A comparative study of two structurally related analogs of Asp1-Ile5-angiotensin II

Summary Two new synthetic analogs of Asp¹-Ile⁵-angiotensin II, Sar¹-Ile⁵-Ile⁸-angiotensin II and (N,N-dimethyl) Gly¹-Ile⁵-Ile⁸-angiotensin II which are chemically related, have a potent competitive antagonistic action against Asp¹--amid-Val⁵-angiotensin II on the rabbit isolated aorta, rat ascending colon, isolated perfused heart of cat and in vivo on the blood pressure and intestinal motility of the chloralose-anesthetized cat. Compared to Sar¹-Ile⁵-Ile⁸-angiotensin II, (N,N-dimethyl) Gly¹-Ile⁵-Ile⁸-angiotensin II has equal antagonistic potency on the isolated preparations but a lower potency in vivo. The duration of the antagonistic effect of (N,N-dimethyl) Gly¹-Ile⁵-Ile⁸-angiotensin II in all preparations investigated has been found to be significantly shorter than that of Sar¹-Ile⁵-Ile⁸-angiotensin II. It is assumed that replacement of sarcosine with N,N-dimethyl glycine at the NH₂-terminal position, enhances the degradation of the analog by aminopeptidase and therefore decreases its half-life.     

Sar1-Ile8-angiotensin II decreases urinary PGE2 and PGF2α excretion in rabbits on dietary sodium restriction

Urinary PGE₂ and PFG_2α excretion (U_PGV) were determined prior, during and after 48 h treatment with angiotensin-II-blocker, Sar¹-Ile⁸-angiotensin II (200 μg/kg/3 h), in rabbits maintained on chronic low (LS) or high (HS) salt diets. U_PGE2 V and F_2α were significantly (P < 0.001) higher in LS than in HS. Sar¹-Ile-⁸-angiotensin II decreased the elevated U_PGE2 V and U_PGF2α V in LS from 2.60 ± 0.084 μg/24 h to 1.13 ± 0.21 μg/24 h and from 6.63 ± 0.61 to 3.77 ± 0.86 μg/24 h respectively. In animals with HS intake, angiotensin II blockade failed to effect urinary prostaglandin excretion.     

Effect of three angiotensin II antagonists, [Sar1, Thr8]-, [Sar1, Ile8]-and [Sar1, Ala8]angiotensin II on blood pressure and endocrine factors in normal subjects

Summary The biological effects of 1-Sarcosine, 8-Threonine angiotensin II ([Sar¹, Thr⁸]ANG II) on blood pressure, plasma aldosterone concentration (PAC) and plasma renin activity (PRA) were investigated in six normal subjects on an unrestricted diet, and compared with those of 1-Sarcosine, 8-Isoleucine ANG II ([Sar¹, Ile⁸]ANG II) and 1-Sarcosine, 8-Alanine ANG II ([Sar¹, Ala⁸]ANG II). All three ANG II analogues (A II A) showed agonistic pressor activity, that of [Sar¹, Ile⁸]ANG II being greater than that of [Sar¹, Thr⁸]ANG II or [Sar¹, Ala⁸]ANG II. The antagonistic effect of [Sar¹, Thr⁸]ANG II on blood pressure was less than [Sar¹, Ile⁸]ANG II or [Sar¹, Ala⁸]ANG II. Both [Sar¹, Ile⁸]ANG II and [Sar¹, Ala⁸]ANG II increased PAC and blocked the steroidogenic action of ANG II, while [Sar¹, Thr⁸]ANG II showed little effect on PAC. All three A II A caused similar suppression of PRA and showed no inhibitory effect on the decrease in PRA produced by ANG II. These results indicate that [Sar¹, Thr⁸] ANG II is an A II A with weak agonistic pressor action and that it has vascular selective properties. It is also suggested that ANG II receptors in a variety of target organs are heterogeneous.     

Effect of an angiotensin antagonist,Sar1-Ala8-angiotensin II on physiological thirst

Initially it was shown that infusion of Sar¹-Ala⁸-angiotensin II (P113) into the third ventricle (50–100 μg/ml at 1.1 ml/hr) effectively abolished the large water intake induced 1–2 min after beginning an intracarotid infusion of angiotensin II at 800 ng/min which causes an unphysiologically high concentration of angiotensin II in cerebral arterial blood. Infusion of P113 (50–100 μg/ml at 1.1 ml/hr) into the third brain ventricle for 20 min prior to and during presentation of water to sheep after 48 hr water deprivation did not reduce water intake. Water intake associated with rapid food intake or carotid artery infusion of hypertonic NaCl was similarly unaffected by intraventricular administration of P113. While high concentrations of angiotensin II are dipsogenic in sheep, these results cast doubt on a contributory role for angiotensin II in thirst caused by water depletion or rapid food intake in the sheep.     

DIFFERENT POTENCIES OF [ASP1, ILE5]- AND [ASN1 VAL5]-ANGIOTENSIN II IN STIMULATING ALDOSTERONE PRODUCTION FROM RAT ADRENAL ZONA GLOMERULOSA CELLS IN VITRO

1. Rat adrenal zona glomerulosa cells were incubated in Krebs Ringer bicarbonate medium containing bovine serum albumin. Aldosterone production was measured by radioimmunoassay. 2. Both [Asp¹, Ile⁵]-angiotensin II ('free acid') and (Asn¹, Val⁵]-angiotensin II ('amide'), (10^?5 mol/1), stimulated aldosterone output but the free acid compound produced a larger increase in steroid output than the amide. 3. This difference in steroidogenic activity could not be explained by differences in purity or initial concentration or by stimulation of the contaminating fasciculata cells. 4. However, serial bioassays of the media revealed that the amide form of angiotensin was degraded more rapidly than the free acid form. 5. The different potency of these two forms of angiotensin II could explain some of the reported discrepancies of the action of angiotensin II on rat adrenal tissue or cells in vitro.     

Editorial

Analogues of the Type I angiotensin (ANG) antagonist, [Sar¹,Ile⁸]ANG II, in which the N-terminal dipeptide was modified were synthesized by the solid phase method and purified by reversed-phase HPLC. Antagonist potencies (pA₂) of the peptides were determined on the rat isolated uterus using ANG II as the agonist. Substitution of the Arg residue occupying position 2 of [Sar¹,Ile⁸]ANG II (pA₂ 8.1) by Gly, Ala, Nle, Phe, Pro or Sar reduced the antagonist potency to pA₂= 7.0, 6.8, 6.7, 6.8, 5.8 and 5.3, respectively. Deletion of the N-terminal Sar residue in these same peptides gave pA₂= 6.8, 5.7, 5.5, 5.9, 6.1 and 7.5, respectively. The characteristically long duration of action of [Sar¹,Ile⁸] was absent for all of these analogues including (des¹, Sar², Ile⁸]ANG II. These findings demonstrate that the antagonist potencies of Type I angiotensin antagonists for smooth muscle receptors, and also the long duration of action, are dependent on the location of positive charges within the peptide and on the conformation of the molecule in determining favorable electrostatic interactions with the receptor. A model is proposed in which the two positively charged loci on the angiotensin molecule (N-terminus and Arg) interact with two corresponding anionic binding sites on the smooth muscle receptor. The possibility that the prolonged duration of action of [Sar¹, Ile⁸]ANG II results from binding to a different site on the angiotensin receoptor from that occupied by ANG II is discussed in relation to the present findings.     

Influence of methylation of the histidine ring of [Sar1]angiotensin II on conformation and biological activity

[Sar¹His(1-Me)⁶]ANG II and [Sar¹His(3–Me)⁶]ANG II were synthesized by the solid-phase method and purified by reversed-phase HPLC. ¹H-NMR spectroscopy at 400 MHz demonstrated the presence of two major conformers for both peptides in DMSO, representing cis and trans isomers (ratio 1:3 and 1:4, respectively) due to restricted rotation at the His-Pro bond. The contractile activities of these peptides in the rat isolated uterus assay were <0.1 and 27% of that of ANG II, respectively. The bioactivities of these analogues were mirrored in their NMR spectra: the inactive analogue [Sar¹His(1-Me)⁶]ANG II showed perturbations of the Sar and His residues which were not present for [Sar¹His(3-Me)⁶]ANG II and the reference agonist [Sar¹] ANG II. The high activity of the analogue methylated at His N₃ suggests that an ionizable imidazole proton is not an absolute requirement for expression of biological activity by angotensin ligands, and that the imidazole group in the molecule may function in an ion dipole-based mechanism when an intramolecular proton transfer (charge relay) mechanism is not available. The biological activity of the ligand appears to depend on the degree of proton transfer from Tyr-OH to the imidazole acceptor, wherein complete (formal) proton transfer represents 100%, activity.     

