Angiotensin II receptors in the fowl aorta.

In the domestic fowl, angiotensin II (ANG II) causes an in vivo depressor response and in vitro relaxation of aortic rings which appear to be a direct action of ANG II on the blood vessels. Thus, we determined whether binding sites specific to ANG II exist in the membrane fraction of the fowl aorta. The particulate fraction of aortas from adult female fowl, Gallus gallus, exhibits high specific binding to ANG II ligand. 125I-[Ile5]ANG II (0.5 nM) binding to the above fraction (30 micrograms protein) in 50 mM Tris (pH 7.2), 10 mM MgCl2, and 0.2% bovine serum albumin at 12 degrees (1) is rapid, saturable, and reversible; (2) increases as a function of ligand or membrane concentration, time, and temperature; and (3) optimally fits to a two-site (high and low affinity) model. The equilibrium dissociation constant (0.15 +/- 0.03 nM) and binding site concentration (28.7 +/- 8.1 fmol/mg protein) of the high affinity site as well as association (0.055 nM-1.min-1) and dissociation (0.0122 min-1) rate constants are similar to those of mammalian vascular ANG II receptors. Both 125I-[Ile5]ANG II and 125I-[Val5]ANG II are competitively displaced by unlabeled ANG II. These results suggest that specific ANG II receptors exist in the fowl aorta.

Identification and characterization of the rabbit angiotensin II myocardial receptor.

The octapeptide, angiotensin II, elicits a positive inotropic response in myocardial tissue by activating slow calcium channels. Pharmacological studies suggest that the inotropic action of angiotensin II is receptor mediated. The current investigation was performed to characterize the binding of 125I-angiotensin II to the putative receptor in a plasma membrane-sarcoplasmic reticulum preparation of the rabbit left ventricle. In experiments performed at 18 degrees C, steady state binding occurred at 45 minutes and saturable binding was 80-85% of the total binding. Analysis of the binding data indicated that the 125I-angiotensin II interacted with a single class of sites with a Kd = 4.5 +/- 0.8 nM and exhibited a binding capacity of 53.5 +/- 8 fmol/mg protein. The potency order for the competitive binding of analogues and antagonists of angiotensin II paralleled that observed for in vitro contractile force development in bioassay systems utilizing left atrial tissue. The binding of 125I-angiotensin II was stimulated 2-fold in the presence of the divalent cations of calcium and magnesium (10 mM). Guanine nucleotides modified the binding of 125I-angiotensin II to the rabbit myocardial particulate fraction. Guanine triphosphate and nonhydrolyzable analogues of guanine triphosphate increased the dissociation rate of the bound 125I-angiotensin II and decreased hormone binding to the receptor at equilibrium. In the absence of magnesium, guanine nucleotides had no effect on the dissociation rate of 125I-angiotensin II. 125I-Angiotensin II binding to a rabbit myocardial particulate fraction was found to have high affinity, to be saturable, reversible, specific, and modulated by guanine nucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for the role of phosphorylase kinase, protein kinase C, and other Ca2+-sensitive protein kinases in the response of hepatocytes to angiotensin II and vasopressin.

Angiotensin II, catecholamines, and vasopressin can stimulate the phosphorylation of 10 hepatic cytosolic proteins via a Ca2+-linked, cyclic AMP-independent mechanism. To explore the role of known Ca2+-sensitive protein kinases in this response, [32P]PO4(3-)-labeled hepatocytes were stimulated with various agonists, the cytoplasmic proteins were separated on two-dimensional gels, and the resulting autoradiographs were computer analyzed. The role of phosphorylase kinase was examined using hepatocytes from gsd/gsd rats which are deficient in this enzyme. The phosphorylation state of phosphorylase was not increased by glucagon, angiotensin II, or vasopressin in hepatocytes from the gsd/gsd animals. The phosphorylation state of all other substrates was changed by glucagon or the Ca2+-linked hormones to the same extent in gsd/gsd hepatocytes as in normal Wistar controls, suggesting that phosphorylase kinase plays a restricted role in the hormone response. The role of the Ca2+- and phospholipid-sensitive protein kinase (protein kinase C) was examined by stimulating hepatocytes with phorbol esters which are thought to activate protein kinase C by substituting for diacylglycerol. Phorbol esters increased the phosphorylation state of 3 of the 10 substrates affected by angiotensin II or vasopressin, but did not stimulate Ca2+ fluxes in hepatocytes. Treatment of hepatocytes with the Ca2+ ionophore A23187 mimicked the effect of the Ca2+-linked hormones on the phosphorylation of the other 7 substrates. The results demonstrate that at least three Ca2+-sensitive protein kinases are involved in the response of hepatocytes to Ca2+-linked hormones. Since these kinases can be activated independently by phorbol esters or A23187, the results imply that hormones such as vasopressin generate two intracellular messengers, diacylglycerol and Ca2+ ion.

