Angiotensin II antagonists versus drinking induced by bombesin or eledoisin in pigeons

The angiotensin II analogues Sar¹,Ile⁸-,Sar¹,Leu⁸- and, to a lesser extent, Sar¹,Ala⁸- and Sar¹-Gly⁸-angiotensin II proved to inhibit angiotensin-induced drinking in the pigeon, but did not significantly affect drinking induced by eledoisin or bombesin. The results suggest that the dipsogenic response elicited by bombesin or eledoisin, although almost identical to that evoked by angiotensin II, is mediated by the activation of central receptors different from those of angiotensin-induced drinking. These findings are consistent with the hypothesis that several peptidergic mechanisms are involved in the regulation of water intake.     

Reduction of blood clearance rate of [Val5] angiotensin II by [Sar1, ILE8] angiotensin II in sheep

The present study examines the effect of [Sar¹, Ile⁸] angiotensin II ([Sar¹, Ile⁸] ANG II) on the blood clearance rate of [Val⁵] angiotensin II ([Val⁵] ANG II) in conscious, sodium-replete sheep. Animals were infused simultaneously with [Val⁵] ANG II and [Sar¹, Ile⁸] ANG II at a rate of 42 nmol/h and 6 μmol/h respectively. Blood [Val⁵] ANG II was quantitatively determined with care taken in separating [Val⁵] ANG II from [Sar¹, Ile⁸] ANG II prior to radioimmunoassay. The blood clearance rate of [Val⁵] ANG II calculated from infusion rate/blood concentration was significantly different before and during [Sar¹, Ile⁸] ANG II infusion, being 141 ± 13 L/h (n = 12) and 95 ± 10 L/h (n = 12) respectively. Plasma renin concentration remained suppressed after the commencement of [Sar¹, Ile⁸] ANG II infusion. $\text{In-vitro}$ studies showed no significant decrease in the rate of degradation of [Val⁵] ANG II in blood in the presence of [Sar¹, Ile⁸] ANG II. Possible interpretation of this reduction of blood clearance rate of [Val⁵] ANG II by 45 ± 15 L/h (n = 6) was discussed.     

NMR studies on angiotensin II: Histidine and phenylalanine ring stacking and biological activity

The conformations of angiotensin II and the antagonist [Sar¹, Ile⁸]angiotensin II in dimethylsulfoxide have been examined by high resolution proton magnetic resonance spectroscopy at 400MHz. The chemical shifts for the aromatic protons of the phenylalanine residue in angiotensin II are consistent with shielding and restricted rotation for this side-chain. The chemical shifts for the histidine C₂ and C₄ protons in angiotensin II also indicate shielding, whereas these same protons in the antagonist [Sar¹, Ile⁸]angiotensin II do not demonstrate this shielding influence. These findings suggest a stacking interaction for the histidine and phenylalanine side-chains in angiotensin II which is important for activating angiotensin receptors.     

Effects of angiotensin II on the spontaneous activity of rostral ventrolateral medullary cardiovascular neurons and blood pressure in spontaneously hypertensive rats

The interactive role of rostral ventrolateral medulla (RVL) cardiovascular neurons and brain angiotensin II (Ang II) in regulating the arterial blood pressure was examined by recording simultaneously the spontaneous activity of these spinal projecting neurons and the arterial blood pressure in the pentobarbital-anesthetized spontaneously hypertensive rat (SHR) and its normotensive control, the Wistar Kyoto rat (WKY). It was found that Ang II elicited dose-dependent excitatory responses in a subpopulation of RVL cardiovascular neurons, followed by a subsequent increase in blood pressure. These effects of Ang II were significantly greater in SHR than in WKY. The effects were attenuated or abolished by co-administration of Ang II antagonist, [Sar¹, Ile⁸]-Ang II. Pre-administration of [Sar¹, Ile⁸]-Ang II to RVL using bilateral microinjection attenuated the blood pressure effects of intracerebroventricularly administered Ang II by as much as 70%. These results indicated that spinal projecting RVL cardiovascular neurons are important in mediating the pressor action of Ang II. The enhanced sensitivity and responsiveness of RVL cardiovascular neurons to Ang II may be pertinent to the genesis of hypertension in adult SHR.     

