To study cellular mechanisms influencing vascular reactivity, vascular smooth muscle cells (VSMC) were obtained by enzymatic dissociation of the rat mesenteric artery, a highly reactive, resistance-type blood vessel, and established in primary culture. Cellular binding sites for the vasoconstrictor hormone angiotensin II (All) were identified and characterized using the radioligand 125 1-angiotensin 11 . Freshly isolated VSMC, and VSMC maintained in primary culture for up to 3 wk, exhibited rapid, saturable, and specific ' 25 1-All binding similar to that seen with homogenates of the intact rat mesenteric artery . In 7-d primary cultures, Scatchard analysis indicated a single class of high-affinity binding sites with an equilibrium dissociation constant (Kd) of 2.8 ± 0.2 nM and a total binding capacity of 81 .5 ± 5 .0 fmol/mg protein (equivalent to 4.5 x 104 sites per cell) . Angiotensin analogues and antagonists inhibited 125 1-All binding to cultured VSMC in a potency series similar to that observed for the vascular All receptor in vivo . Nanomolar concentrations of native All elicited a rapid, reversible, contractile response, in a variable proportion of cells, that was inhibited by pretreatment with the competitive antagonist Sarl ,lleB-All . Transmission electron microscopy showed an apparent loss of thick (12-18 nm Diam) myofilaments and increased synthetic activity, but these manifestations of phenotypic modulation were not correlated with loss of 1251-All binding sites or hormonal responsiveness . Primary cultures of enzymatically dissociated rat mesenteric artery VSMC thus may provide a useful in vitro system to study cellular mechanisms involved in receptor activation-response coupling, receptor regulation, and the maintenance of differentiation in vascular smooth muscle .The interaction of vasoactive hormones, such as the octapeptide, angiotensin II (All), with vascular smooth muscle cells (VSMC) has important implications for normal vascular physiology (1) and the pathophysiology of hypertension (2). Although pharmacologic studies in whole animals, isolated vascular strips, and blood vessel homogenates have identified receptors for a number of vasoactive substances, the cellular localization of these receptors, biochemical correlates of their activation, and factors regulating their expression have not been well defined, in part due to the structural complexity of vascular tissues . Selective isolation and culture of the cellular components of blood vessels provides a potentially useful approach to this problem (3-12).Large fibroelastic arteries, such as the aorta, have been a preferred source of VSMC for culture (3,7,8) because the inner one-third of the medial layer of these vessels normally does not contain other cell types. However, once established in

The sensitivity of blood vessels to the vasoconstrictor effects of the hormone angiotensin II appears to be modulated by the activity of the renin-angiotensin system. Elevation of circulating angiotensin II levels by sodium depletion or renal artery stenosis is associated with a diminished pressor response to infused angiotensin II (refs 1-3). Conversely, the vasocontrictor response to the hormone is enhanced when endogenous angiotensin II levels are reduced by sodium loading or nephrectomy. The mechanisms of these varying effects are not known, but physiological and pharmacological experiments suggest involvement of the vascular smooth receptor for angiotensin II (refs 5-8). Modification of the interaction between angiotensin II and its vascular receptor, resulting in altered responsiveness to the hormone, could occur either via 'prior occupancy' of receptors by elevated levels of endogenous angiotensin II resulting in fewer free receptors available to respond to circulating angiotensin II (ref. 5), or, elevated levels of angiotensin II could result in a decrease in receptor affinity for the hormone or a decrease in total receptor number in the vascular smooth muscle cell. We now report the first direct evidence, by radioligand binding assay, that angiotensin II regulates the number of its own receptors in resistance vasculature.

-Hill, 1975SUMMARY To test the hypothesis that impaired cardiac performance in some patients with pressureoverload hypertrophy is due to inappropriately high wall stress, rather than depressed contractility, the importance of hemodynamic and geometric factors was assessed in 14 patients with isolated aortic stenosis and various degrees of left ventricular failure (ejection fraction range 0.19-0.85). There was poor correlation between either aortic valve area, peak left ventricular systolic pressure, or left ventricular mass, and measures of ventricular function. In contrast, there were close correlations between circumferential wall stress and both ejection fraction (r= 0.96) and velocity of fiber shortening (r = 0.91) in patients with aortic stenosis. Forcevelocity-shortening relationships in six normal control subjects fell on the same regression line as that defined by the patients with aortic stenosis, while force-velocity-shortening relationships of patients with primary myocardial failure clearly differed. A major determinant of wall stress was the ratio of left ventricular wall thickness to cavity radius (h/R). Patients with h/R ratios > 0.36 had higher values for ejection fraction (0.61 ± 0.06 vs 0.36 i 0.07,p < 0.05), Vcf (0.79 ± 0.10 vs 0.39 ± 0.04 sec ',p < 0.05) and stroke work index (71 ± 10 vs 45 9 g-m/m2, p < 0.005) than those with lower ratios.The results indicate that left ventricular wall thickness and geometry are closely correlated with ventricular performance in patients with pressure-overload hypertrophy due to aortic stenosis. Poor cardiac performance in some such patients may be due to inadequate hypertrophy (or inappropriate geometry) rather than to depression of myocardial contractility.MYOCARDIAL FUNCTION in pressure-overload hypertrophy is a controversial subject. It is generally agreed that the development of hypertrophy in response to a pressure overload is associated at least initially with wall thickening commensurate with the

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.