alpha(2)-adrenergic agonist modulation of [S-35]GTP gamma S binding to guanine-nucleotide-binding-proteins in human platelet membranes

alpha(2)-adrenoceptor (alpha(2)-AR)-regulated binding of the labelled GTP a nalog, guanosine 5'-O-(3-[S-35]thiotriphosphate) ([S-35]GTP gamma S), to gu anine-nucleotide-binding proteins (G proteins) was studied in human platele t membranes. Under optimal conditions, the potent alpha(2)-AR agonist, 5-br omo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14304), increased the bindin g of [S-35]GTP gamma S up to approximately 1.8 fold, with half-maximal incr ease at 60 nM and was competitively inhibited by the alpha(2)-AR antagonist Rauwolscine. The actions of both UK 14304 and Rauwolscine were modulated b y monovalent and divalent cation levels, as well as by the concentrations o f GDP. [S-35]GTP gamma S binding induced by UK 14304 had a Kd value of 4.5 +/- 0.3 nM and a Bmax value of 4.15 +/- 0.40 pmol/mg protein. The rank orde r of potencies of adrenergic ligands tested in stimulating [S-35]GTP gamma S binding and inhibiting forskolin-stimulated c-AMP accumulation was UK 143 04> Guanabenz acetate> Oxymetazoline hydrochloride> BHT 920 dihydrochloride > p-Aminoclonidine hydrochloride> Clonidine hydrochloride. The data present ed indicate that enhancement of [S-35]GTP gamma S binding by alpha(2)-AR in human platelet membranes provides a simple functional measure for receptor activation and can be used for determination of potencies and efficacies o f ligands at the alpha(2)-AR.