Analysis of angiotensins I,II, III,and iodinated derivatives by high-performance liquid chromatography

Angiotensins I, II, and III were separated by reversed-phase high-performance liquid chromatography on an octadecylsilyl column. The peptides were isocratically eluted with 50 mm NaH₂PO₄-25% ($\text{v}\text{v}$) acetonitrile, pH 6.0. The retention times were 3.3, 6.0, and 9.6 min for angiotensin II, III, and I, respectively. ¹²⁵I-Angiotensins II, III, and I eluted with retention times of 5.4, 16.8, and 19.9 min, respectively, under the same chromatographic conditions used for the unlabeled angiotensins. The effect of iodination of the tyrosine residue on the retention time was also demonstrated by chromatographic comparison of tyrosine and diiodotyrosine. Saralasin (Sar¹, Ala⁸-angiotensin II), a partial agonist of angiotensin II, and des-Asp¹, Ile⁸-angiotensin II, an inhibitor of angiotensin III, eluted with retention times of 2.5 and 3.9 min, respectively.     

Role of angiotensin II in oral contraceptive hypertension in anesthetized rats

Rats fed a diet containing an oral contraceptive estrogen-progestogen mixture (Enovid) for 26 weeks developed a significant (P<0.01) elevation in mean arterial pressure (145 ± 6 mm Hg) when compared to a control group of rats fed the same diet without oral contraceptive steroids (mean arterial pressure averaged 117 ± 6 mm Hg). Infusion of [Sar¹, Ala⁸] angiotensin II, a competitive antagonist of angiotensin II, reduced the mean arterial pressure (P<0.01) in the oral contraceptive-treated rats to 117 ± 9 mm Hg, while infusion of this angiotensin II analogue into the control rats did not lower the arterial pressure. These studies provided evidence that angiotensin II may be involved in the hypertension produced by Enovid treatment in rats.     

The Arrestin-selective Angiotensin AT1 Receptor Agonist [Sar1,Ile4,Ile8]-AngII Negatively Regulates Bradykinin B2 Receptor Signaling via AT1-B2 Receptor Heterodimers

The renin-angiotensin and kallikrein-kinin systems are key regulators of vascular tone and inflammation. Angiotensin II, the principal effector of the renin-angiotensin system, promotes vasoconstriction by activating angiotensin AT₁ receptors. The opposing effects of the kallikrein-kinin system are mediated by bradykinin acting on B₁ and B₂ bradykinin receptors. The renin-angiotensin and kallikrein-kinin systems engage in cross-talk at multiple levels, including the formation of AT₁-B₂ receptor heterodimers. In primary vascular smooth muscle cells, we find that the arrestin pathway-selective AT₁ agonist, [Sar¹,Ile⁴,Ile⁸]-AngII, but not the neutral AT₁ antagonist, losartan, inhibits endogenous B₂ receptor signaling. In a transfected HEK293 cell model that recapitulates this effect, we find that the actions of [Sar¹,Ile⁴, Ile⁸]-AngII require the AT₁ receptor and result from arrestin-dependent co-internalization of AT₁-B₂ heterodimers. BRET₅₀ measurements indicate that AT₁ and B₂ receptors efficiently heterodimerize. In cells expressing both receptors, pretreatment with [Sar¹,Ile⁴,Ile⁸]-AngII blunts B₂ receptor activation of G_q/11-dependent intracellular calcium influx and G_i/o-dependent inhibition of adenylyl cyclase. In contrast, [Sar¹,Ile⁴,Ile⁸]-AngII has no effect on B₂ receptor ligand affinity or bradykinin-induced arrestin3 recruitment. Both radioligand binding assays and quantitative microscopy-based analysis demonstrate that [Sar¹,Ile⁴,Ile⁸]-AngII promotes internalization of AT₁-B₂ heterodimers. Thus, [Sar¹,Ile⁴,Ile⁸]-AngII exerts lateral allosteric modulation of B₂ receptor signaling by binding to the orthosteric ligand binding site of the AT₁ receptor and promoting co-sequestration of AT₁-B₂ heterodimers. Given the opposing roles of the renin-angiotensin and kallikrein-kinin systems in vivo, the distinct properties of arrestin pathway-selective and neutral AT₁ receptor ligands may translate into different pharmacologic actions.     

A new approach to angiotensin antagonists: Methylation of the tyrosine hydroxyl in angiotensin II

[Sar¹, Tyr(Me)⁴] angiotensin II, synthesized by the solid phase method and purified by ion-exchange chromatography and reversed-phase HPLC, was found to inhibit the contractile response to angiotensin II in the rat isolated uterus and inhibit the pressor response to angiotensin II in the vagotomized ganglion-blocked rat. In the rat isolated uterus Schild plots gave a pA₂ of 8.1, and a slope of 0.9 indicative of competitive inhibition. In the rat pressor assay, infusion of the analogue at a rate of 500ng/kg/min caused a parallel displacement of the dose-response curve to ANG II to the right. In contrast, the classical angiotensin inhibitor [Sar¹, Ile⁸] ANG II appeared to demonstrate non-competitive inhibition in both the rat isolated uterus and the pressor assays. The phenolic hydroxyl or phenoxide anion of Tyr⁴ in angiotensin II appears to be critical for the activation of angiotensin receptors in smooth muscle. Alkylation of the tyrosine residue in angiotensin analogues provides a new route for the synthesis of potent competitive antagonists of angiotensin.     

Effects of an angiotensin II antagonist; [sarcosine 1, isoleucine 8] angiotensin II, on blood pressure, plasma renin activity and plasma aldosterone concentration in hypertensive and normotensive subjects taking oral contraceptives.

To examine the involvement of renin-angiotensin-aldosterone system in the etiology of oral contraceptive induced hypertension, normal women (Group I), normotensive (Group II) and hypertensive (Group III) women taking Ovulen (R) were infused with a competitive angiotensin II (AII) antagonist, [1-sarcosine, 8-isoleucine] angiotensin II. The angiotensin II antagonist was infused at a rate of 600 ng/kg/min for 30 min 1.5 hrs after intravenous injection of 40 mg of furosemide. Blood pressure was monitored and pre-infusion and post-infusion plasma renin activity (PRA) and plasma aldosterone concentration (PAC) were determined. Pre-infusion PRA and PAC showed no significant differences among these three groups. In response to the AII antagonist infusion blood pressure rose in Groups I and II, but blood pressure responses in Group III were variable. Four out of the total 6 subjects had pressor responses and only one subject had a significant blood pressure reduction. In both Groups I and II, PRA decreased and PAC rose after infusion of the antagonist. In Group III, PRA decreased to a lesser degree and PAC showed no consistent change. These data suggest that the renin-angiotensin-aldosterone system in hypertensive women or oral contraceptives is different from that of the normotensive users. However, the pathophysiology of oral contraceptive induced hypertension is not homogenous and angiotensinogenic hypertension is uncommon.     

Identification and characterization of angiotensin II receptors in cardiac sarcolemma

We have used [¹²⁵I] angiotensin II to investigate the presence of specific angiotensin II receptors in beef heart sarcolemmal membranes. The observed binding is saturable, reversible and specific. The apparent equilibrium dissociation constant is 2.23 ± 0.15 ($\text{x}$ ± SEM) and the maximal number of binding sites per mg membrane protein is 32.8 ± 5.4 fmol ($\text{x}$ ± SEM). The specific binding is 80–100% of the total [¹²⁵I] angiotensin II bound and is directly proportional to membrane protein concentration over the range of 33–173 μg protein per ml. Angiotensin II and its antagonists competed for binding in a potency order of (agent, K_i): angiotensin II, 0.9nM > Sar¹ Ala³, 7 nM > Sar¹-Ile³, 51 nM > Sar¹-Leu³, 427nM > angiotensin I, 1709 nM. The ability to characterize and quantify these receptors should now provide a method for investigating the mechanisms underlying the effects of angiotensin II on myocardial tissues.     

