Sustained diacylglycerol accumulation resulting from prolonged G protein-coupled receptor agonist-induced phosphoinositide breakdown in hepatocytes.
Studies in various cells have led to the idea that agonist-stimulated diacylglycerol (DAG) generation results from an early, transient phospholipase C (PLC)-catalyzed phosphoinositide breakdown, while a more sustained elevation of DAG originates from phosphatidylcholine (PC). We have examined this issue further, using cultured rat hepatocytes, and report here that various G protein-coupled receptor (GPCR) agonists, including vasopressin (VP), angiotensin II (Ang.II), prostaglandin F2alpha, and norepinephrine (NE), may give rise to a prolonged phosphoinositide hydrolysis. Preincubation of hepatocytes with 1-butanol to prevent conversion of phosphatidic acid (PA) did not affect the agonist-induced DAG accumulation, suggesting that phospholipase D-mediated breakdown of PC was not involved. In contrast, the GPCR agonists induced phosphoinositide turnover, assessed by accumulation of inositol phosphates, that was sustained for up to 18 h, even under conditions where PLC was partially desensitized. Pretreatment of hepatocytes with wortmannin, to inhibit synthesis of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate (PIP2), prevented agonist-induced inositol phosphate and DAG accumulation. Upon VP stimulation the level of PIP) declined, but only transiently, while increases in inositol 1,4,5-trisphosphate (InsP3) and DAG mass were sustained, suggesting that efficient resynthesis of PIP2 allowed sustained PLC activity. This was confirmed when cells were pretreated with wortmannin to prevent resynthesis of PIP2. Furthermore, metabolism of InsP3 was rapid, compared to that of DAG, with a more than 20-fold difference in half-life. Thus, rapid metabolism of InsP3 and efficient resynthesis of PIP2 may account for the larger amount of DAG generated and the more sustained time course, compared to InsP3. The results suggest that DAG accumulation that is sustained for many hours in response to VP, Ang.II, NE, and prostaglandin F2alpha in hepatocytes is mainly due to phosphoinositide breakdown.
Nilssen
Laila Sortvik
LS
Department of Pharmacology, Medical Faculty, University of Oslo, PO Box 1057 Blindern, N-0316 Oslo, Norway. l.d.s.nilssen@labmeb.uio.no
Dajani
Olav
O
Christoffersen
Thoralf
T
Sandnes
Dagny
D
D020001:1-Butanol / Q000494:pharmacology; D000730:Androstadienes / Q000494:pharmacology; D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D004075:Diglycerides / Q000378:metabolism*; D015237:Dinoprost / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D009638:Norepinephrine / Q000494:pharmacology; D010120:Oxytocics / Q000494:pharmacology; D010712:Phosphatidic Acids / Q000378:metabolism; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism*; D010739:Phospholipase D / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D043562:Receptors, G-Protein-Coupled / Q000819:agonists* / Q000378:metabolism; D010738:Type C Phospholipases / Q000378:metabolism; D014662:Vasoconstrictor Agents / Q000494:pharmacology; D014667:Vasopressins / Q000494:pharmacology; D000077191:Wortmannin
C011439:1,2-diacylglycerol; D000730:Androstadienes; D004075:Diglycerides; D010120:Oxytocics; D010712:Phosphatidic Acids; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols; D047428:Protein Kinase Inhibitors; D043562:Receptors, G-Protein-Coupled; D014662:Vasoconstrictor Agents; C037178:phosphatidylinositol 4-phosphate; D014667:Vasopressins; D000804:Angiotensin II; D020001:1-Butanol; D015237:Dinoprost; D010738:Type C Phospholipases; D010739:Phospholipase D; D009638:Norepinephrine; D000077191:Wortmannin