The present study examined the existence of the adenosine A(1), A(2A), and A(2B) receptors and the effect of receptor activation on cAMP accumulation and protein phosphorylation in primary rat skeletal muscle cells. Presence of mRNA and protein for all three receptors was demonstrated in both cultured and adult rat skeletal muscle. NECA (10(-9)-10(-4) M) increased the cAMP concentration in cultured muscle cells with an EC50 of (95% confidence interval) = 15 (5.9-25.1) muM, whereas CGS 21680 (10(-9)-10(-4) M) had no effect on cAMP accumulation. Concentrations of [R]-PIA below 10(-6) M had no effect on cAMP accumulation induced by either isoproterenol or forskolin. NECA resulted in phosphorylation of CREB with an EC50 of (95% confidence interval)= 1.7 (0.40-7.02) muM, whereas ERK1/2 and p38 phosphorylation was unchanged. The results show that, although the A(1), A(2A), and A(2B) receptors are all present in skeletal muscle cells, the effect of adenosine on adenylyl cyclase activation and phosphorylation of CREB is mainly mediated via the adenosine AM receptor. (C) 2003 Elsevier Science (USA). All rights reserved.