Conformation and activity in angiotensin II peptides

Angiotensin II and its competitive inhibitor [Sar¹, Ile⁸]-angiotensin II, as well as several analogs of these two compounds specifically chosen for their well-defined pharmacological properties, were studied by circular dichroism and nuclear magnetic resonance methods at various pH values in aqueous solution and in d₆-dimethylsulfoxide. The results were compared with their biological activities. This allowed us to establish relationships between conformation and pressor activity, explaining most of the properties of angiotensin II, its inhibitor, and the analogs successively substituted in positions 3 and 5.     

Angiotensin stimulates oxytocin release

A sensitive and specific radioimmunoassay for oxytocin (OT) was developed to study the effect of angiotensin II (ANG II) upon neurohypophyseal OT release in conscious rats. ANG II injected into the lateral cerebral ventricle (i.c.v.) produced within 60 seconds a steep increase in plasma OT concentration from a control value of 10.46 ± 1.35 fmol/ml to 88.95 ± 5.06 fmol/ml and 119.56 ± 11.46 fmol/ml following 10 and 100 ng i.c.v. ANG II, respectively. Inhibition of OT release by simultaneous application of the ANG II antagonist {Sar¹, Ile⁸}-ANG II suggests that ANG II acted via specific angiotensin receptors in the brain.     

Requirement of membrane-proximal amino acids in the carboxyl-terminal tail for expression of the rat AT1a angiotensin receptor

A series of deletion mutants was created to analyze the function of the membrane-proximal region of the cytoplasmic tail of the rat type 1a (AT_1a) angiotensin receptor. In transiently transfected COS-7 cells, the truncated mutant receptors showed a progressive decrease in surface expression, with no major change in binding affinity for the peptide antagonist, [Sar¹,Ile⁸]angiotensin II. In parallel with the decrease in receptor expression, a progressive decrease in angiotensin II-induced inositol phosphate responses was observed. Alanine substitutions in the region 307–311 identified the highly conserved phenylalanine³⁰⁹ and adjacent lysine residues as significant determinants of AT_1a receptor expression.     

Interaction of Cyclic AMP Derivatives with the Angiotensin II Receptor

Abstract

The effect of cyclic AMP and of its derivatives was studied on ¹²⁵I-angiotensin II and ¹²⁵I- (Sar¹, Ala⁸) -angiotensin II binding to rat adrenal membrane receptors. Dibutyryl cyclic AMP, 8-bro-mo-cyclic AMP and cyclic AMP inhibited both agonist and antagonist binding in a specific and dose-dependent way, with K₁ of 1.3 mM, 6.8 mM and about 30 mM, respectively. Scatchard analysis of binding data indicated that the nucleotides interacted directly with the membrane receptor for angiotensin II. These results suggest that cyclic AMP may act extracellularly and affect receptor-mediated events.     

Biological activity of des-(Asp1, Arg2, Val3)-angiotensin II in man

When des-(Asp¹, Arg², Val³)-angiotensin II was infused iv at rates of 308–5,550 pmol/kg·min for 10–120 min into 5 normal men and 2 patients with Bartter's syndrome, no significant change was observed in blood pressure (BP), plasma renin activity (PRA) or plasma aldosterone (PA), and the lowest dose did not inhibit a captopril-induced increase in PRA in the normal men, although des-(Asp¹, Arg²)-angiotensin II was reported in the same 5 normal men to cause a decrease in PRA and an increase in PA in this dose range and a rise in BP at 2,220 and 5,550 pmol/kg·min. However, an iv infusion of the pentapeptide at 9,000 pmol/kg·min for 15 min significantly raised BP in the 5 normal men but not in patients with Bartter's syndrome. BP returned to the pretreatment level 60 min after the cessation of the infusion, although the duration of the pressor actions of angiotensin II, angiotensin III and des-(Asp¹, Arg²)-angiotensin II were reported to be within 5 min in man. At the same dose level none of the 7 examined subjects showed any significant change in PRA or PA. Des-(Asp¹, Arg², Val³, Tyr⁴)-angiotensin II was infused iv at a rate of 41,480 pmol/kg·min into one of the normal men, but it caused no significant change in BP, PRA or PA. These results suggest that the pentapeptide and probably the tetrapeptide do not possess renin-suppressing and steroidogenic actions in man but the pentapeptide does elecit a minimal pressor action with a prolonged duration.     

