Pertussis Toxin‐Insensitive G Protein Mediates Carbachol Activation of Phospholipase D in Rat Pheochromocytoma PC12 Cells

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Abstract
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, 4a-phorbol 12,13-didecanoate, did not. Although a protein kinase C (PKC) inhibitor, staurosporine (5 μM), 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) (OTPGmS), 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 GTPΓS- induced [3H]PEt formation in permeabilized cells but did not inhibit the formation by PMA, and staurosporine (5 μM) had no effect on [3H]PEt formation by GTPγS. Pretreatment of cells with pertussis toxin (10–200 ng/ml) for 15 h failed to suppress CCh-induced [3H]PEt formation, although the pertussis toxin-sensitive G protein(s) in membranes was completely ADP-ribosylated under the same conditions. From these results, we conclude that the mechanisms of PMA- and CCh-stimulated PLD activation are different from each other and that CCh-induced PLD activation is independent of PKC and mediated, at least in part, via a pertussis toxin-insensitive G protein.

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