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 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 [32P]ADP-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.
Nieuwland
R
R
Department of Haematology, University Hospital Utrecht, The Netherlands.
Wijburg
O L
OL
van Willigen
G
G
Akkerman
J W
JW
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000246:Adenosine Diphosphate Ribose / Q000097:blood; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology*; D001798:Blood Proteins / Q000378:metabolism; D002463:Cell Membrane Permeability; D004789:Enzyme Activation; D005033:Ethylmaleimide / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000502:physiology*; D006801:Humans; D037342:Pertussis Toxin; D010750:Phosphoproteins; D010766:Phosphorylation; D011493:Protein Kinase C / Q000097:blood*; D011942:Receptors, Adrenergic, alpha / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D001798:Blood Proteins; D010750:Phosphoproteins; D011942:Receptors, Adrenergic, alpha; D010566:Virulence Factors, Bordetella; C039077:platelet protein P47; D000246:Adenosine Diphosphate Ribose; D037342:Pertussis Toxin; D011493:Protein Kinase C; D019204:GTP-Binding Proteins; D013755:Tetradecanoylphorbol Acetate; D005033:Ethylmaleimide; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid