Aldosterone receptors in A6 cells: physicochemical characterization and autoradiographic study.
The A6 cell line is derived from the kidney of Xenopus laevis. Aldosterone increases sodium transport across A6 cell epithelia. In the present study, aldosterone binding characteristics were studied in A6 cell cytosol. Both type I (mineralocorticoid) and type II (glucocorticoid) receptors are present in the cytosolic fraction of these cells. Aldosterone and corticosterone had a high affinity for type I sites (Kd = 1.25 and 0.16 nM, respectively) and a lower affinity for type II sites (Kd = 39 and 10 nM, respectively). Testosterone and estradiol did not compete for aldosterone binding. RU 26988, a highly specific glucocorticoid agonist, competed with aldosterone for type II but not for type I sites. Hydrodynamic parameters of both type I and type II corticosterone receptor complexes were identical. Their Stokes radius was approximately 6 nm, as estimated by high-performance size-exclusion chromatography, and their sedimentation coefficient determined by ultracentrifugation on glycerol gradients was approximately 9s. The molecular mass calculated from these parameters was approximately 200 kDa, a value that is very close to the value estimated for nontransformed mineralocorticoid and glucocorticoid receptors of other species. The [3H]aldosterone labeling of intact A6 cells was examined by autohistoradiography. At every concentration tested (2, 20, and 50 nM), all cells were found to be specifically labeled in both cytoplasm and nucleus. At 20 nM, in the presence of an excess of RU 26988, labeling was also detected. At every concentration the labeling data was compatible with a Gaussian distribution, indicating that A6 cells correspond to a homogeneous population with regard to aldosterone binding and that probably both type I and type II sites are present in the same cells.
Claire
M
M
Faculté de Pharmacie, Institut National de la Santé et de la Recherche Médicale U300, Montpellier, France.
Machard
B
B
Lombes
M
M
Oblin
M E
ME
Bonvalet
J P
JP
Farman
N
N
D000450:Aldosterone / Q000378:metabolism*; D000818:Animals; D001345:Autoradiography; D001667:Binding, Competitive; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003345:Corticosterone / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D007668:Kidney; D007700:Kinetics; D011965:Receptors, Glucocorticoid / Q000302:isolation & purification / Q000378:metabolism*; D018161:Receptors, Mineralocorticoid; D014316:Tritium; D014982:Xenopus laevis
D011965:Receptors, Glucocorticoid; D018161:Receptors, Mineralocorticoid; D014316:Tritium; D000450:Aldosterone; D003345:Corticosterone