Effect of phorbol ester treatment on receptor-mediated versus G-protein-activator-mediated responses in platelets. Evidence for a two-site action of phorbol ester at the level of G-protein function.
In platelets, and in several other cell systems, pre-treatment with protein kinase C activators such as phorbol 12-myristate 13-acetate (PMA) results in the inhibition of receptor-mediated responses, suggesting that protein kinase C may play an important role in the termination of signal transduction. In the present study, we have attempted to locate the site of action of phorbol ester by comparing thrombin-induced (i.e. receptor-mediated) platelet activation with that induced by guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and NaF, two agents which by-pass the receptor and initiate platelet responses by directly modulating G-protein function. After a 10 s pre-treatment with PMA (16 nM), dense-granule secretion induced by thrombin (0.2 unit/ml), GTP[S] (40 microM) and NaF (30 mM) was potentiated, resulting in a greater than additive response to agent plus PMA. However, after a 5 min pre-treatment, thrombin-induced secretion alone was inhibited, whereas PMA plus GTP[S]/NaF-induced release remained greater than additive. [32P]Phosphatidate formation in response to all three agents, in contrast, was inhibited by 50-70% in PMA (5 min)-treated platelets. That secretion induced by these agents is a protein kinase C-dependent event was demonstrable by using staurosporine, a protein kinase C inhibitor which at concentrations of 1-10 nM inhibited (70-90%) PMA-induced as well as thrombin- and NaF-induced secretion and protein phosphorylation. In membranes from PMA-treated platelets, thrombin-stimulated GTPase activity was significantly enhanced compared with that in untreated membranes (59% versus 82% increase over basal activity). The results suggest that inhibition of receptor-mediated responses by PMA may be directed towards two sites relating to G-protein activation: (i) receptor-stimulated GTPase activity and (ii) G-protein-phospholipase C coupling. Furthermore, the lack of inhibition of NaF- and GTP[S]-induced secretion by PMA suggests that different mechanisms may be involved in thrombin-induced and G-protein-activator-induced secretion.
Krishnamurthi
S
S
Thrombosis Research Unit, Rayne Institute, King's College School of Medicine & Dentistry, London, U.K.
Wheeler-Jones
C P
CP
Kakkar
V V
VV
D000470:Alkaloids / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects* / Q000201:enzymology; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D010712:Phosphatidic Acids / Q000096:biosynthesis; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D012701:Serotonin / Q000378:metabolism; D012969:Sodium Fluoride / Q000494:pharmacology; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D013917:Thrombin / Q000494:pharmacology; D013997:Time Factors; D010738:Type C Phospholipases / Q000378:metabolism
D000470:Alkaloids; D010712:Phosphatidic Acids; D012701:Serotonin; D006160:Guanosine Triphosphate; D012969:Sodium Fluoride; D011493:Protein Kinase C; D010738:Type C Phospholipases; D013917:Thrombin; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate