Activation of mitogen-activated protein kinase by muscarinic receptors in astroglial cells: Role in DNA synthesis and effect of ethanol

Mitogen-activated protein kinase (MAPK) can be phosphorylated by mitogens b inding to G-protein-coupled receptors and is considered a major pathway inv olved in cell proliferation. In this study, we report on the activation of MAPK by muscarinic acetylcholine receptors in astroglial cells, namely the 1321N1 human astrocytoma cell line, primary rat cortical astrocytes, and fe tal human astrocytes. Carbachol caused a rapid and transient phorphorylatio n of MAPK (ERK1/2) in all cell types, with an increase in MAPK activity, wi thout changing the levels of MAPK proteins. Human astrocytoma cells were us ed to characterize the effect of carbachol on MAPM Experiments with M-2- an d M-3-receptor subtype-selective antagonists, and with pertussis toxin, ind icated that the M-3 subtype is responsible for activating MAPK in glial cel ls. Pretreatment of cells with the protein kinase C (PKC) inhibitor bisindo lylmaleimide I, or downregulation of PKC by 24-h treatment with the phorbol ester TPA inhibited carbachol-induced MAPK activation. Additional experime nts with PKC alpha- or PKC epsilon -specific compounds indicated that the e psilon isozyme of PKC is primarily involved in MAPK activation by carbachol . Chelation of calcium also inhibited MAPK activation by carbachol. Two MEK (MAPK kinase) inbibitors inhibited carbachol-induced DNA synthesis but onl y at concentrations that exceeded those sufficient to block carbachol-induc ed MAPK activation. Ethanol (less than or equal to 200 mM) had no effect on MAPK when present alone and did not affect carbachol-induced MAPK activati on under various experimental conditions, although it inhibits carbachol-in duced DNA synthesis at low concentrations (10-100 mM). These results sugges t that activation of MAPK by carbachol may be necessary but not sufficient for its mitogenic effect in astroglial cells, and that does not represent a target for ethanol-induced inhibition of DNA synthesis elicited by muscari nic receptors. (C) 2001 Wiley-Liss, Inc.