Characteristics of ANP-sensitive guanylate cyclase in inner medullary collecting duct cells.
Our previous characterization of equilibrium binding kinetics of atrial natriuretic peptide (ANP) to the surface of inner medullary collecting duct (IMCD) cells suggested the existence of a single class of high-affinity receptors, functionally coupled to increases in cellular guanosine 3',5'-cyclic monophosphate (cGMP). We have now sought to understand the mode of regulation of this signal transduction system by studying the particulate guanylate cyclase (PGC) enzyme from these cells. PGC activity with and without ANP in membranes, made by homogenization and high-speed centrifugation of suspensions of IMCD cells, was linear up to 5 min and was stimulated by ANP [143 +/- 21 (ANP) vs. 38 +/- 7 (control) pmol/mg protein, n = 3, P less than 0.02]. Vmax increased more than threefold with ANP [130 +/- 19 (ANP) vs. 35 +/- 4 (control) pmol.mg protein-1.min-1, n = 4, P less than 0.005] without significant change in the Km [0.68 +/- 0.17 (ANP) vs. 0.55 +/- 0.08 (control) mM] of the enzyme. Half-maximal stimulation of guanylate cyclase activity occurred at 5 x 10(-10) M ANP, a concentration consistent with our binding data, and with physiological effect. PGC required divalent cations for basal activity and for ANP-stimulated activity; Mg2+ and Mn2+ were most potent in this respect, and Ca2+ was without effect. Both basal and stimulated PGC activities were inhibited in response to changes in the NaCl, but not urea concentration of the assay system. We conclude that binding to the single 120-130 kDa ANP receptor in IMCD cells results in stimulation of PGC by increasing its Vmax and thereby elevating intracellular cGMP, the likely mediator of ANP action in these cells.
Gunning
M
M
Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts.
Silva
P
P
Brenner
B M
BM
Zeidel
M L
ML
D000818:Animals; D009320:Atrial Natriuretic Factor / Q000494:pharmacology*; D002413:Cations, Divalent / Q000494:pharmacology; D006162:Guanylate Cyclase / Q000378:metabolism*; D007679:Kidney Medulla; D007684:Kidney Tubules / Q000201:enzymology*; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000201:enzymology*; D009994:Osmolar Concentration; D011817:Rabbits; D017461:Receptors, Atrial Natriuretic Factor; D011956:Receptors, Cell Surface / Q000378:metabolism
D002413:Cations, Divalent; D011956:Receptors, Cell Surface; D009320:Atrial Natriuretic Factor; D006162:Guanylate Cyclase; D017461:Receptors, Atrial Natriuretic Factor