Prostaglandin F2 alpha-stimulated phospholipase D activation in osteoblast-like MC3T3-E1 cells: involvement in sustained 1,2-diacylglycerol production.
In [3H]myristic acid-labelled osteoblast-like MC3T3-E1 cells, prostaglandin F2 alpha (PGF2 alpha)-induced PLD activity was assessed by measuring the [3H]phosphatidylethanol (PEt) formation in the presence of ethanol. Inhibition of the increase in intracellular Ca2+ concentration ([Ca2+]i) by U73122, an inhibitor of phosphoinositide-specific phospholipase C (PI-PLC), or chelation of extracellular Ca2+ with EGTA or of intracellular Ca2+ with BAPTA, suppressed PGF2 alpha-induced phospholipase D (PLD) activation. Neither protein kinase C (PKC) inhibitors nor PKC down-regulation with phorbol 12-myristate 13-acetate affected PGF2 alpha-induced [3H]PEt formation. In permeabilized cells, guanosine 5'-[gamma-thio]triphosphate enhanced PGF2 alpha 's potency in [3H]PEt formation in the presence of Ca2+. The pretreatment of intact cells with pertussis toxin failed to inhibit PGF2 alpha-induced [3H]PEt formation. PGF2 alpha caused a biphasic production of [3H]1,2-diacylglycerol ([3H]1,2-DAG) in [3H]glycerol-labelled cells. The initial transient phase was decreased by U73122, whereas the late sustained phase was decreased by ethanol and the phosphatidic acid phosphohydrolase inhibitor, propranolol. From these results, it was suggested that PGF2 alpha-induced PLD activation was mediated by the dual control of the [Ca2+]i increase due to PI-PLC activation and activation of pertussis-toxin-insensitive G-protein, but not mediated by PKC, and also that PLD activation was involved in the late sustained 1,2-DAG generation in MC3T3-E1 cells.
Sugiyama
T
T
Department of Oral and Maxillo-Facial Surgery, Gifu University School of Medicine, Japan.
Sakai
T
T
Nozawa
Y
Y
Oka
N
N
D016475:3T3 Cells; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D004075:Diglycerides / Q000096:biosynthesis; D015237:Dinoprost / Q000494:pharmacology*; D004789:Enzyme Activation; D006150:Guanine Nucleotides / Q000494:pharmacology; D051379:Mice; D010006:Osteoblasts / Q000187:drug effects / Q000201:enzymology*; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010739:Phospholipase D / Q000187:drug effects / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism
C011439:1,2-diacylglycerol; D004075:Diglycerides; D006150:Guanine Nucleotides; D015237:Dinoprost; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D010739:Phospholipase D; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D002118:Calcium