© 1998 by European Society of Cardiology
Copyright © 1998, European Society of Cardiology
Phospholipid source and molecular species composition of 1,2-diacylglycerol in agonist-stimulated rat cardiomyocytes
aDepartment of Biochemistry, Cardiovascular Research Institute COEUR, Faculty of Medicine and Health Sciences, Erasmus University Rotterdam, P.O.Box 1738, 3000 DR Rotterdam, The Netherlands
bPhysiologisches Institut der Universität München, Pettenkoferstrasse 12, 80336 München, Germany
* Corresponding author. Tel: +31 (10) 408 7335; Fax: +31 (10) 436 0615; E-mail: lamers@bc1.fgg.eur.nl
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-1, 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 [14C]glycerol or double-labelled with [14C]16:0 and [3H]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-1 or phorbolester, but not phenylephrine. The results of the double-labelling experiments were consistent with the latter finding: the ratio [3H]20:4 over [14C]16:0 in 1,2-diacylglycerol decreased from 1.70 in the control to 1.40 during 10-min endothelin-1 or phorbolester stimulation, but not during phenylephrine stimulation. The [14C]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-1 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.
KEYWORDS ET-1, endothelin-1; PMA, phorbol 12-myristate 13-acetate; PHE, phenylephrine; AngII, angiotensin II; PtdIns(4,5)P2, phosphatidylinositol-4,5-bisphosphate; Ins(1,4,5)P3, inositol-1,4,5-trisphosphate; 1,2-DAG, 1,2-diacylglycerol; TAG, triacylglycerol; PKC, protein kinase C; PLC, PLD and PLA2, respectively phospholipase C, D and A2; PtdChol, PtdIns, PtdEtn, PtdSer, respectively phosphatidylcholine, -inositol, -ethanolamine, -serine; (HP)TLC, (High-performance) thin-layer chromatography; HPLC and GLC, respectively, high-performance and gas–liquid chromatography; DPH, diphenyl-1,3,5 hexatrien; DNBC, 3,5-dinitrobenzoylchloride; stearic acid, 18:0; arachidonic acid, 20:4n6; palmitic acid, 16:0.
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