Phospholipid source and molecular species composition of 1,2-diacylglycerol in agonist-stimulated rat cardiomyocytes

Objective: The aim was to investigate the consequences of simultaneous stimulation of phospholipase C and D by agonists for the molecular species composition of 1,2-diacylglycerol and phospholipids in cardiomyocytes. Methods: Serum-free cultured neonatal rat cardiomyocytes were stimulated by endothelin-l, phenylephrine or phorbolester. The molecular species of 1,2-diacylglycerol (in mol%) and those derived from phosphatidylcholine and phosphatidylinositol were analyzed by high-performance liquid chromatography and their absolute total concentration (nmol per dish) by gas-liquid chromatography. Phospholipids were labelled with [C-14]glycerol or double-labelled with [C-14]16:0 and [H-3]20:4n6 for measurements of respectively, the amount of or relative rate of label incorporation into 1,2-diacylglycerol. Results: The major molecular species of 1,2-diacylglycerol in unstimulated cells was found to be 18:0/20:4 (57 mol%). The same species was observed predominantly in phosphatidylinositol (73 mol% compared to 11 mol% in phosphatidylcholine). A significant decrease (about 10 mol%) was found for the 18:0/20:4 species of 1,2-diacylglycerol during stimulation (10-40 min) with endothelin-l or phorbolester, but not phenylephrine. The results of the double-labelling experiments were consistent with the latter finding: the ratio [H-3]20:4 over [C-14]16:0 in 1,2-diacylglycerol decreased from 1.70 in the control to 1.40 during 10-min endothelin-l or phorbolester stimulation, but not during phenylephrine stimulation. The [C-14]glycerol incorporation into 1,2-diacylglycerol remained relatively constant under agonist-stimulated conditions as did the total concentration of 1,2-diacylglycerol. Conclusions: 1,2-Diacylglycerol present in unstimulated cardiomyocytes is likely derived from phosphatidylinositol. During stimulation with endothelin-l and phorbolester, but not phenylephrine, phosphatidylcholine becomes an increasingly important source for 1,2-diacylglycerol due to sustained activation of phospholipase D. The 1,2-diacylglycerol level remains relatively constant during agonist stimulation which strongly indicates that particular molecular species of 1,2-diacylglycerol more than its total concentration determine the activation of protein kinase C isoenzymes. (C) 1998 Elsevier Science B.V. All rights reserved.