Angiotensin II-induced phosphatidylcholine hydrolysis in cultured vascular smooth-muscle cells. Regulation and localization.
In cultured vascular smooth-muscle cells (VSMC), angiotensin II (AngII) induces a biphasic, sustained increase in diacylglycerol (DG) of unclear origin. To determine whether hydrolysis of phosphatidylcholine (PC) is a possible source of DG, we labelled cellular PC with [3H]choline, and measured the formation of intra- and extra-cellular [3H]choline and [3H]phosphocholine after stimulation with AngII. AngII induced a concentration-dependent release of choline from VSMC that was significant at 2 min and was sustained over 20 min. In contrast, accumulation of choline inside the cells was very slight. AngII also increased the formation of [3H]myristate-labelled phosphatidic acid, and, in the presence of ethanol, of [3H]phosphatidylethanol, characteristic of a phospholipase D (PLD) activity. Extracellular release of choline was partially inhibited by removal of extracellular Ca2+ (54 +/- 9% inhibition at 10 min) or inhibition of receptor processing by phenylarsine oxide (79 +/- 8% inhibition at 20 min). The protein kinase C activator phorbol myristate acetate also stimulated a large release of choline after a 5 min lag, which was unaffected by the Ca2+ ionophore ionomycin, but was additive with AngII stimulation. Down-regulation of protein kinase C by a 24 h incubation with phorbol dibutyrate (200 nM) decreased basal choline release, but had no effect on AngII stimulation. We conclude that AngII induces a major PC hydrolysis, probably mainly via PLD activation. This reaction is partially dependent on Ca2+ and is independent of protein kinase C, and appears to be mediated by cellular processing of the receptor-agonist complex. Our results are consistent with a preferential hydrolysis of PC from the external leaflet of the plasmalemma, and raise the possibility that PC hydrolysis occurs in specialized 'signalling domains' in VSMC.
Lassègue
B
B
Department of Medicine, Emory University School of Medicine, Atlanta, GA 30322.
Alexander
R W
RW
Clark
M
M
Griendling
K K
KK
D000804:Angiotensin II / Q000494:pharmacology*; D000818:Animals; D001013:Aorta, Thoracic; D001152:Arsenicals / Q000494:pharmacology; D002478:Cells, Cultured; D002794:Choline / Q000378:metabolism; D004533:Egtazic Acid / Q000494:pharmacology; D006868:Hydrolysis; D007700:Kinetics; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism*; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010712:Phosphatidic Acids / Q000378:metabolism; D010713:Phosphatidylcholines / Q000378:metabolism*; D010714:Phosphatidylethanolamines / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D001152:Arsenicals; D009227:Myristic Acids; D010712:Phosphatidic Acids; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; C029341:oxophenylarsine; D019814:Myristic Acid; D000804:Angiotensin II; D015240:Phorbol 12,13-Dibutyrate; D004533:Egtazic Acid; D011494:Protein Kinases; D002794:Choline; D013755:Tetradecanoylphorbol Acetate