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

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Abstract
To study the physiology of the high affinity vascular smooth muscle receptor for angiotensin II [A], 125I-A binding sites were characterized in a particulate fraction prepared from rat mesenteric arteries. 125I-A binding was saturable at physiological concentrations of hormone and was of high affinity. Scatchard analysis indicated a single class of binding sites with an Kd of 0.91 .+-. 0.11 (SD) nM, and a total binding capacity of 53.7 .+-. 3.0 f[femto]mol/mg protein. Parallel studies with 3H-A yielded a similar Kd (1.18 .+-. 0.48 nM) and total binding capacity (56.8 .+-. 9.2 fmol/mg protein). 125I-A associated with binding sites rapidly (half-time for association = 4 min at 37.degree. C), and reversibly. Kinetic analysis of binding at 22.degree. C by 2 independent methods yielded comparable values for both the association rate (4.0 and 6.8 .times. 105/M per s) and dissociation rate (3.2 and 3.8 .times. 10-4/s) constants. Kd calculated from kinetic analysis (0.56 and 0.80 nM) were in close agreement with those obtained from steady state Scatchard analysis. Analogs and antagonists of A 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. 125I-A binding was stimulated 2- to 3-fold in the presence of 1 mM divalent cations (Mn2+ > Mg2+ > Ca2+) and reversibly inhibited by EDTA and ethylene glycol-bis(.beta.-aminoethyl ether)N,N,N'',N''-tetraacetic acid. Dithiothreitol (5 mM), a sulfhydryl-reducing agent that was reported to block vasoconstrictor response to A, inhibited 125I binding by 45%. The present study defines specific A binding sites in a muscular artery representative of the resistance vasculature. These binding sites, unlike those previously described in conductance-type vessels (aorta), apparently have a sufficiently high affinity to interact with physiological levels of hormone.