[3H]Yohimbine binding to human platelet membranes: evidence for high and low affinity binding sites with negative cooperativity.

Evidence is presented that [3H]yohimbine binding to human platelet membranes does not follow the simple mass action kinetics. Although [3H]yohimbine binding was saturable and stereospecific, Scatchard analysis of the equilibrium binding data produced a curvilinear plot. Competitive displacement of [3H]yohimbine from the binding sites by unlabeled yohimbine and other alpha 2-antagonists produced shallow inhibition curves. Further, the apparent Hill coefficients of equilibrium binding and competitive displacement data were found to be less than unity. Factors such as binding to nonreceptor sites or to alpha1-adrenoceptors, dopamine receptors, or 5-HT receptors that may explain the curvilinear curve were excluded. The rank order for inhibiting [3H]yohimbine was rauwolscine greater than yohimbine greater than phentolamine greater than clonidine much greater than prazosin, suggesting that the binding sites had the characteristics of alpha2-adrenoreceptors. The affinity of the alpha2-antagonist for the receptor was enhanced by Na+ but not by guanine nucleotide, suggesting that the binding of the antagonist is modulated only by Na+. Graphic analysis of the specific binding data resulted in two components: one with high affinity and low capacity sites, and second with low affinity and high capacity sites. The experiments on dissociation kinetics, however, suggest that the observed deviation of [3H]yohimbine binding from the simple mass action kinetics is most likely due to negative cooperative interactions among alpha2-adrenoceptor sites.