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°C for 5 hours with 15 μg 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.