Identification and quantification of human kidney atrial natriuretic peptide receptors

Abstract

Kahana L, Yechiely H, Mecz Y, Lurie A. Identification and quantification of human atrial natriuretic peptide receptors. Eur J Endocrinol 1995;132:465–71. ISSB 0804–4643 The present study determined 125I-label atrial natriuretic peptide (ANP) binding sites in human kidney glomerular and papillary membranes. The membranes were prepared from non-malignant renal tissue obtained at nephrectomy of patients with renal carcinoma. To evaluate the proportion of ANP receptor classes ANP-R1 (ANPR-A, -B) versus ANP-R2 (ANPR-C), competitive binding studies were performed using [125I]-ANP in the presence of increasing concentrations of ANP or an internally ring-deleted analog, des(Gln116, Ser117, Gly118, Leu119, Gly120)ANP(102–121), called C-ANP, which binds selectively to ANPR-C receptors. Analysis of the competitive binding curve with ANP in glomerular membranes suggested the presence of one group of high-affinity receptors with dissociation constant Kd = 26 ± 12 pmol/l and density Bmax = 101 ± 47 nmol/kg protein. A decrease of 10–30% in Bmax with no change in Kd was obtained in the presence of excess (10−6 mol/l) C-ANP, suggesting the existence of a small amount of a second class of receptors, the ANPR-C class. The densities of ANPR-A, -B versus ANPR-C receptors in human glomeruli, calculated from competitive inhibition experiments, were 75 ± 42 and 22 ± 16 nmol/kg protein (N = 8). Autoradiography of the sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions showed two bands: a highly labeled 130kD band and a weakly labeled 66kD band, both displaced by ANP. Only the 66-kD band was displaced by the C-ANP analog. Human papilla membrane, as shown by competition binding studies and SDS gel electrophoresis, presented only one class of receptors with Kd = 40 ± 23 pmol/l (mean ± SD, N = 3) and Bmax = 17 ± 6.3 nmol/kg protein. No displacement of [125I]ANP with C-ANP analog could be detected. The Kd of the binding sites in human kidney papilla was not statistically different (p > 0.05) from the Kd values of the two receptor subtypes for ANP in glomeruli. Thus, human kidney, much like that of the rat, presents: two classes, the high- (ANPR-A, -B) and low (ANPR-C)-molecular-weight receptors, in glomerular membranes; only one class, the high-molecularweight receptors, in papillary membranes. In contrast to rat kidney, the high-molecular-weight ANPR-A, -B is the dominant class in human glomerular membranes. Luna Kahana, Endocrine Laboratory, Carmel Medical Center, 7 Michal Street, Haifa 34362, Israel