Stimulation of phospholipase D activity in human neuroblastoma (LA-N-2) cells by activation of muscarinic acetylcholine receptors or by phorbol esters: relationship to phosphoinositide turnover.
We have investigated the coupling of muscarinic acetylcholine receptors (mAChR) to phospholipid hydrolysis in a human neuroblastoma cell line, LA-N-2, by measuring the formation of 3H-inositol phosphates (3H-IP) and of [3H]phosphatidylethanol ([3H]PEt) in cells prelabeled with [3H]inositol and [3H]oleic acid. The muscarinic agonist carbachol (CCh) stimulated the phospholipase C (PLC)-mediated formation of 3H-IP in a time- and dose-dependent manner (EC50 = 40-55 microM). In addition, in the presence of ethanol (170-300 mM), CCh elevated levels of [3H]PEt [which is regarded as a specific indicator of phospholipase D (PLD) activity] by three- to sixfold. The effect of CCh on PEt formation also was dose dependent (EC50 = 50 microM). Both effects of CCh were antagonized by atropine, indicating that they were mediated by mAChR. Incubation of LA-N-2 cells with the phorbol ester phorbol 12-myristate 13-acetate (PMA, 0.1 microM; 10 min) increased [3H]PEt levels by up to 10-fold. This effect was inhibited by the protein kinase C (PKC) inhibitor staurosporine (1 microM) or by pretreatment for 24 h with 0.1 microM PMA, by 74% and 65%, respectively. In contrast, the effect of CCh on PEt accumulation was attenuated by only 28% in the presence of staurosporine (1 microM). In summary, these results suggest that, in LA-N-2 neuroblastoma cells, mAChR are coupled both to phosphoinositide-specific PLC and to PLD. PKC is capable of stimulating PLD activity in these cells; however, it is not required for stimulation of the enzyme by mAChR activation.
Sandmann
J
J
Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139.
Wurtman
R J
RJ
D000470:Alkaloids / Q000494:pharmacology; D002217:Carbachol / Q000494:pharmacology; D020404:Glycerophospholipids; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D009447:Neuroblastoma / Q000201:enzymology* / Q000473:pathology; D010712:Phosphatidic Acids / Q000037:antagonists & inhibitors / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism*; D011976:Receptors, Muscarinic / Q000378:metabolism*; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D000470:Alkaloids; D020404:Glycerophospholipids; D007295:Inositol Phosphates; D010712:Phosphatidic Acids; D011976:Receptors, Muscarinic; C051521:phosphatidylethanol; D002217:Carbachol; D010739:Phospholipase D; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate