alpha(2)-adrenoceptors in opossum kidney cells couple to stimulation of mitogen-activated protein kinase independently of adenylyl cyclase inhibition

We have compared the effects of adrenaline on activation of mitogen-activated protein kinase (MAP kinase), cyclic AMP accumulation and [H-3]thymidine uptake in OK cells, a cell line derived from proximal tubules of the opossum kidney. Effects of serotonin and the direct protein kinase C activator, phorbol-12-myristate-13-acetate (PMA), were also studied. Adrenaline transiently (peak at 5 min, return to baseline by 30 min) and concentration-dependently (EC50 between 10 and 100 - nM) stimulated MAP kinase activity. Maximal stimulation was approximately 100% above basal and was similar to the effects of 1 mu M serotonin or 1 mu M PMA. MAP kinase activation by adrenaline was inhibited by 10 mu M phentolamine or 1 mu M yohimbine but not significantly affected by 100 nM prazosin or 200 nM pindolol. The selective alpha(2)-adrenoceptor agonist UK 14,304 (10 mu M) also stimulated MAP kinase activity. Activation of the 42 and 44 kDa ERK forms of MAP kinase was demonstrated by immunoblot analysis. The effect of adrenaline and UK 14,304 on MAP kinase was inhibited by pertussis toxin pretreatment and by the MAP kinase kinase inhibitor, PD 98059 (100 mu M). Stimulation of MAP kinase activity was independent of cellular cAMP levels and was not affected by protein kinase C down-regulation. Adrenaline, UK 14,304, serotonin, and PMA stimulated [H-3]thymidine uptake, an effect inhibited by PD 98059. We conclude that adrenaline stimulates MAP kinase activity in OK-cells via alpha(2)-adrenoceptors and pertussis sensitive G proteins. While this occurs independently of cellular cAMP levels and protein kinase C, it involves the MEK1 form of MAP kinase kinase and the ERK forms of MAP kinase. This activation results in enhanced cellular proliferation as assessed by [H-3]thymidine uptake.