INHIBITION OF LIGAND-BINDING TO THROMBOXANE A(2) PROSTAGLANDIN H-2 RECEPTORS BY DIETHYLPYROCARBONATE - PROTECTION BY RECEPTOR LIGANDS AND REVERSAL BY HYDROXYLAMINE/

The potential of histidines to modulate the binding of agonists and an tagonists to human platelet thromboxane A(2) (TXA(2)) receptors was in vestigated. TXA(2) receptors were purified from crude platelet membran es via affinity and wheat germ lectin chromatography. Radioligand bind ing studies were conducted using the TXA(2), mimetic [I-125]BOP (I-BOP = [1S-(1 alpha,2 beta(5Z),3 alpha(1E,3R),4 tenyl)7-oxabicyclo-[2.2.1 ]heptan-2-yl]-5-heptenoic acid) and the TXA(2) receptor antagonist [I- 125]SAP (I-SAP = nylamino)-bicyclo-[3.1.1]hept-2-yl]-(5Z)-heptenoic ac id). The histidine modifying reagent diethylpyrocarbonate (DEPC) produ ced a concentration (30-100 mu M) dependent inhibition of binding of b oth [I-125]BOP and [I-125]SAP. DEPC treatment significantly (P < 0.05, N = 6) decreased the affinity of the receptor for [I-125]SAP (K-d = 2 .4 +/- 0.4 and 5.4 +/- 0.4 nM, control and DEPC, respectively) without significantly decreasing the B-max. The effects of DEPC were reversed by hydroxylamine. The inhibition of [I-125]BOP and [I-125]SAP binding produced by DEPC was reduced significantly by prior incubation of the purified receptors with the TXA(2) receptor agonist U-46619 or the TX A(2) receptor antagonist SQ 29548. The results strongly support the no tion that one or more histidines reside in a domain that can modulate ligand binding to the TXA(2) receptor.