Binding characteristics of ET receptors in retinal pericytes and effects of high glucose incubation

Abstract

Purpose. Autoregulation and control of blood flow within the retina is dependent on vascular endothelial release of vasoconstrictors and dilators. The effector cells are the pericytes and vascular smooth muscle cells of the retinal microvascular bed. Many studies show that the responses of these effector cells are affected by diabetes or artificially elevated glucose. In particular, the contractile response of pericytes to endothelin-1 (ET-1) is markedly reduced. ET-1 and related isopeptides exert their effects by binding and activating specific cell surface receptors (ETRs). Pericytes express two subtypes of ETRs, ET A and ET B. The purpose of this study was to assess the dynamics of [ 125 I]ET-1 binding to ET A and ET B receptors when cultured in normal and high glucose concentrations. Methods. Bovine retinal pericytes (BRP) were exposed to normal (5 mM) or high glucose (25 mM) concentrations for up to 14 days. Displacement of binding and competitive inhibition of binding was carried out in perictye membranes. mRNA expression for ETR subtypes was examined using northern blotting. The expression of G protein a subunits (Ga s and Ga q) was detected by western blotting. Results. Highly specific binding of [ 125 I]ET-1 was seen with BRP membranes. In competition assays, ET-3 was found to displace [ 125 I]ET-1 binding to BRP membranes in a biphasic manner, indicating the presence of two classes of binding sites. The presence of both ETR subtypes was supported by competition experiments with selective ligands. The expression of Ga s and Ga q subunits decreased in pericytes incubated with 25 mM glucose. Conclusions. No significant differences in binding parameters were evident after culture in high glucose concentrations. However northern blot analysis which confirmed the presence of ET B receptor mRNA did identify an increase in the expression of this receptor.