IMPORTANCE OF CIRCULATING ANGIOTENSIN II FOR ELEVATION OF ARTERIAL PRESSURE DURING ACUTE HYPERCAPNIA IN ANAESTHETIZED DOGS

1. In anaesthetized dogs acute hypercapnia produced by ventilation with 10% CO₂ in air caused release of vasoactive substances and changes of arterial pressure. Catecholamines, angiotensin II, bradykinin and prostaglandin-like substances were bioassayed continuously in arterial blood using the blood-bathed organ technique of Vane. 2. Acute hypercapnia in control dogs causes responses of rat stomach strip, chick rectum and rat colon indicating initial release of noradrenaline, followed by angiotensin II, and occasionally release of prostaglandin-like substances into the circulation. 3. The initial transient, pressor response to hypercapnia coincided with the appearance of noradrenaline, and the secondary progressive rise in arterial pressure was accompanied by increased circulating angiotensin II. 4. In dogs treated with the angiotensin converting enzyme inhibitor SQ 20881 (1–2 mg/kg, i.v.), hypercapnia did not cause the secondary elevation of arterial pressure despite pronounced elevation of noradrenaline levels in the circulation. Increased arterial concentrations of bradykinin of less than 1 ng/ml, assayed by a strip of cat jejunum, were detected during hypercapnia in four out of six dogs treated with SQ 20881, but not in untreated dogs. 5. Intravenous infusion of the angiotensin antagonist [Sar¹-Ile⁸]-angiotensin II (0.5–1.0 μg/kg per min) attenuated the secondary pressor response to hypercapnia and abolished the contractions of rat colon produced by hypercapnia and angiotensin II. 6. The results indicate that adrenergic stimulation may be responsible for the transient pressor response on induction of hypercapnia, but elevation of angiotensin II activity in the circulation is of major importance for mediating the secondary progressive rise in arterial pressure during the later stage of acute respiratory acidosis. 7. After inhibition of angiotensin converting enzyme, hypercapnia reduces arterial pressure, not only because the vasoconstrictor effect of angiotensin has been removed, but also because inhibition of pulmonary kininase may allow vasodilator kinins to reach the arterial circulation.     

Pharmacological studies of neurotensin,several fragments and analogues in the isolated perfused rat heart

Neurotensin (NT) induced a dose-dependent increase of the coronary perfusion pressure (CPP) in the isolated perfused rat heart. This effect was not modified by pretreating the organ with methysergide (8.5 × 10^?6 M), atropine (3.4 × 10^?6 M), a mixture of phentolamine (3.1 × 10^?5 M), 8-leucine-angiotensin II (1.0 × 10^?6 M), indomethacin (2.8 × 10^?5 M) or a mixture of cimetidine (2.6 × 10^?5M) and diphenhydramine (2.9 × 10^?5 M) thus suggesting the existence of specific NT receptors in the coronary vessels of rat. The structure-activity studies performed using several NT fragments and analogues in the isolated perfused rat heart led us to the following conclusions: (1) the minimum structure required for the full expression of the biological activity of NT is H-Arg⁹-Pro¹⁰-Tyr¹¹-Ile¹²-Leu¹³-OH; (2) the amino acid Tyr in position 11 appears to play a key role in the process of NT receptor activation. The replacement of Tyr¹¹ with Tyre(Me) gave a compound which inhibits selectively the increase in coronary perfusion pressure induced by NT, but still exhibits some NT-like activity, specially when used in concentrations higher than 10^?6M. [Tyre(Me)¹¹]NT did not antagonize the stimulant effects of NT in rat stomach and guinea pig atria, thus suggesting that the receptors mediating the constrictor effect of NT in coronary vessels of the rat are pharmacologically different form those subserving the stimulant effect of NT in rat stomach strip and guinea pig atria.     

Influence of the adrenal gland on the pressor effect and antagonistic potency of angiotensin II analogs.

