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 antagonists to human platelet thromboxane A(2) (TXA(2)) receptors was investigated. TXA(2) receptors were purified from crude platelet membranes via affinity and wheat germ lectin chromatography. Radioligand binding studies were conducted using the TXA(2), mimetic [I-125]BOP (I-BOP = [1S-(1 alpha,2 beta(5Z),3 alpha(1E,3R*),4 alpha)]-7-[3(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)7-oxabicyclo-[2.2.1]heptan-2-yl]-5-heptenoic acid) and the TXA(2) receptor antagonist [I-125]SAP (I-SAP = 7-[(1R,2S,3S,5R)-6,6-dimethyl-3-(4-iodobenzene-sulfonylamino)-bicyclo-[3.1.1]hept-2-yl]-(5Z)-heptenoic acid). The histidine modifying reagent diethylpyrocarbonate (DEPC) produced a concentration (30-100 mu M) dependent inhibition of binding of both [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 TXA(2) receptor antagonist SQ 29548. The results strongly support the notion that one or more histidines reside in a domain that can modulate ligand binding to the TXA(2) receptor.