IDENTIFICATION OF ENDOTHELIN RECEPTOR SUBTYPES IN RAT RETINA USING SUBTYPE-SELECTIVE LIGANDS

We investigate the existence of endothelin receptor subtypes using subtype selective ligands and the presence of immunoreactive (IR) endothelin (ET)-3 (IR-ET-3) by radioimmunoassay (RIA) in rat retina. Scatchard transformation of saturation binding experiments with [I-125]ET-3 revealed specific binding sites with a K-d and B-max values of 42 +/- 12 pM and 111 +/- 24 fmol/mg of protein, respectively. The K-d was similar to that obtain in previous studies using [I-125]ET-1. However, the B-max was 65% of that obtained with [I-125]ET-1. Competitive experiments in the presence of the cyclic pentapeptide BQ123 (selective for ET(A) receptor) and Sarafotoxin 6C (selective for ET(B) receptor), demonstrated the existence of ET(A) and ET(B) receptors in a ratio of 35:65. The order of potency of ET family peptides was ET-3 = ET-1 > S6C for ET(B) receptor and ET-1 > ET-3 > BQ123 for ET(A) receptor. Cross-linking of [I-125]ET-1 to retinal membranes with disuccinimidyl suberate and SDS-PAGE followed by autoradiography resulted in the labeling of two bands with apparent molecular masses of 52 and 34 kDa. Similar results were obtained using [I-125]ET-3, suggesting that ET(A) and ET(B) receptors have similar molecular mass. The 34 kDa band is a proteolytic degradation product of the 52 kDa band. The concentration of IR-ET-3 was 1212 +/- 153 fmol/g wet weight in rat retina. All these data suggest that ETs may play a role in neurotransmission or neuromodulation in the retina, operating on both ET(A) and ET(B) receptor subtypes present in this tissue.