In ganglion blocked vagotomized rats, several 1,8-substituted angiotensin II analogs (250 ng/kg/min, i.v.) antagonized the pressor effect of angiotensin II. Dose ratios measured at the ED20 levels were: [Sar1,Ile8]-28; [Gac1,Ile8]- 19;[MeAla1,Ile8i1- 16;[MeIle1,Ile8]- 10;[sar1,Ala8]- 9;[me2Gly1,Ile8]- 4. Elimination of aspajtic acid in position 1 of [Ile8]-angiotensin II significantly reduced the antagonistic potency of the analog. No antagonistic effect was observed with [Phe4,Ile8] and [Ala4,Ile8]-angiotensin II even when infused at 6 mug/kg/min. During infusion, a partial rise in blood pressure was observed with all the above 1,8-substituted angiotensin II analogs. Phentolamine (100 mug/rat) injected 30 min after the start of the analog infusion reduced and sometimes abolished the pressor effect. However, phenoxybenzamine )Pbz, 2 mg/kg) injected 30 min prior to the analog infusion diminished but did not completely abolish the initial pressor effect. In adrenalectomized rats, the pressor effect was reduced by approximately 50 percent and disappeared completely 15-30 min after start of the infusion. Under these conditions, dose ratios of [Sar1,Ile8]-,[MeAla1,Ile8]- and [Gac1,Ile8]-angiotensin II were significantly reduced. Noradrenaline, 83 ng/kg/min. increased the ED20 value of angiotensin II(ratio 1.79) in normal rats but did not do so in adrenalectomized rats. In these rats no regular correlation was found between the angiotensin II ED20 values and initial blood pressure. These data indicate that under the present experimental conditions, the low pressor effect observed with these angiotensin II antagonists appears to be due to both adrenal catecholamine release and a direct vasoconstrictor effect. Variations in antagonistic activity of angiotensin II analogs, apart from changes introduced in the molecule, may be the manifestation of a complex interaction between angiotensin II, its antagonists, and the sympathoadrenal system.     

Effect of cyclopiazonic acid on contractions produced by tachykinin NK1 and NK2 receptor agonists in the circular muscle of guinea-pig colon

1 This study aimed to assess the effect of cyclopiazonic acid (CPA), an inhibitor of sarcoplasmic reticulum calcium (Ca) pump, against contractile responses produced by selective tachykinin NK₁ and NK₂ receptor agonists, [Sar⁹]substance P (SP) sulfone and[βAla⁸]neurokinin A (NKA) (4–10), respectively, on the circular muscle of guinea-pig colon. All experiments were performed in the presence of atropine (1 μm ) and indomethacin (10 μm ). 2 In organ bath experiments, a submaximally equieffective concentration of the two agonists (10 nm ) was selected: [Sar⁹]SP sulfone (10 nm ) produced a biphasic contraction, the two amplitudes averaging 75 ± 2 and 43 ± 3% of the maximal response to KCl (80 mm ) at 1 and 15 min from application of the agonist, respectively. CPA (3 μm for 60 min) slightly reduced the phasic response to [Sar⁹]SP sulfone (16 ± 4% inhibition) and markedly suppressed the tonic component (89 ± 3% inhibition). 3 The contraction produced by [βAla⁸]NKA (4–10) (10 nm ) was more sustained than that induced by the NK₁ receptor agonist: it averaged 69 ± 5 and 73 ± 4% of the response to KCl at 1 and 15 min from application of the agonist, respectively. CPA slightly and evenly depressed the response to [βAla⁸]NKA (4–10) (18 ± 7 and 21 ± 5% inhibition at 1 and 15 min). 4 In the presence of tachykinin NK₁ and NK₂ receptor antagonists (SR 140333 and MEN 10627, respectively, 1 μm each) and of l -nitroarginine (100 μm ), KCl (40 mm ) produced a distinct phasic and tonic contraction which was suppressed by 1 mm nifedipine. CPA (3 μm ) did not affect the phasic contraction to KCl but abolished the tonic component of the response. 5 In the presence of 1 μm nifedipine, the response to [βAla⁸]NKA (4–10) was slightly depressed (32 ± 6% inhibition) in its early component only, while the response to [Sar⁹]SP sulfone was abolished. CPA produced a slight inhibition (15 ± 9 and 33 ± 10% at 1 and 15 min, respectively) of the nifedipine-resistant response to [βAla⁸]NKA (4–10), an effect similar to that observed in the absence of nifedipine. Therefore, a large part of the response to [βAla⁸]NKA (4–10) persisted in the presence of both CPA and nifedipine. 6 In the sucrose gap, a prolonged superfusion with [Sar⁹]SP sulfone (0.1 μm for 5 min) produced sustained depolarization with superimposed spikes and contraction. CPA (3 μm ) produced transient depolarization and contraction. In the presence of CPA, the initial responses (depolarization, spikes and contraction) to [Sar⁹]SP sulfone were unaffected but the sustained component of contraction was absent; the latter effect was accompanied by a suppression of spikes while the sustained depolarization was present. 7 We conclude that, during sustained depolarization produced by the NK₁ receptor agonist, blockade of the sarcoplasmic reticulum Ca pump by CPA produces a faster Ca-dependent inactivation of Ca channels, thereby eliminating spikes and abolishing the tonic component of contraction. Ca mobilization/reuptake from a CPA-sensitive store seems to be of minor importance for regulating the NK₂ receptor-mediated contractile responses.     

