IDENTIFICATION OF ENDOTHELIN RECEPTOR SUBTYPES IN HUMAN RENAL-CORTEX AND MEDULLA USING SUBTYPE-SELECTIVE LIGANDS

High affinity and high density endothelin (ET)-binding sites were identified in membranes prepared from human kidney cortex and medulla. Saturation binding experiments performed in membranes prepared from cortex and medulla using [I-125]ET-1 and [I-125]ET-3 revealed that the proportion of [I-125]ET-3-binding sites was 30-35% less than that of [I-125]ET-1-binding sites. The apparent dissociation constants and maximum binding for [I-125]ET-1 and [I-125]ET-3 to membranes from cortex were 91 +/- 5 pm and 165 +/- 10 fmol/mg protein, and 117 +/- 9 pM and 110 +/- 7 fmol/mg protein, respectively, whereas in medulla they were 139 +/- 10 pm and 360 +/- 11 fmol/mg protein, and 142 +/- 11 pm and 245 +/- 15 fmol/mg protein, respectively. In the presence of 10 nm sarafotoxin-6c, which is selective for ET(B) receptors, [I-125]ET-1 binding was decreased by 65-70%, whereas [I-125]ET-3 binding was totally abolished, suggesting that 65-70% of [I-125]ET-1 binding and 100% of [I-125]ET-3 binding was to ET(B) receptors. This was further confirmed by the use of a cyclic pentapeptide [cyclo(D-Trp,D-Asp,L-Pro,D-Val,L-Leu)] (BQ123), which is selective for ETA receptors. In the presence of 1-mu-M BQ123, [I-125]ET-1 binding was decreased by 25-30%, whereas [I-125]ET-3 binding was unaffected, confirming that 30-35% of ET receptors belong to the ET(A) subtypes, and that [I-125]ET-1 bound to both ET(A) and ET(B) receptors with the same high affinity, but [I-125]ET-3 bound only to ET(B) receptors with high affinity. These results suggest that human kidney cortex and medulla contain ET(A) and ET(B) receptors in a ratio of 30:70, and that sarafotoxin-6c and BQ123 are valuable tools in identifying the subtype of ET receptors in various tissues.