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

To study the physiology of the high affinity vascular smooth muscle receptor for angiotensin II, we have characterized 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 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/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. I-Angiotensin II binding was stimulated 2to 3-fold in the presence of 1 nun divalent cations (Mn > Mg* > Ca) 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 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. Circ Res 47; 278-2861980