Inhibition of tachykinin-induced hypotension in dogs by CP-96,345, a selective blocker of NK-1 receptors

Summary The effects of substance P, neurokinin A, neurokinin B, [Sar⁹, Met(O₂)¹¹]-substance P, [Nle¹⁰]neurokinin A (4–10) and senktide {succinyl-[Asp⁶, McPhe⁸]-substance P (6–11)} on blood pressure and heart rate were studied in anesthetized dogs. Dose-dependent decreases in blood pressure and increases in heart rate were caused by each peptide except senktide. The latter elicited weak hypotensive or hypertensive responses at high doses. The order or potency was as follows: [Sar⁹, Met(O₂)¹¹]-substance P substance P > neurokinin ß₁ > neurokinin B > [Nle¹⁰]-neurokinin A (4–10) » senktide.CP-96,345, [(2S,3S)-cis-2-(diphenylmethyl)-N-[(2-methoxyphenyl)-methyl]-1-azabicyclo[2.2.2]octan-3-amine] a selective NK-1 tachykinin receptor blocker, inhibited substance P-induced hypotension in a dose-related manner. R responses to each of the other peptides were inhibited by CP-96,345, 1.0 mg/kg (excluding senktide against which CP-96,345 was not tested). CP-96,344 (1.0 mg/kg i.v.) the 2R-3R enantiomer of CP-96,345 which does not block NK-1 receptors, had no effect on substance P-induced hypotension. We conclude that tachykinin-induced hypotension in dogs is mediated by NK-1 tachykinin receptors. Send offprint requests to J. W. Constantine at the above address     

Nitric oxide regulates angiotensin II receptors in vascular smooth muscle cells

The objective of this study was to determine whether an enhanced generation of nitric oxide (NO) causes regulation of angiotensin II receptors in vitro using rat vascular smooth muscle cells in culture. Chronic treatment of cells with a series of NO-generating drugs, sodium nitroprusside, S-nitroso-N-acetylpenicillamine and isosorbide dinitrate for 18h dose and time-dependently decreased [¹²⁵I]-angiotensin II binding to cells without any significant change in affinity. Induction of nitric oxide synthase following lipopolysaccharide (10 and 100 ng/ml) treatment of cells for 18 h increased basal nitric oxide synthase activity with a concomitant increase of nitrite and cyclic cGMP levels in the conditioned media. LPS treatment significantly (P < 0.05) decreased [¹²⁵I]-angiotensin II binding to these cells, an effect that was significantly (P < 0.05) attenuated in the presence of N^G-nitro-L-arginine methyl ester. In contrast, treatment of cells with atrial natriuretic factor, dibutyryl cGMP, 8-bromo-cGMP, NaNO₂ or NaNO₃ failed to significantly alter the affinity or number of [¹²⁵I]-angiotensin II binding sites. These results suggest that NO regulates angiotensin II receptors in vitro through a cGMP-independent mechanism.     

Tachyphylactic properties of angiotensin II analogs with bulky and hydrophobic substituents at the N‐terminus

Abstract: Tachyphylaxis, defined as the acute loss of response of some smooth muscles upon repeated stimulations with angiotensin II (Ang II), has been shown to be dependent mainly on the N‐terminal region of the ligand. To further study the structural requirements for the induction of tachyphylaxis we have synthesized Ang II analogs containing the bulky and very lipophilic substituents 9‐fluorenylmethyloxycarbonyl (Fmoc) and 9‐fluorenylmethyl ester (OFm) at the α‐amino (N^α‐Fmoc‐Ang II) or the β‐carboxyl ([Asp(OFm)¹]‐Ang II) groups of the Asp¹ residue, respectively. In binding assays with Chinese hamster ovary cells transfected with the AT₁ Ang II receptor, N^α‐Fmoc‐Ang II bound with high affinity, whereas [Asp(OFm)¹]‐Ang II showed lower affinity. In biological assays, these two analogs were full agonists and showed 30 and 3%, respectively, of the Ang II potency in contracting the guinea‐pig ileum smooth muscle. The two analogs induced tachyphylaxis, in spite of the lack of a free amino group in N^α‐Fmoc‐Ang II. Thus, analogs with Fmoc‐ or OFm‐type groups coupled to the Asp¹ residue, whether at the amino or carboxyl functions, induce tachyphylaxis through an unreported mechanism. Based in these findings and those available from the literature, an alternate molecular interaction mode between Ang II N‐terminal portion and the AT₁ receptor is proposed to explain the tachyphylactic phenomenon.     

