Identification of endothelin receptor subtypes in rat ciliary body using subtype-selective ligands

The endothelins are important vasoactive ocular peptides and there is some evidence that they may modulate intraocular pressure. We investigated the existence and localization of endothelin receptor subtypes using subtype selective ligands in rat ciliary body. Scatchard transformation of saturation binding experiments revealed that the K-D, and B-max,,, for [I-125]ET-1,, and [I-125]ET-3 to, membranes from ciliary body were 41.7 +/- 9 pM and 236 +/- 20 fmol mg(-1) protein and 37.8 +/- 0.4 pM and 160 +/- 2.0 fmol mg(-1) protein, respectively. Competitive experiments in the presence of cyclic pentapeptide BQ123 (selective for ETA receptors) and BQ3020 (selective for ETB receptors), demonstrated the existence of ETA and ETB receptors in a ratio of 35:65. Cross-linking of [I-125]ET-1 and [I-125]ET-3 to, ciliary body membranes resulted in the labeling of two bands with apparent molecular masses of 52 and 34 kDa, suggesting that ETA and ETB receptors have similar molecular mass. The 34 Kda band is a proteolytic degradation product of the 52 Kda band. Autoradiographic results show that specific [I-125]ET-1 binding sites, displaced by BQ123 and BQ3020, are localized to the ciliary epithelium, supporting the idea that ETA and ETB subtype receptors exist in this tissue. (C) 1998 Academic Press Limited.