Effects of N-ethylmaleimide on adenosine receptors of rat fat cells and human platelets

Abstract

Summary N-Ethylmaleimide (NEM) differentially modified R_i adenosine receptors in rat fat cells and R_a adenosine receptors in human platelets. Pretreatment of rat fat cell membranes with NEM inhibited the binding of the agonist (−)N⁶-phenylisopropyl[³H]adenosine ([³H]PIA), but did not affect the binding of the antagonist 1,3-diethyl-8-[³H]phenylxanthine ([³H]DPX). The IC₅₀-value for inhibition of [³H]PIA binding was 0.067 mM. Saturation of [³H]PIA binding revealed that NEM converts the high affinity form of the R_i receptor into a low affinity form. NEM also decreased the potency of agonists to displace [³H]DPX binding, as shown by a 74-fold shift of the K _i-value for (−)PIA, whereas antagonist-induced displacement remained unchanged. In addition, low concentrations of NEM (0.01–0.1 mM) attenuated the (−)PIA-induced inhibition of adenylate cyclase activity of rat fat cells. At higher concentrations (0.1–1 mM) NEM reduced basal and stimulated adenylate cyclase activities in rat fat cells and human platelets, presumably by inactivation of the catalytic unit. Radioligand binding of 5′-N-ethylcarboxamido[³H]-adenosine ([³H]NECA) to R_a adenosine receptors of human platelet membranes was not changed by NEM at low radioligand concentrations. Saturation analysis of [³H]-NECA binding showed that NEM led to an apparent increase of agonist affinity with a concomitant decrease in total [³H]NECA binding sites. These results suggest that NEM reduces the affinity of R_i adenosine receptors, probably by affecting the inhibitory guanine nucleotide binding protein (N_i), whereas [³H]NECA binding sites are inversely affected. The inhibitory action of NEM is not selective with respect to N_i, since at higher concentrations NEM inhibits the catalytic unit of the adenylate cyclase.