Angiotensin II does not inhibit vagally-induced bradycardia or gastric contractions in the anaesthetized ferret

The effects of angiotensin II (AII) and the analogue Sar1-Leu8-angiotensin II on the changes in heart rate and gastric pressure induced by vagal stimulation were investigated in the urethane-anaesthetized ferret. AII at high doses (500 ng kg-1 i.v.) increased blood pressure and decreased gastric pressure whereas Sar1-Leu8-AII at the same dose had a much smaller effect on blood pressure and no effect on gastric pressure. Intravenous injections or infusions of AII or its analogue did not alter the effect of vagal stimulation on heart rate or gastric motility. Our results do not provide support for the proposal that AII modulates parasympathetic activity at peripheral sites (Potter, 1982a, b).     

ANGIOTENSIN II RECEPTOR DENSITY IN BOVINE OVARIAN FOLLICLES RELATES TO TISSUE RENIN AND FOLLICULAR SIZE

1. The purpose of this study was to investigate angiotensin II (AII) receptors in isolated bovine ovarian follicles and the relationship of their density to follicular concentrations of prorenin, active renin, oestradiol and progesterone. 2. Displacement of [¹²⁵I]-[Sar¹-Ile⁵-Ile⁸]-AII binding by the AII receptor antagonists PD 123319 and Losartan (DuP 753) confirmed that follicular AII receptors are of subtype 2 (AT₂ receptor). 3. The dissociation constant (K_d) for [Ile⁵]-AII (human AII) was 0.84 (range 0.51–1.47) nmol/ L. The receptor density varied between 90 and 5990 (mean 1640) fmol/ mg membrane protein. 4. The follicular AII receptor density correlated positively with follicular diameter (Spearman's rho= 0.518; P<0.003) and tissue weight (Spearman's rho= 0.636; P<0.0001), and negatively with the active renin concentration in the follicular wall (Spearman's rho=-0.399; P<0.02). The AII receptor density did not correlate with the follicular fluid concentrations of prorenin, active renin, oestradiol (E₂, progesterone (P₄) or the E₂/P₄ ratio. The follicular fluid concentrations of prorenin correlated negatively with the E₂/P₄ ratio (Spearman's rho= -0.716; P<0.00001). 5. The inverse relationship between AII receptor density and the high active renin concentrations in the follicular wall suggests an active regulated tissue renin-angiotensin system. A high AII receptor density is a general feature of large bovine ovarian follicles.     

Assessment of biological activity of novel peptide analogues of angiotensin IV

Objectives Angiotensin IV (Ang IV) is a metabolite of angiotensin II which acts on specific AT₄ receptors identified as the enzyme insulin regulated aminopeptidase (IRAP). The transduction process of these receptors is unresolved, but Ang IV inhibits the aminopeptidase activity. Ang IV improves cognition in animal models thus there is a desire to develop metabolically stable analogues for further development. Methods Peptide analogues of Ang IV were obtained commercially or synthesised. Each peptide was tested in vitro for its ability to inhibit the aminopeptidase activity (IRAP) of mouse brain homogenates and for its effects on isolated rat uterine smooth muscle. Key findings [Des‐Val¹]‐Ang IV, acetylated‐Ang IV‐amide, Ang IV‐amide and [des‐His⁴]‐Ang IV all inhibited IRAP. [Sar¹, Ile⁸]‐Angiotensin II (10 µm ) had an effect greater than that of Ang IV or any of the other analogues studied. In isolated uterine smooth muscle, angiotensins II and IV induced contractions, which could be antagonised by an AT₁‐receptor antagonist. None of the novel peptides induced uterine smooth muscle contractions, but [Sar¹, des Arg²‐Gly⁸]‐angiotensin II showed significant antagonism of the contractile effects of angiotensin II and carboxyamide‐terminated Ang IV‐NH₂ showed antagonism of Ang IV‐induced contractions. Conclusions This study provides five novel inhibitors of IRAP worthy of assessment in behavioural models of learning and memory. The analogues are devoid of AT₁ receptor agonist properties, and the carboxyamide analogue presents an opportunity to elucidate the mechanism of action of Ang IV as, like Ang IV, it inhibits IRAP, but antagonises the effects of Ang IV on isolated smooth muscle.     

