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.     

Biological activity of the novel cyclic angiotensin II analogue [Sar1,Lys3,Glu5]ANG II

Summary This study was undertaken to investigate the biological activity of the cyclic amide-linked analogue of angiotensin II (ANG II), [Sar¹,Lys³,Glu⁵]ANG II, in both ex vivo and in vivo experiments. This constrained analogue was designed on the basis of a recently suggested conformational model for ANG II-induced receptor activation, which is characterized by a Tyr-Ile-His backbone bend and the clustering of the three aromatic rings (Tyr, His, Phe). After [Sar¹,Lys³,Glu⁵]ANG II was found to have contractile activity (15% of ANG II in the rat uterus assay), it was administered in anesthetized rabbits where it produced an immediate and dose-dependent increase in blood pressure, which peaked within minutes, was sustained as long as the drug was given, and was gradually returned to baseline after discontinuation of the drip. The blood pressure response to the cyclic analogue was of less magnitude compared to that elicited by an isovolemic and equimolar solution of ANG II. These data confirm the importance of a properly oriented ring cluster, allowing the charge-relay conformation proposed for ANG II.     

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.     

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     

Effects of ANG II and III and angiotensin receptor blockers on nasal salt gland secretion and arterial blood pressure in conscious Pekin ducks (Anas platyrhynchos)

The vertebrate renin-angiotensin system controls cardiovascular, renal and osmoregulatory functions. Angiotensin II (ANG II) is the most potent hormone of the RAS but in some vertebrate animals angiotensin III (Val⁴-ANG III) may be a hormone. We studied the effects of some angiotensins and mammalian ANG II receptor antagonists on nasal salt gland function and arterial blood pressure in conscious white Pekin ducks. Nasal salt gland fluid secretion (NFS) was induced by a 10 ml · kg⁻¹ bw i.v. injection of a NaCl solution (1000 mosmol · kg⁻¹ H₂O) and maintained by a continuous i.v. infusion of the same solution at a rate of 0.97 ml · min⁻¹. There was a positive linear correlation between nasal fluid [Na⁺] and osmolality, between [Na⁺] and [K⁺], and also between the rate of NFS and [Na⁺] and [K⁺]. [Asp¹,Val⁵]-ANG II (1 nmol · kg⁻¹ i.v.) inhibited NFS but did not change ionic concentrations. Val⁴-ANG III (1 or 5 nmol · kg⁻¹) and ANG I (1-7) (20 nmol · kg⁻¹) had no effect on NFS. [Sar¹, Ile⁸]-ANG II (SARILE) acted as an ANG II receptor agonist and resulted in a prolonged and complete inhibition of NFS. The AT₁ receptor antagonist, losartan (DuP 753) and the AT₂ receptor antagonist, PD 123319 both failed to block the inhibitory effect of [Asp¹, Val⁵]-ANG II on the nasal salt glands. [Asp¹,Val⁵]-ANG II (2 nmol · kg⁻¹ i.v.) increased mean arterial blood pressure (MABP), whereas the same dose of [Asn¹,Val⁵]-ANG II (teleost) had only 30% of the pressor potency of the avian ANG II. Neither 1 nor 5 nmol · kg⁻¹ of Val⁴-ANG III i.v. nor 20 nmol · kg⁻¹ of ANG I (1-7) had any measurable effect on MABP. SARILE blocked completely the pressor response to [Asp¹,Val⁵]-ANG II but the AT₁ antagonists losartan and CGP 48933 and the AT₂ antagonist PD 123319 all failed to block the pressor response to [Asp¹,Val⁵]-ANG II. These results have substantiated an important role of the nasal salt gland in potassium regulation and highlighted a pharmacological dimorphism of saralasin, namely agonist and antagonist to angiotensin II-mediated inhibition of nasal salt gland function and pressor response, respectively. Using specific nonpeptidergic angiotensin II receptor antagonists, we have confirmed the distinct pharmacology of the avian angiotensin II receptors in a nongallinaceous species and the absence of significant angiotensin I (1-7) and angiotensin II effects on the cardiovascular system and nasal salt gland. Accepted: 6 November 1997     

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.     

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.     

Angiotensin II induces phosphatidic acid formation in neonatal rat cardiac fibroblasts: Evaluation of the roles of phospholipases C and D