Effects of angiotensin II and its blockers Sar1-lle8-angiotensin II and DuP 753 on drinking in ducks in relation to properties of subfornical organ neurons

Properties of systemically applied angiotensin II in stimulating water intake of normally hydrated ducks were studied and the results compared with properties of angiotensin II-responsive neurons of the subfornical organ which are considered as targets for circulating angiotensin, II acting as a dipsogen. Following intravenous infusion of hypertonic saline (2000 mosmol·kg^-1 at 0.3 ml·min^-1 for 1 h), intravenous infusion of 0.3 ml·min^-1 isotonic saline with angiotensin II (200 ng·min^-1), starting 1 h later, stimulated drinking in each case at an angiotensin II plasma level of about 1400 pg·ml^-1. Without hypertonic priming, the same angiotensin II infusion did not stimulate drinking in each experiment; however, if effective, repeated infusions of ANGII induced stable dipsogenic responses. Angiotensin II infusions did not alter plasma levels of antidiuretic hormone. Sar¹-Ile⁸-angiotensin II, a non-selective angiotensin II antagonist, acted weakly as a partial agonist when injused at a dose 200-fold higher than angiotensin II and effectively blocked the dipsogenic action of angiotensin II; this corresponds to the inhibition of angiotensin II-induced excitation by Sar¹-Ile⁸-angiotensin II observed in duck subfornical organ neurons. DuP 753 (losartan), an angiotensin II antagonist specifically blocking AT₁ receptors in mammals, had equivocal effects on angiotensin II-induced drinking in ducks at rates 50- and 200-fold higher than angiotensin II, which corresponds to the weak inhibitory action of this compound on angiotensin II-induced neuronal excitation in the duck SFO. Blood pressure was only marginally elevated by the applied angiotensin II dose and Sar¹-Ile⁸-angiotensin II had no effect.Abbreviations ANGII angiotensin II - AVT arginine vasotocin - DuP 753 losartan - EDTA ethylene diamine tetra-acetic acid - HR heart rate - ICV intracerebroventricular - IV intravenous - MAP mean arterial pressure - SARILE Sar¹-Ile⁸-angiotensin II - SFO subfornical organ     

Angiotensin II-induced fluid phase endocytosis in human cerebromicrovascular endothelial cells is regulated by the inositol-phosphate signaling pathway

The involvement of the early signaling messengers, inositol tris-phosphate (IP₃), intracellular calcium, [Ca²⁺]_i, and protein kinase C (PKC), in angiotensin II (AII)-induced fluid phase endocytosis was investigated in human brain capillary and microvascular endothelial cells (HCEC). AII (0.01–10 μM) stimulated the uptake of Lucifer yellow CH, an inert dye used as a marker for fluid phase endocytosis, in HCEC by 50–230%. AII also triggered a fast accumulation of IP₃ and a rapid increase in [Ca²⁺]_i in cells loaded with the Ca²⁺-responsive fluorescent dye fura-2. The prompt AII-induced [Ca²⁺]_i spike was not affected by incubating HCEC in Ca²⁺-free medium containing 2 mM EGTA or by pretreating the cultures with the Ca²⁺ channel blockers, methoxyverapamil (D600; 50 μM), nickel (1 mM), or lanthanum (1 mM), suggesting that the activation of AII receptors on HCEC triggers the release of Ca²⁺ from intracellular stores. The AII-triggered increases in IP₃, [Ca²⁺]_i, and Lucifer yellow uptake were inhibited by the nonselective AII receptor antagonist, Sar¹, Val⁵, Ala⁸-AII (SVA-AII), and by the phospholipase C (PLC) inhibitors, neomycin and U-73122. By contrast, the protein kinase C (PKC) inhibitors, staurosporine and calphostin C, failed to affect any of these AII-induced events. This study demonstrates that increased fluid phase endocytotosis induced by AII in human brain capillary endothelium, an event thought to be linked to the observed increases in blood-brain barrier permeability in acute hypertension, is likely dependent on PLC-mediated changes in [Ca²⁺]_i and independent of PKC. © 1996 Wiley-Liss, Inc.     

