Sodium concentration in human renal medulla regulates endothelin-1 binding to the endothelin type B receptor

Autocrine-paracrine activities of endothelin-1 (ET-1) in the kidney are mediated by two receptor subtypes, the ETA, involved in sodium retention, and the ETB subtype, which reduces Na+-K+-ATPase activity in the inner medullary collecting ducts and antagonizes AVP mediated water reabsorption. Renal ET-1 synthesis in the inner medullary collecting ducts is enhanced during low sodium diet and increased renal medulla osmolarity. The present study was planned to investigate whether sodium influences also ET-1 binding to human medullary endothelin receptors. Medulla specimens were obtained from 5 patients who underwent nefrectomy for localized renal tumor without evidences of impaired renal function. After tissue homogenization, plasma membranes were isolated by fractional centrifugation and resuspended in Tris 50 mM, PMSF 1 mM, pH 7.4. In binding studies [125I]-ET-1 (2,000 Ci/mmol) was used as the radiolabeled ligand and ET-1 or ETA and ETB selective antagonists (PD155080 and BQ788) as cold displacers. Kinetics experiments were performed in the presence of increasing concentrations of NaCl (0, 77, 154, 231 and 308 mM, final concentration). Kinetics data were analyzed according to Weiland and Molinoff. Competition experiments with ETA and ETB selective antagonists revealed a large prevalence (more than 90%) of ETB receptor subtypes in human renal medulla. RT-PCR studies performed with specific primers confirmed the prevalence of ETB receptors in renal medulla. In the absence of NaCl, kinetics of [125I]-ET-1 binding showed a slow pattern of association reaching equilibrium after 90 min. [125I]-ET-1 binding was only partially displaceable by the addition of a large excess of unlabeled ET-1. Increasing concentration of NaCl in the binding medium progressively rose the association rate of [125I]-ET-1 which plateaued at 231 mM NaCl (at 308 mM +112% vs NaCl-free buffer, p<0.05). Conversely, the dissociation rate decreased and the kinetically derived dissociation constant (Kd) was reduced with a resultant 4-fold increase in ET-1 binding affinity. ET-1 receptor density also linearly increased with increased NaCl concentrations (at 308 mM +45% vs NaCl-free buffer, p<0.05). In conclusion, the NaCl induced effects on the affinity and density of ETB receptors in human renal medulla might influence the activity of the peptide during physiologic changes in medulla osmolarity.