Early intracellular signalling pathway of ethanol in vascular smooth muscle cells

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Abstract
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 (p44mapk and p42mapk) 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 on growth and on early intracellular events normally induced by growth factors in VSMCs. Measurement of cytosolic Ca2+ and pH in cell monolayers was performed using fura‐2/AM and BCECF/AM, respectively. The effect of ethanol on VSMCs growth was assessed by [3H]‐thymidine incorporation, by cell counting and by determination of the caspase 3 activity. Stimulation of ERK1 and ERK2 was examined by the chemiluminescence Western blotting method. The expression of c‐fos was quantitated by Northern blotting. Determination of inositolphosphates was performed after labelling of VSMCs with myo‐[2‐3H]‐inositol and separation of inositolphosphates by HPLC. Ethanol (0.3–1.0% v v−1, 17–170 mM) induced a dose‐dependent maximal stimulation of p44mapk/p42mapk at 30 min and expression of c‐fos mRNA with a maximum at 120 min. Intracellular events upstream to MAP kinase, like an increase in [Ca2+]i, activation of the Na+/H+ exchanger and formation of phosphoinositol metabolites were also markedly activated by ethanol. Treatment of VSMCs with ethanol for 3–5 min induced an increase in DNA synthesis whereas treatment of the cells for more than 30 min was toxic. Caspase 3 activity was not modulated by ethanol treatment of VSMCs. We may postulate that the activation of these mitogenic signals including the elevation of DNA synthesis reflects a cell effort to protect itself against the toxic effects of ethanol. British Journal of Pharmacology (1999) 128, 1761–1771; doi:10.1038/sj.bjp.0702969