CHARACTERIZATION OF ENDOTHELIN-A (ET(A)) AND ENDOTHELIN-B (ET(B)) RECEPTORS IN CULTURED BOVINE RETINAL PERICYTES

Purpose. The endothelins are a family of structurally similar vasoacti ve peptides. It has been shown recently that cultured retinal microvas cular endothelial cells secrete endothelin-1 (ET-1) and that correspon ding pericytes bear receptors and are responsive to this peptide. Thes e findings suggest a role for ET-1 in the autoregulation of retinal bl ood flow. There are at least two known subtypes of ET receptors, ET(A) and ET(B). The purpose of this study was to characterize endothelin r eceptor subtypes on cultured bovine retinal pericytes (BRP). Methods. To characterize the specific binding sites for ET-1 and ET-3 on monola yers of BRP, a radioligand binding assay was performed using [I-125] E T-1 and [I-125] ET-3. Competition binding studies with ET-1 and ET-3 w ere used to assess the heterogeneity of the ET-receptor population on BRP. Also, [I-125] ET-1 and ET-3 were covalently linked to their corre sponding receptors and analyzed on sodium dodecyl sulfate-polyacrylami de gel electrophoresis (SDS-PAGE) followed by autoradiography. Results . [I-125] ET-1 and [I-125] ET-3 showed specific binding to BRP and sub sequent Scatchard analysis for both labels showed upward concavity, im plying two-site ligand binding. Unlabeled ET-1 was found to displace [ I-125] ET-1 with greater efficiency than ET-3, indicating the presence of the ET(A) receptor subtype. Conversely, [I-125] ET-3 was displaced by ET-1 and ET-3 with equal potency, indicating a component of ET(B) in the receptor population. Preincubation with BQ123, an ET(A) selecti ve antagonist, decreased the binding of [I-125] ET-1 but had no effect on [I-125] ET-3 binding curves. Affinity cross-linking of the recepto rs showed two distinct protein bands on SDS-PAGE of 66 and 45 kd, corr esponding to ET(A) and ET(B). Conclusions. These results show that BRP possess ET(A) and ET(B) receptor subtypes. The function of ET(B) on B RP may be to modulate the vasoconstrictive effect of ET-1 caused throu gh ET(A).