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

We investigate the existence of endothelin receptor subtypes using sub type selective ligands and the presence of immunoreactive (IR) endothe lin (ET)-3 (IR-ET-3) by radioimmunoassay (RIA) in rat retina. Scatchar d transformation of saturation binding experiments with [I-125]ET-3 re vealed 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 simil ar 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 experimen ts in the presence of the cyclic pentapeptide BQ123 (selective for ET( A) receptor) and Sarafotoxin 6C (selective for ET(B) receptor), demons trated 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-linki ng of [I-125]ET-1 to retinal membranes with disuccinimidyl suberate an d SDS-PAGE followed by autoradiography resulted in the labeling of two bands with apparent molecular masses of 52 and 34 kDa. Similar result s were obtained using [I-125]ET-3, suggesting that ET(A) and ET(B) rec eptors have similar molecular mass. The 34 kDa band is a proteolytic d egradation product of the 52 kDa band. The concentration of IR-ET-3 wa s 1212 +/- 153 fmol/g wet weight in rat retina. All these data suggest that ETs may play a role in neurotransmission or neuromodulation in t he retina, operating on both ET(A) and ET(B) receptor subtypes present in this tissue.