Early intracellular signalling pathway of ethanol in vascular smooth muscle cells

1 ERKs belong to MAP kinase family and are activated by several growth and stress factors. Although ethanol has been shown to modulate ERK1 and ERK2 ( p44(mapk) and p42(mapk)) activity, it can also act as an antiproliferative agent in various mammalian cells. Since the nature of the antiproliferative effect of ethanol in VSMCs has not been defined, we examined its effects o n growth and on early intracellular events normally induced by growth facto rs in VSMCs. 2 Measurement of cytosolic Ca2+ and pH in cell monolayers was performed usi ng fura-2/AM and BCECF/AM, respectively. The effect of ethanol on VSMCs gro wth was assessed by [H-3]-thymidine incorporation, by cell counting and by determination of the caspase 3 activity. Stimulation of ERK1 and ERK2 was e xamined by the chemiluminescence Western blotting method. The expression of c-fos was quantitated by Northern blotting. Determination of inositolphosp hates was performed after labelling of VSMCs with myo-[2-H-3]-inositol and separation of inositolphosphates by HPLC. 3 Ethanol (0.3-1.0% v v(-1), 17-170 mM) induced a dose-dependent maximal st imulation of p44(mapk)/p42(mapk) at 30 min and expression of c-fos mRNA wit h a maximum at 120 min. Intracellular events upstream to MAP kinase, like a n increase in [Ca2+](i), activation of the Na+/H+ exchanger and formation o f phosphoinositol metabolites were also markedly activated by ethanol. Trea tment of VSMCs with ethanol for 3-5 min induced an increase in DNA synthesi s whereas treatment of the cells for more than 30 min was toxic. Caspase 3 activity was not modulated by ethanol treatment of VSMCs. 4 We may postulate that the activation of these mitogenic signals including the elevation of DNA synthesis reflects a cell effort to protect itself ag ainst the toxic effects of ethanol.