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 sp ecies composition of 1,2-diacylglycerol and phospholipids in cardiomyo cytes. Methods: Serum-free cultured neonatal rat cardiomyocytes were s timulated by endothelin-l, phenylephrine or phorbolester. The molecula r species of 1,2-diacylglycerol (in mol%) and those derived from phosp hatidylcholine and phosphatidylinositol were analyzed by high-performa nce liquid chromatography and their absolute total concentration (nmol per dish) by gas-liquid chromatography. Phospholipids were labelled w ith [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 la bel incorporation into 1,2-diacylglycerol. Results: The major molecula r 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 ph osphatidylinositol (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 endo thelin-l or phorbolester, but not phenylephrine. The results of the do uble-labelling experiments were consistent with the latter finding: th e 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 phorboleste r stimulation, but not during phenylephrine stimulation. The [C-14]gly cerol incorporation into 1,2-diacylglycerol remained relatively consta nt under agonist-stimulated conditions as did the total concentration of 1,2-diacylglycerol. Conclusions: 1,2-Diacylglycerol present in unst imulated cardiomyocytes is likely derived from phosphatidylinositol. D uring stimulation with endothelin-l and phorbolester, but not phenylep hrine, phosphatidylcholine becomes an increasingly important source fo r 1,2-diacylglycerol due to sustained activation of phospholipase D. T he 1,2-diacylglycerol level remains relatively constant during agonist stimulation which strongly indicates that particular molecular specie s of 1,2-diacylglycerol more than its total concentration determine th e activation of protein kinase C isoenzymes. (C) 1998 Elsevier Science B.V. All rights reserved.