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-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.
Eskildsen-Helmond
Y E
YE
Department of Biochemistry, Faculty of Medicine and Health Sciences, Erasmus University Rotterdam, The Netherlands.
Hahnel
D
D
Reinhardt
U
U
Dekkers
D H
DH
Engelmann
B
B
Lamers
J M
JM
D000316:Adrenergic alpha-Agonists / Q000494:pharmacology*; D000818:Animals; D002478:Cells, Cultured; D004075:Diglycerides / Q000737:chemistry* / Q000378:metabolism; D019332:Endothelin-1 / Q000494:pharmacology; D004789:Enzyme Activation; D007527:Isoenzymes / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism*; D010656:Phenylephrine / Q000494:pharmacology; D010703:Phorbol Esters / Q000494:pharmacology; D010713:Phosphatidylcholines / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D013268:Stimulation, Chemical
C011439:1,2-diacylglycerol; D000316:Adrenergic alpha-Agonists; D004075:Diglycerides; D019332:Endothelin-1; D007527:Isoenzymes; D010703:Phorbol Esters; D010713:Phosphatidylcholines; D010716:Phosphatidylinositols; D010743:Phospholipids; D010656:Phenylephrine; D011493:Protein Kinase C; D010739:Phospholipase D