Pertussis toxin-insensitive G protein mediates carbachol activation of phospholipase D in rat pheochromocytoma PC12 cells.
In the present study, an activation mechanism for phospholipase D (PLD) in [3H]palmitic acid-labeled pheochromocytoma PC12 cells in response to carbachol (CCh) was investigated. PLD activity was assessed by measuring the formation of [3H]phosphatidylethanol ([3H]PEt), the specific marker of PLD activity, in the presence of 0.5% (vol/vol) ethanol. CCh caused a rapid accumulation of [3H]-PEt, which reached a plateau within 1 min, in a concentration-dependent manner. The [3H]PEt formation by CCh was completely antagonized by atropine, demonstrating that the CCh effect was mediated by the muscarinic acetylcholine receptor (mAChR). A tumor promoter, phorbol 12-myristate 13-acetate (PMA), also caused an increase in [3H]-PEt content, which reached a plateau at 30-60 min after exposure, but an inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate, did not. Although a protein kinase C (PKC) inhibitor, staurosporine (5 microM), blocked PMA-induced [3H]PEt formation by 77%, it had no effect on the CCh-induced formation. These results suggest that mAChR-induced PLD activation is independent of PKC, whereas PLD activation by PMA is mediated by PKC. NaF, a common GTP-binding protein (G protein) activator, and a stable analogue of GTP, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), also stimulated [3H]PEt formation in intact and digitonin-permeabilized cells, respectively. GTP, UTP, and CTP were without effect. Furthermore, guanosine 5'-O-(2-thiodiphosphate) significantly inhibited CCh- and GTP gamma S-induced [3H]PEt formation in permeabilized cells but did not inhibit the formation by PMA, and staurosporine (5 microM) had no effect on [3H]PEt formation by GTP gamma S.(ABSTRACT TRUNCATED AT 250 WORDS)
Kanoh
H
H
Department of Biochemistry, Gifu University School of Medicine, Japan.
Kanaho
Y
Y
Nozawa
Y
Y
D000818:Animals; D002217:Carbachol / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D019204:GTP-Binding Proteins / Q000187:drug effects / Q000502:physiology*; D016716:PC12 Cells; D037342:Pertussis Toxin; D010739:Phospholipase D / Q000378:metabolism* / Q000502:physiology; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D010566:Virulence Factors, Bordetella; D002217:Carbachol; D037342:Pertussis Toxin; D010739:Phospholipase D; D019204:GTP-Binding Proteins; D013755:Tetradecanoylphorbol Acetate