Identification and Characterization of Endothelin Receptor Subtype B in Rat Retina

Abstract: The presence of immunoreactive (IR) endothelin (ET)-1 and ET-1 receptors in rat retina has been studied by radioimmunoassay and receptor assay, respectively. The specific binding of ¹²⁵I-ET-1 to rat retinal particulate preparations was saturable. Apparent equilibrium conditions were established within 120–140 min. Scatchard analysis of binding data indicated a single class of high-affinity binding sites with a K _D of 35 ± 11 p M and a B_max of 168 ± 60 fmol/mg of protein. ¹²⁵I-ET-1 binding to retinal particulate preparations was not inhibited by 1 μ M concentrations of somatostatin, atrial natriuretic factor, brain natriuretic peptide, thyroid-stimulating hormone, growth hormone, or insulin. The three endothelin isoforms, ET-1,-2, and-3, had similar affinity for the receptor. Cross-linking of ¹²⁵I-ET-1 to retinal particulate preparations with disuccinimidyl suberate resulted in the labeling of two bands with apparent molecular masses of 52 and 34 kDa. We have established a highly sensitive and specific radioimmunoassay for ET-1. The concentration of IR-ET-1 in rat retina was 35 ± 10 fmol/g wet weight. The demonstration of specific high-affinity ETB receptors and the presence of IR-ET-1 suggest that the peptide may act as a neurotransmitter or neuro-modulator in the retina.