Activation of protein kinase C inhibits hormonal stimulation of the GTPase activity of Gi in human platelets

Abstract

The elTwt of 12,tetradecanoyl phorbol 13.n~.etate on the GTPase activity of Gi was invextil|ated. Treatment with TPA did not alter basal GTPa=e activity of membrane= or |he stimulalory effect or prosta~,landin E= (ptltatiwly via Gs), In contrast, the phorbo| c~ter markedly diminished ~timuh. don of GTPase by al~ents who~e receptor= are coupled to Gt such as epinephrine (:c.adrener=ic action), plntele! a¢tivatintt Etctof or thrombin. Pertus~is toxin catalyzed ADP.ribo~ylation was abe decrca,,ed in membranes from TPA-treated platelets as con'~pared to the ~ontrok, It is sulll|ested th,tt the =Iteration in tile hormonal activation of tile OTPau activity of Oi is ~¢ondary to a perturbation in the receptor.Gi interaction.Protei=~ kin=us C; Enzyme activation Gi; GTPase activity; ==.Adreno~cptor !. INTRODUCTION MATERIALS AND METHODSBlood was obtained from healthy men and women wile had taken ~o medication during the previous 2 weeks. Platelet.rich plasma was obtained by centrifuBation. After a preequillbration period of 5 rain at 37"C, the platelets were challenged with I ~,M TPA, or vehicle for I rain; the platelets were centrifuged and homogenized. A crude membrane preparation was obtained as described by Hoffman et zd.GTPase activity was assayed according to Cas~el and $elinger [8], in a mixture containing 2 mM MgCI.,, t m~ 3.isobutyl-l-nl¢th)'lxanthine, O. I mM cyclic AMP, 5 mM creatine phosphate, 1,2 mg/ml ere=line kinase, 0.2% bovine serum albumin, I mM dithiothreitol, 0,1 ntM EDTA, I #M GTP, 50 mM triethanolamine-HCI, pH 7,4° and [~,nPJGTP (0,2 ,~Ci/tube), Tile reaction was initiated by the addition of the membranes (10 #g of protein) and was carried out for l0 rain or the times indicated at 25=C in a final volume of 0.1 ml. The release of [~zP]Pi was determined as described by Aktories and Jakobs [9]. Protein was quantified by the method of Lowry et ~tl.[10]. ADP-ribosylation was assayed in a mixture containing 250 mM potassium phosphate buffer pH 7.5, 10 mM arginine, 5 mM MgCl=, 1 mM A'rP, 10 mM thymidine, 0.'75 mM NADP, 0,1 mM GTP, I0 #M NAP and [nP]NAD (10 ~Ci/tube), The reaction was carried out for 60 min at 30°C for 1 h. Pertussis toxin was activated with 20 rnM DTT for l0 rain at 37°C. The reacti:~, was started by the addition of the membranes (100/.tg); following tt :' reaction, 1 ml of phosphate buffer was added and the membranes were pelleted by centrifugation. The pellet was dissolved and subjected to SDS-PAGE. The gels were fixed, dried and exposed to X.ray film at -72°C [I 1].tmmunoprecipitation of Gi,-, was achieved using a specific polyclonal antibody generated against the decapeptide corresponding to a Gi (KNNLKDCGLF) [13], essentially as described for Gs~ [12]. RESULTSUnder the conditions employed, basal GTPase activity was linear as a function of time of incubation and of similar magnitude in membranes from control and TPA-treated platelets (Fig. 1). Epinephrine (100/~M) and prostaglandin Et (PGEt) (1 #M) increased the rate of GTPase activity (Fig. I) . The effect of epinephrinePublished by E...