4,4'-Diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS) stimulates human platelets via α2A-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 μM), measured as 32P incorporation in pleckstrin, is completely inhibited by guanosine 5'-(2-O-thio)diphosphate (200 μM), an inhibitor of heterotrimeric G-proteins. Also pertussin toxin (4 μgml), which ADP-ribosylates the α-subunits of Gi's and Go, prevents pleckstrin phosphorylation by DIDS. N-Ethylmaleimide (50 μM), which uncouples Gi from α2A-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 3PADP-ribosylation of a 41 kDa protein fraction previously shown to contain the α-subunits ofGiα-1, Giα-2 and Giα-3. Thus, the α2A-adrenergic receptor activates PKC via a G-protein of the Gi-family.
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 microM), measured as 32P incorporation in pleckstrin, is completely inhibited by guanosine 5'-(2-O-thio)diphosphate (200 microM), an inhibitor of heterotrimeric G-proteins. Also pertussin toxin (4 micrograms/ml), which ADP-ribosylates the alpha-subunits of Gi's and Go, prevents pleckstrin phosphorylation by DIDS. N-Ethylmaleimide (50 microM), which uncouples Gi 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 32PADP-ribosylation of a 41 kDa protein fraction previously shown to contain the alpha-subunits of Gi alpha-1, Gi alpha-2 and Gi alpha-3. Thus, the alpha 2A-adrenergic receptor activates PKC via a G-protein of the Gi-family.
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