CHARACTERIZATION OF THE ENZYMATIC-ACTIVITY FOR BIPHASIC COMPETITION BY GUANOXABENZ 2,6-DICHLOROBENZYLIDENE-AMINO)-3-HYDROXYGUANIDINE) AT ALPHA(2)-ADRENOCEPTORS - I - DESCRIPTION OF AN ENZYMATIC ACTIVITY IN SPLEEN MEMBRANES

The mechanism for formation of high-affinity binding of (2,6-dichlorob enzylidene-amino)-3-hydroxyguanidine (guanoxabenz) to alpha(2)-adrenoc eptors was studied in particulate fractions from the rat spleen. The p roportion of apparent high versus low-affinity alpha(2)-adrenoceptor b inding sites increased with increasing incubation time and was also au gmented by Mg2+ ions. The formation of high-affinity guanoxabenz bindi ng seemed to be inhibited by a series of N-hydroxyguanidine analogs to guanoxabenz, as well as by a series of metabolic inhibitors that incl uded allopurinol, 1-chloro-2,dinitrobenzene, 5,5'-dithiobis-(2-nitrobe nzoic acid), cibacron blue, phenyl-p-benzoquinone, didox, and trimidox . The formation of guanoxabenz high-affinity binding was also inhibite d in a time- and concentration dependent fashion by preincubating the membranes with the LW03 N-hydroxyguanidine analogue of guanoxabenz. Mo reover, when the spleen membranes were extensively washed for 30 min w ith buffers at 25 degrees, the guanoxabenz high-affinity binding disap peared. However, when these washed membranes were supplemented with xa nthine, the apparent affinity of guanoxabenz increased four to five-fo ld. Taken together, all data were compatible with the theory that the formation of high-affinity binding was dependent on the generation of a guanoxabenz metabolite that showed an approximate 100-fold greater a ffinity for the alpha(2)-adrenoceptors than guanoxabenz itself. Becaus e the most potent blocker of the formation of high-affinity binding wa s allopurinol (apart from some N-hydroxyguanidine analogs to guanoxabe nz) and since the activity could be restored with xanthine, a likely c andidate responsible for the metabolic activation is xanthine oxidase. (C) 1998 Elsevier Science Inc.