Hormonal stimulation of diacylglycerol formation in hepatocytes. Evidence for phosphatidylcholine breakdown.
The molecular species of 1,2-diacylglycerol in control and agonist-stimulated rat hepatocytes were analyzed by high performance liquid chromatography. Twelve species were identified which were increased nonuniformly by 100 nM vasopressin. Most species were increased 2-3-fold, but some (C16:0/C20:4 and C18:0/C20:4) were increased 3-6-fold. Selectively greater increases in the latter two species were also induced by ATP, angiotensin II, and A23187 ionophore, however, phorbol ester caused uniform increases. Calcium depletion of the cells with chelator resulted in a uniform 2-fold effect of vasopressin on 1,2-diacylglycerol species, with greater increases in C16:0/C20:4 and C18:0/C20:4 being restored by Ca2+ readdition. Comparison of the increases in 1,2-diacylglycerol species caused by the Ca2+-mediated agents with the molecular species present in rat hepatocyte phospholipids supports the concept that phosphatidylcholine is a major source of the 1,2-diacylglycerol that accumulates. In hepatocytes incubated for 5 min to 2 h with 1-O-[3H]alkyl-2-lyso-sn-glycero-3-phosphocholine, the label was incorporated mainly into phosphatidylcholine, and subsequent incubation with vasopressin, angiotensin II, ATP, epinephrine, A23187, and phorbol ester caused formation of [3H]alkyl-acylglycerol, but not [3H]alkyl-phosphatidic acid. The time course and concentration dependence of the vasopressin effect were similar to those reported previously for total 1,2-diacylglycerol (Bocckino, S. B., Blackmore, P. F., and Exton, J. H. (1985) J. Biol. Chem. 260, 14201-14207). Calcium depletion induced by chelator inhibited the effect of vasopressin, and readdition of Ca2+ largely restored the effect. In cells incubated with [14C]lyso-phosphatidylcholine, [3H]phosphatidylcholine, or [14C]phosphatidylethanolamine for 5 or 30 min to label hepatocyte phosphatidylcholine, vasopressin also induced the formation of labeled 1,2-diacylglycerol, but not phosphatidic acid. In contrast, in hepatocytes prepared from rats injected intraportally with [3H]alkyl-lyso-glycerophosphocholine 20 h previously, the hormone induced the rapid formation of both labeled 1,2-diacylglycerol and phosphatidic acid. In summary, these isotopic data indicate that a rapidly labeled pool of phosphatidylcholine is hydrolyzed to 1,2-diacylglycerol and a slowly labeled pool is broken down to both 1,2-diacylglycerol and phosphatidic acid in hepatocytes stimulated by Ca2+-mobilizing agents. It is concluded from both the analyses of molecular species of 1,2-diacylglycerol and the labeling experiments that phosphatidylcholine is a major source of the 1,2-diacylglycerol that accumulates in hepatocytes stimulated with Ca2+-mobilizing agonists and that the mechanisms responsible may involve both Ca2+ and protein kinase C.
Augert
G
G
Howard Hughes Medical Institute Laboratory, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
Bocckino
S B
SB
Blackmore
P F
PF
Exton
J H
JH
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D001127:Arginine Vasopressin / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D004075:Diglycerides / Q000378:metabolism*; D004533:Egtazic Acid / Q000494:pharmacology; D004837:Epinephrine / Q000494:pharmacology; D005227:Fatty Acids / Q000032:analysis; D005934:Glucagon / Q000494:pharmacology; D005989:Glycerides / Q000378:metabolism*; D006728:Hormones / Q000494:pharmacology*; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D010713:Phosphatidylcholines / Q000378:metabolism*; D010743:Phospholipids / Q000096:biosynthesis; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D004075:Diglycerides; D005227:Fatty Acids; D005989:Glycerides; D006728:Hormones; D010713:Phosphatidylcholines; D010743:Phospholipids; D000804:Angiotensin II; D001127:Arginine Vasopressin; D000001:Calcimycin; D004533:Egtazic Acid; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D005934:Glucagon; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D004837:Epinephrine