Endocrinology, Vol 112, 2007-2014, Copyright © 1983 by Endocrine Society

ARTICLES

Angiotensin II-binding sites in rat and primate isolated renal tubular basolateral membranes

GP Brown and JG Douglas

In the kidney, angiotensin II influences reabsorptive processes by a direct tubular effect(s). The receptors mediating the response may be located on either the luminal (brush border) and/or the contraluminal (basolateral) membranes of the tubular epithelial cells. In the studies reported here, we identify specific [125I]angiotensin II-binding sites in rat and baboon tubular basolateral membranes. Specific binding was saturable, largely reversible, and proportional to membrane protein concentration. Structural specificity was confirmed by the use of angiotensin analogs and structurally unrelated polypeptides. The latter did not compete with radioligand for binding. Scatchard analyses of binding inhibition data indicated a single class of high affinity sites in rat (Kd = 2.2 +/- 0.2 nM; n = 12) and two classes of sites in baboon [Kd = 1.32 (n = 1) and 0.6 +/- 0.1 nM; n = 2) basolateral membranes. The binding site concentrations were 929 +/- 138 fmol/mg protein (rat) and 463 and 439 +/- 120 fmol/mg protein (baboon). Rat binding sites were affected by the addition of cations, as chloride salts, to the incubation medium. Na+ (100-200 mM) decreased the Kd from 4.2 +/- 0.4 nM in the absence of cations to 2.7 +/- 0.3 nM (n = 4). Mg2+ (4 mM) had no effect on Kd, but increased the binding site concentration from 556 +/- 84 to 915 +/- 166 fmol/mg protein. In contrast, 2 mM Ca2+ increased the Kd to 5.3 +/- 0.6 nM, and Mg2+ and Ca2+ added together affected neither Kd nor number of binding sites. Bound (eluted from membranes) and free (in medium) radioactivity, after incubation with membranes to steady state, were 54-68% (n = 3) and 85% (n = 2) intact [125I] angiotensin II, respectively, as determined by rebinding to fresh membranes. These data are inconsistent with binding to a degradative enzyme and indicate the presence of specific [125I] angiotensin II- binding sites in renal tubular basolateral membranes.

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