Identification and characterization of the high affinity vascular angiotensin II receptor in rat mesenteric artery.

Abstract

SUMMARY To study the physiology of the high affinity vascular smooth muscle receptor for angiotensin II, we have characterized 125 I-angiotensin II binding sites in a participate fraction prepared from rat mesenteric arteries. 1-Angiotensin II binding was saturable at physiological concentrations of hormone, and was of high affinity. Scatchard analysis indicated a single class of binding sites with an equilibrium dissociation constant (Kd) of 0.91 ± 0.11 (SD) nM, and a total binding capacity of 53.7 ± 3.0 fmol/mg protein. Parallel studies with 3 H-angiotensin II yielded a similar Kj (1.18 ± 0.48 nM) and total binding capacity (56.8 ± 9.2 fmol/mg protein).m I-Angiotensin II associated with binding sites rapidly (ti/2 for association = 4 minutes at 37°C), and reversibly. Kinetic analysis of binding at 22°C by two independent methods yielded comparable values for both the association rate (4.0 and 6.8 x 10 5 /M per sec) and dissociation rate (3.2 and 3.8 x 10~Vsec) constants. Equilibrium dissociation constants calculated from kinetic analysis (0.56 and 0.80 nM) were in close agreement with that obtained from steady state Scatchard analysis. Analogues and antagonists of angiotensin II competed for binding in a potency series which exactly paralleled that observed for bioassay systems utilizing pressor response in vivo and vascular smooth muscle contraction in vitro.m I-Angiotensin II binding was stimulated 2-to 3-fold in the presence of 1 nun divalent cations (Mn 2+ > Mg 2 * > Ca 2+ ) and reversibly inhibited by EDTA and EGTA. Dithiothreitol (5 mil), a sulfhydryl-reducing agent that has been reported to block vasoconstrictor response to angiotensin II, inhibited 125 I-angiotensin II binding by 45%. The present study defines specific angiotensin II binding sites in a muscular artery representative of the resistance vasculature. We conclude that these binding sites, unlike those previously described in conductancetype vessels (aorta), have a sufficiently high affinity to interact with physiological levels of hormone.

The binding of [125I]‐angiotensin to rat renal epithelial cell membranes

“…The members of the renin angiotensin system also competed for the binding, although with a much reduced potency compared with the native hormone; thus angiotensin I was nine times less potent, angiotensin III 14 times, and renin substrate 40 times less potent. This order of potency is similar to that described in other studies of angiotensin binding (Brown, Douglas & KrontinsLitowitz, 1980;Gunther et al, 1980 (Glossmann, Baukal & Catt, 1974b), cerebral cortex (Bennett & Snyder, 1980) and mesenteric artery (Gunther et al, 1980). It also suggests that a direct action of the hormone upon epithelial transport as proposed by Munday et al (1972) and Harris & Young (1977) is at least part of the mechanism by which antinatriuresis and antidiuresis is affected.…”

“…Fitting of initial rate binding data to pseudo first order and second order rate equations gave an association rate constant of 11.8 x 106M-IS -1 and a dissociation rate of 6.5 x 10-3 s -1 and thus an affinity constant of 0.55 nM, this value being in good agreement with that produced by saturation analysis. (Bennett & Snyder, 1980), adrenal cortex (Glossmann, Baukal & Catt, 1974a) and vascular tissue (Gunther, Gimborne & Alexander, 1980 Figure 4. It can be seen that sodium has little influence upon the rate of association of [125J]-angiotensin II but markedly alters the rate of dissociation (6.5 x 10'3s' in the presence of sodium compared with 27.0 x 10-'s-1 in the absence of sodium).…”

Functional angiotensin II receptors in cultured vascular smooth muscle cells.

“…The 125 1-AII binding sites identified in cultured rat mesenteric artery VSMC are essentially similar to those of the All receptors present in particulate fractions prepared from the same blood vessel in vivo (13) . The affinity of these cellular binding sites (Kd, 2.8 nM by Scatchard analysis; 2.6 nM by kinetic analysis) is sufficiently high to permit interaction with All at concentrations present in rat blood under various physiological conditions (17).…”

Mechanism of decreased vascular reactivity to angiotensin II in conscious, potassium-deficient rats.

“…Smooth muscle from two sources was employed for studies of All receptors. Mesenteric arteries were used because these vessels are reactive to All and are representative ofresistance vessels in the body (13). However, it is difficult to prepare large quantities of smooth muscle membranes from these vessels.…”

“…The mesenteric arteries and branches were bluntly dissected away from veins, fat, and bowel by using a rubber policeman and fine forceps. Adherent fat was then removed from the arteries with a loose-fitting Potter Elvehjeim mechanical homogenizer, as described by Gunther et al (13).…”