Characterization of endothelin A (ETA) and endothelin B (ETB) receptors in cultured bovine retinal pericytes.
OBJECTIVE
The endothelins are a family of structurally similar vasoactive peptides. It has been shown recently that cultured retinal microvascular endothelial cells secrete endothelin-1 (ET-1) and that corresponding pericytes bear receptors and are responsive to this peptide. These findings suggest a role for ET-1 in the autoregulation of retinal blood flow. There are at least two known subtypes of ET receptors, ETA and ETB. The purpose of this study was to characterize endothelin receptor subtypes on cultured bovine retinal pericytes (BRP).


METHODS
To characterize the specific binding sites for ET-1 and ET-3 on monolayers of BRP, a radioligand binding assay was performed using [125I] ET-1 and [125I] ET-3. Competition binding studies with ET-1 and ET-3 were used to assess the heterogeneity of the ET-receptor population on BRP. Also, [125I] ET-1 and ET-3 were covalently linked to their corresponding receptors and analyzed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography.


RESULTS
[125I] ET-1 and [125I] ET-3 showed specific binding to BRP and subsequent Scatchard analysis for both labels showed upward concavity, implying two-site ligand binding. Unlabeled ET-1 was found to displace [125I] ET-1 with greater efficiency than ET-3, indicating the presence of the ETA receptor subtype. Conversely, [125I] ET-3 was displaced by ET-1 and ET-3 with equal potency, indicating a component of ETB in the receptor population. Preincubation with BQ123, an ETA selective antagonist, decreased the binding of [125I] ET-1 but had no effect on [125I] ET-3 binding curves. Affinity cross-linking of the receptors showed two distinct protein bands on SDS-PAGE of 66 and 45 kd, corresponding to ETA and ETB.


CONCLUSIONS
These results show that BRP possess ETA and ETB receptor subtypes. The function of ETB on BRP may be to modulate the vasoconstrictive effect of ET-1 caused through ETA.
McDonald
D M
DM
Department of Ophthalmology, Queen's University of Belfast, Northern Ireland.
Bailie
J R
JR
Archer
D B
DB
Chakravarthy
U
U
D000818:Animals; D001667:Binding, Competitive; D002196:Capillaries / Q000166:cytology / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D065128:Endothelin Receptor Antagonists; D016232:Endothelins / Q000378:metabolism; D010456:Peptides, Cyclic / Q000494:pharmacology; D011869:Radioligand Assay; D044022:Receptor, Endothelin A; D044023:Receptor, Endothelin B; D017466:Receptors, Endothelin / Q000378:metabolism*; D012171:Retinal Vessels / Q000166:cytology / Q000378:metabolism*
D065128:Endothelin Receptor Antagonists; D016232:Endothelins; D010456:Peptides, Cyclic; D044022:Receptor, Endothelin A; D044023:Receptor, Endothelin B; D017466:Receptors, Endothelin; C072247:cyclo(Trp-Asp-Pro-Val-Leu)