that Ca2+ binding to the cytoplasmic leaflet, in combination with membrane alterations secondary to the metabolic insult, combine to destabilize the sarcolemma (20). This Ca2+ binding to the negatively charged phosphatidylserine results in the expression of the bilayer destabilizing effect of phosphatidylethanolamine. This Ca2+ binding is greatly diminished by lowered pH, resulting in an attenuation of cell lysis.