Secretion of Immunoreactive endothelin-1 by capillary and microvascular endothelium of human brain

Modulation of immunoreactive endothelin-1 (IR-ET-1) production by vasoactive substances was investigated in cultured endothelial cells (EC) derived from capillaries and microvessels of human brain. Peptides, catecholamines, thrombin, protein kinase C-activating phorbol ester, and calcium ionophore enhanced the secretion of IR-ET-1. The known vasoconstrictive peptides, angiotensin II (Ang II) and arginine-vasopressin (AVP) dose-dependently stimulated the endothelial secretion of IR-ET-1. The angiotensin and vasopressin-inducible production of IR-ET-1 was completely inhibited by their respective receptor antagonists [Sar¹, Ala⁸]-angiotensin II and [1–6 (-mercapto-,-cyclopentamethylene propionic acid), 2-O-methyl-tyrosine]. The results indicate that the peptide-stimulated secretion of IR-ET-1 is receptor-mediated in EC which have specific angiotensin II and arginine-vasopressin receptors. These findings represent the first demonstration of IR-ET-1 production by capillary and microvascular endothelium of human brain.     

Synthesis,characterization, and biological activity of Nα-(N-fluoresceinthiocarbamoyl)-(Asp1, Ile5)-angiotensin II

A fluorescent analog of angiotensin II was synthesized by reacting fluorescein 5′-isothiocyanate with (Asp¹, Ile⁵)-angiotensin II. N^α-(N-Fluoresceinthiocarbamoyl)-(Asp¹, Ile⁵)-angiotensin II was purified by chromatography on DEAE-cellulose and Sephadex G-25. Analysis of the analog by thin-layer chromatography, thin-layer electrophoresis, and reversed-phase high-performance liquid chromatography indicated that the analog was free of angiotensin II and fluorescein 5′-isothiocyanate. N-Terminal sequence analysis demonstrated that fluorescein 5′-isothiocyanate reacted with the N-terminal aspartic acid residue of angiotensin II. N^α-(N-Fluoresceinthiocarbamoyl)-(Asp¹, Ile⁵)-angiotensin II has an absorption maximum at 492 nm, and the value of the molar extinction coefficient, ?, is 7.7 × 10⁴m^?1 cm^?1. The fluorescence emission maximum occurs at 520 nm. Infusion of the analog (0.69 μg/min/kg body wt) directly into the renal artery of an anesthetized rat reduced the blood flow by 12 to 27% within 2 min. Infusion of angiotensin II (0.48 μg/min/kg body wt) reduced renal arterial blood flow by 35 to 53% within 2 min. Saralasin, a partial agonist and antagonist of angiotensin II, inhibited the biologic effect of the fluorescent analog and angiotensin II by 75 and 70%, respectively. The purity, spectral properties, and in vivo biologic activity of N^α-(N-fluoresceinthiocarbamoyl)-(Asp¹, Ile⁵)-angiotensin II indicate that this analog should facilitate characterization of angiotensin II receptors.     

Conformational analysis of cyclic angiotensin II analogues

Summary Conformationally restricted cyclic analogues of angiotensin II (ANG II), Asp¹-Arg²-Val³-Tyr⁴-Val⁵-His⁶-Pro⁷-Phe⁸, with a link between positions 3 and 5 have considerable biological activity. It is proposed that the spatial arrangement of the pharmacophore groups of Tyr⁴, His⁶ and Phe⁸ side chains and the C-terminal carboxyl group in ANG II and active analogues is similar. Conformational analysis of ANG II and two cyclic analogues c[Sar¹, Lys³,Glu⁵]ANG II and c[Sar¹,Hcy³,Mpt⁵]ANG II was performed, and a geometrical comparison of the low-energy conformations of these compounds allowed one to propose a model of receptor-bound conformation in terms of the spatial arrangement of the pharmacophore groups. This model is characterised by the close spatial location of the His⁶-Phe⁸ side chains and the Tyr⁴ C-terminal carboxyl group and is stabilised by the electrostatic interaction of Arg² and the C-terminal carboxyl group.Abbreviations ANG II angiotensin II - Hcy homocysteine - Mpt trans-4-mercaptoproline