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 [125I]ET-1 and [125I]ET-3 revealed that the proportion of [125I]ET-3-binding sites was 30-35% less than that of [125I]ET-1-binding sites. The apparent dissociation constants and maximum binding for [125I]ET-1 and [125I]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 ETB receptors, [125I]ET-1 binding was decreased by 65-70%, whereas [125I]ET-3 binding was totally abolished, suggesting that 65-70% of [125I]ET-1 binding and 100% of [125I]ET-3 binding was to ETB 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 microM BQ123, [125I]ET-1 binding was decreased by 25-30%, whereas [125I]ET-3 binding was unaffected, confirming that 30-35% of ET receptors belong to the ETA subtypes, and that [125I]ET-1 bound to both ETA and ETB receptors with the same high affinity, but [125I]ET-3 bound only to ETB receptors with high affinity. These results suggest that human kidney cortex and medulla contain ETA and ETB 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.
Nambi
P
P
Department of Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406.
Pullen
M
M
Wu
H L
HL
Aiyar
N
N
Ohlstein
E H
EH
Edwards
R M
RM
D002462:Cell Membrane / Q000378:metabolism; D016232:Endothelins / Q000378:metabolism*; D006801:Humans; D007457:Iodine Radioisotopes; D007672:Kidney Cortex / Q000378:metabolism*; D007679:Kidney Medulla / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008875:Middle Aged; D010456:Peptides, Cyclic / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism*; D017466:Receptors, Endothelin
D016232:Endothelins; D007457:Iodine Radioisotopes; D008024:Ligands; D010456:Peptides, Cyclic; D011956:Receptors, Cell Surface; D017466:Receptors, Endothelin; C072247:cyclo(Trp-Asp-Pro-Val-Leu)