ALPHA(2A)-ADRENERGIC RECEPTORS ACTIVATE PROTEIN-KINASE-C IN HUMAN PLATELETS VIA A PERTUSSIS-TOXIN-SENSITIVE G-PROTEIN

4,4'-Diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS) stimulates human platelets via alpha(2A)-adrenergic receptor-mediated activation of protein kinase C (PKC) independent of the phospholipase C pathway. Here we show, that in permeabilized platelets activation of PKC by DIDS (20 mu M), measured as P-32 incorporation in pleckstrin, is completely inhibited by guanosine 5'-(2-O-thio)diphosphate (200 mu M), an inhibitor of heterotrimeric G-proteins. Also pertussin toxin (4 mu g/ml), which ADP-ribosylates the alpha-subunits of G(i)'s and G(o), prevents pleckstrin phosphorylation by DIDS. N-Ethylmaleimide (50 mu M), which uncouples G(i) from alpha(2A)-adrenoceptors, inhibits pleckstrin phosphorylation by DIDS in intact platelets. Activation of PKC by 55 nM phorbol 12-myristate 13-acetate and 500 nM platelet-activating factor are not disturbed by NEM. DIDS inhibits by 40 +/- 5% (n = 4) the pertussis toxin-catalyzed [P-32]ADP-ribosylation of a 41 kDa protein fraction previously shown to contain the alpha-subunits of G(i) alpha-1, G(i) alpha-2 and G(i) alpha-3. Thus, the alpha(2A)-adrenergic receptor activates PKC via a G-protein of the G(i)-family.