The hepatic angiotensin II receptor. II. Effect of guanine nucleotides and interaction with cyclic AMP production.

Guanine nucleotides were observed to modify the binding of 125I-angiotensin II to rat hepatic plasma membrane receptors. GTP and its nonhydrolyzable analogues greatly increased the dissociation rate of bound 125I-angiotensin II and altered hormone binding to the receptor under equilibrium conditions. In the absence of GTP, 125I-angiotensin II labeled both high affinity sites (Kd1 = 0.46 nM, N1 = 650 fmol/mg) and low affinity sites (Kd2 = 4.1 nM, N2 = 1740 fmol/mg). In the presence of guanine nucleotides, the affinities of the two sites were unchanged, but the number of high affinity sites decreased markedly to 52 fmol/mg. In analogous experiments using the angiotensin II antagonist, 125I-sarcosine1,Ala8-angiotensin II (125I-saralasin), guanine nucleotides minimally affected the interaction of 125I-saralasin with its receptor, increasing the dissociation rate 1.9-fold and the Kd 1.4-fold. The guanine nucleotide inhibition of agonist binding required a cation such as Na+ or Mg2+, with a maximal effect occurring at about 1 mM Mg2+. In liver plasma membranes prepared in EDTA, angiotensin II inhibited basal and glucagon-stimulated adenylate cyclase activities by 30% and 10%, respectively. Angiotensin II also caused a 40% inhibition of glucagon-stimulated cyclic AMP accumulation in intact hepatocytes, with a half-maximal effect occurring at 1 nM. The inhibition by angiotensin II of adenylate cyclase in membranes and of cAMP levels in intact cells could be reversed by the antagonist sarcosine1,Ile8-angiotensin II. Vasopressin caused a smaller 26% inhibition of glucagon-stimulated cyclic AMP accumulation. The ability of angiotensin II to inhibit cyclic AMP synthesis may provide an explanation for the observed effects of guanine nucleotides on 125I-angiotensin II binding to plasma membranes.

The effect of prolactin on human aldosterone-producing adenomas in vitro.

There is evidence for an unidentified aldosterone-stimulating factor of pituitary origin. We measured the effect of ovine PRL (oPRL) on aldosterone secretion by isolated cell suspensions of human aldosterone-producing adenomas (APAs) and compared it to the effects of angiotensins, ACTH, and potassium (K+). In the first APA, the aldosteronotropic action of large doses of oPRL was double that of angiotensin II (AII); the response to ACTH was triple that to AII, while K+ had a small stimulatory effect. Results with the second APA showed that physiological concentrations of oPRL caused a response nearly double that to AII, but, once again, less than the response to ACTH; K+ was inert. ACTH contamination of the oPRL preparation was too minute to account for these findings. We conclude that oPRL possesses aldosterone-stimulating activity in APAs greater than that of angiotensins and potassium, but less that that of ACTH. These data suggest a role for PRL in aldosterone secretion by aldosterone-producing adenomas.

Steroids as potential modulators of angiotensin receptors in bovine adrenal glomerulosa and kidney.

To test the hypothesis that there is feedback inhibition of adrenal angiotensin receptors by substances released in response to the peptides, we measured binding of labeled angiotensins in the presence of various steroids. Approximately half of the 70 steroids tested inhibited binding of labeled angiotensin II and III to intact and broken cells from bovine adrenal glomerulosa and kidney, but the concentrations required for inhibition were relatively high. The most potent inhibitors were 3 alpha, 5 beta tetrahydroaldosterone and tetrahydrodeoxycorticosterone (ID50 = 8 x 10-5 M). Kinetic analysis showed that inhibition was mostly competitive. among steroids whose reduced congeners were tested, potency increased in the sequence: parent steroid less than 5 alpha dihydroderivative less than 5 beta dihydro derivative less than 3 alpha, 5 beta tetrahydro-derivative. Tetrahydrodeoxycorticosterone inhibited aldosteronogenesis by intact cells at concentrations that inhibited angiotensin binding. Steroids differentially inhibited binding of labeled angiotensins in II and III, and discriminated between receptors in adrenal glomerulosa and kidney. The results provide additional evidence for heterogeneity of angiotensin receptors, and lead to the prediction that any normal or pathological inhibition of angiotensin receptors by steroids will be mediated by reduced derivatives.