Synthesis and biological activities of angiotensin II and Sarmesin analogues containing cyclohexylalanine

Analogues of angiotensin II with cyclohexylalanine (Cha) at position 4 or 8, and analogues of the competitive (type II) angiotensin antagonist [Sar¹,Tyr(Me)⁴]ANG II (Sarmesin) with Cha at position 8, have been prepared by the solid phase method and purified by reversed-phase HPLC. Analogues of ANG II with Cha at position 8 in which the position 1 residue was substituted with sarcosine (Sar) or amino-isobutyric acid (Aib) or was deleted (Des), were slowly reversing (Type I) antagonists with ?pA₂” values in the rat isolated uterus assay of ～ 8.5. The additional substitution of Tyr(Me) for Tyr at position 4 of these peptides gave reversible competitive (Type I/II) antagonists with pA₂ values of 6.7, 5.8, and < 5, while substitution of Phe for Tyr gave pA₂ values of 7.4, 6.7, and < 5, respectively. All 19 peptides synthesized in this study had low intrinsic agonist activity in the rat isolated uterus assay except for the type I antagonists [Sar¹,Cha⁸]ANG II (7%), [Aib¹, Cha⁸]ANG II (12%) and [Des¹,Cha⁸]ANG II (20%). These data illustrate that the substitution of Cha at position 8 of ANG II analogues produces potent antagonists; however, Type I antagonists retain significant agonist activity whereas Type I/II antagonists do not. In contrast, substitution of Cha at position 4 in a variety of ANG II analogues resulted in severely diminished biological activity, illustrating that the presence of an aromatic ring quadrupole at position 4 is obligatory for receptor binding and activity.     

Author index to volume 43

The effect of analogs of angiotensin (modified with an Ile-substituted for Phe) was studied in the isolated, retrogradely perfused adrenal of the cat. Continuous differential analysis of norepinephrine and epinephrine output was quantified with an automated trihydroxyindole procedure. [Ile⁸]-angiotensin I and [Ile⁷]-angiotensin III exhibited negligible secretory activity, in contrast to the stimulatory effects of [Ile⁸]-angiotensin II (10–20% activity relative to angiotensin II). [Ile⁸]-angiotensin I blocked angiotensin II-induced catecholamine secretion and a pA₂ value of 8.50 was obtained. [Ile⁷]-angiotensin III was an especially potent antagonist of angiotensin II and a pA₂ value of 10.4 was calculated for this heptapeptide analog. The pA₂ value for [Ile⁸]-angiotensin II, a partial agonist in the adrenal medulla was 9.33. These three analogs were equally effective against secretion induced by the corresponding unsubstituted homologs (Ang I and Ang III). These data suggest that all these angiotensin peptides interact with a common receptor. [Ile⁸]-angiotensin I and [Ile⁷]-angiotensin III had no effect on adrenal medullary responses induced by KCl, nicotine and bradykinin. These structural analogs of angiotensin are pure competitive antagonists of angiotensin in the cat adrenal chromaffin cell.     

Cephalic angiotensin receptors mediating drinking to systemic angiotensin II

In rats implanted with cannulae to allow delivery of solutions to the cerebral ventricular system, pretreatment with 5 μg or 0.5 μg of saralasin acetate (Sar¹ Ala⁸ Angiotensin II), an angiotensin II competitive analog, significantly attenuated drinking to subcutaneous (SC) injections of 500 μg of angiotensin II. However, pretreatment with either SC (5 μg or 20 μg) or with intravenous (5 μg) saralasin had no effect on drinking to SC angiotensin II (500 μg). Intracranial (IC) injections of 5 μg of saralasin had no effect on drinking in response to SC injections of 0.8 cc of a 10 percent NaCl solution and did not attenuate ingestion of a milk solution in a dessert test. On the basis of the specificity and the greater efficacy shown by IC saralasin in attenuating drinking to systemically applied angiotensin II, it was concluded that circulating angiotensin II reaches brain periventricular receptors which mediate drinking.