Phospholipid degradation in hypoxic/reoxygenated cardiomyocytes in response to phospholipase C from Bacillus cereus

Abstract

In the present study, we investigated possible mechanisms behind exogenous phospholipase C-induced glycerol production in irreversibly damaged myocytes. Rat ventricular myocytes were preincubated for 60 min in substrate-free Krebs-Henseleit bicarbonate buffer equilibrated with 95% N 2-5% CO 2 (37°C, pH = 7.4), resulting in exhaustion of cellular high energy phosphates and loss of rod-shaped morphology. At the end of the preincubation period, the incubation vials were divided into two groups; one receiving 10 mU/ml phospholipase C (PC-PLC), whereas the other received an equivalent volume of buffer (control incubations). Incubation was then continued for another 60 min under 95% air-5% CO 2 atmosphere. Samples for measurement of metabolite levels were taken immediately after cell isolation, at the end of the preincubation period and at the end of the normoxic incubation period. During the 60 min incubation period following reoxygenation, glycerol output was markedly higher from PC-PLC treated than from control myocytes. However, the elevated glycerol output from these cells was not accompanied by a simultaneous rise in glycerol-3-phosphate, nor was it inhibited by inclusion of pyruvate in the incubation buffer. On the other hand, glycerol output from PC-PLC treated myocytes was effectively inhibited by a diacylglycerol lipase inhibitor (U-57908, The Upjohn Company). Analysis of cellular lipids revealed a 22% reduction of phospholipid in PC-PLC treated myocytes ( P<0.02), while the content of triacylglycerol, diacylglycerol and unesterified fatty acids increased by 76, 261 and 103%, respectively ( P<0.02). No significant changes were observed for these parameters in control myocytes. It is concluded that output of glycerol from hypoxic-reoxygenated myocytes in response to exogenous PC-PLC is caused entirely by the combined action of PC-PLC and intracellular di- and monoacylglycerol lipase. The results demonstrate that the triacylglycerol depot may serve an important function to scavenge fatty acids liberated durting phospholipid hydrolysis. Finally, the relatively high production of diacylglycerol could make this model an important tool for studies of intracellular signal transduction.