Discrimination and quantification of glomerular receptor subtypes for atrial natriuretic factor (ANF).
Binding sites for atrial natriuretic factor (ANF) were determined on isolated rat glomeruli as well as on glomerular membranes. To define optimal conditions, binding of ANF was investigated varying incubation time, temperature and protein concentration. Binding conditions were found to be best at 4 degrees C for 5 hours with 15 micrograms of glomerular protein. Saturation and affinity cross-linking experiments confirmed the presence of two distinct receptor subtypes-the B-receptor (130 kDa) and the C-receptor (65 kDa). Quantitative differentiation of both ANF binding sites was achieved by competitive displacement with two different unlabeled ANF ligands: a) rANF(99-126) (homologous displacement), b) des(18-22)rANF(4-23)NH2 (heterologous displacement). Intact glomeruli and glomerular membranes did not differ significantly in receptor density for the B-receptor (71 +/- 37 vs. 94 +/- 53 fmol/mg protein) or the C-receptor (976 +/- 282 vs. 966 +/- 167 fmol/mg protein) or in affinity constants for the B-receptor (43 +/- 36 vs. 52 +/- 44 pM) or the C-receptor (876 +/- 377 vs. 307 +/- 36 pM). Glomerular membranes compared to glomeruli showed less nonspecific binding and less intra-assay variation of measuring points done in triplicates. This method of selective displacement should allow to study the influence of various physiological and pathophysiological conditions on the binding properties of B- and C-receptors for ANF.
Kollenda
M C
MC
Dept. of Medicine II, University of Munich, W. Germany.
Scarborough
R M
RM
Gerbes
A L
AL
D000818:Animals; D009320:Atrial Natriuretic Factor / Q000378:metabolism*; D003432:Cross-Linking Reagents; D066298:In Vitro Techniques; D007678:Kidney Glomerulus / Q000378:metabolism*; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D017461:Receptors, Atrial Natriuretic Factor; D011956:Receptors, Cell Surface / Q000378:metabolism*
D003432:Cross-Linking Reagents; D011956:Receptors, Cell Surface; D009320:Atrial Natriuretic Factor; D017461:Receptors, Atrial Natriuretic Factor