Phosphatidic acid has been proposed to contribute to the mitogenic actions of various growth factors. In³²P-labeled neonatal rat cardiac fibroblasts, 100 nM [Sar¹]angiotensin II was shown to rapidly induce formation of³²P-phosphatidic acid. Levels peaked at 5 min (1.5-fold above control), but were partially sustained over 2 h. Phospholipase D contributed in part to phosphatidic acid formation, as³²P- or³H-phosphatidylethanol was produced when cells labeled with [³²P]H₃PO₄ or 1-O-[1,2-³H]hexadecyl-2-lyso-sn-glycero-3-phosphocholine were stimulated in the presence of 1% ethanol. [Sar¹]angiotensin II-induced phospholipase D activity was transient and mainly mediated through protein kinase C (PKC), since PKC downregulation reduced phosphatidylethanol formation by 68%. Residual activity may have been due to increased intracellular Ca²⁺, as ionomycin also activated phospholipase D in PKC-depleted cells. Phospholipase D did not fully account for [Sar¹]angiotensin II-induced phosphatidic acid: 1) compared to PMA, a potent activator of phospholipase D, [Sar¹]angiotensin II produced more phosphatidic acid relative to phosphatidylethanol, and 2) PKC downregulation did not affect [Sar¹]angiotensin II-induced phosphatidic acid formation. The diacylglycerol kinase inhibitor R59949 depressed [Sar¹]angiotensin II-induced phosphatidic acid formation by only 21%, indicating that activation of a phospholipase C and diacylglycerol kinase also can not account for the bulk of phosphatidic acid. Thus, additional pathways not involving phospholipases C and D, such asde novo synthesis, may contribute to [Sar¹]angiotensin II-induced phosphatidic acid in these cells. Finally, as previously shown for [Sar¹]angiotensin II, phosphatidic acid stimulated mitogen activated protein (MAP) kinase activity. These results suggest that phosphatidic acid may function as an intracellular second messenger of angiotensin II in cardiac fibroblasts and may contribute to the mitogenic action of this hormone on these cells. (Mol Cell Biochem141: 135–143, 1994)Abbreviations DAG diacylglycerol - DMSO dimethyl sulfoxide - lysoPC 1-O-hexadecyl-2-lyso-sn-glycero-3-phosphocholine - NRCF newborn rat cardiac fibroblasts - PA phosphatidic acid - PAPase phosphatidic acid phosphohydrolase - PC phosphatidylcholine - PEt phosphatidylethanol - PI phosphatidylinositol - PL (labeled) phospholipids - PLC phospholipase C - PLD phospholipase D Drs. G. W. Booz and M. M. Taher contributed equally to the work described here.     

Design,synthesis and biological evaluation of cyclic angiotensin II analogues with 3,5 side-chain bridges. Role of C-terminal aromatic residue and ring cluster for activity and implications in the drug design of AT1 non-peptide antagonists

The novel amide linked angiotensin II (ANG II) cyclic analogues: gamma, epsilon -cyclo(3, 5)-[Sar(1)-Glu(3)-Lys(5)-Ile(8)] ANG II (I) and gamma, epsilon -cyclo(3, 5)-[Sar(1)-Glu(3)-Lys(5)-Phe(8)] ANG II (II) have been designed, synthesized and bioassayed in anesthetized rabbits in order to unravel structural ring cluster characteristics important for receptor activation. Analogue I with Ile at position 8 was an inhibitor of Angiotensin II while analogue II with Phe at position 8 was found to be an agonist. Similar results were reported for cyclic compounds that have reversed the linking between positions 3 and 5. The overall results show that positions 3 and 5 do not govern the biological activity of the synthetic analogues. It also appears that the aromatic ring cluster (Tyr-His-Phe) in agonist peptides is an essential stereo-electronic feature for Angiotensin II to exert its biological activity. A non-peptide mimetic of ANG II, 1-[2'-[(N-benzyl)tetrazol-5-yl]biphenyl-4-yl]methyl]-2-hydroxymethylbenzimidazole (BZI8) has been designed and synthesized. This molecule is more rigid and much less active than AT(1) non-peptide mimetic losartan probably because it lacks to mimic the orientation of tetrazole and the pharmacophore segments of butyl chain and imidazole ring.     

Signaling Pathways in the Biphasic Effect of ANG II on Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> Exchanger in T84 Cells

The effect of ANG II on pH_i, [Ca²⁺]_i and cell volume was investigated in T84 cells, a cell line originated from colon epithelium, using the probes BCECF-AM, Fluo 4-AM and acridine orange, respectively. The recovery rate of pH_i via the Na⁺/H⁺ exchanger was examined in the first 2 min following the acidification of pH_i with a NH₄Cl pulse. In the control situation, the pH_i recovery rate was 0.118 ± 0.001 (n = 52) pH units/min and ANG II (10⁻¹² M or 10⁻⁹ M) increased this value (by 106% or 32%, respectively) but ANG II (10⁻⁷ M) decreased it to 47%. The control [Ca²⁺]_i was 99 ± 4 (n = 45) nM and ANG II increased this value in a dose-dependent manner. The ANG II effects on cell volume were minor and late and should not interfere in the measurements of pH_i recovery and [Ca²⁺]_i. To document the signaling pathways in the hormonal effects we used: Staurosporine (a PKC inhibitor), W13 (a calcium-dependent calmodulin antagonist), H89 (a PKA inhibitor) or Econazole (an inhibitor of cytochrome P450 epoxygenase). Our results indicate that the biphasic effect of ANG II on Na⁺/H⁺ exchanger is a cAMP-independent mechanism and is the result of: 1) stimulation of the exchanger by PKC signaling pathway activation (at 10⁻¹² – 10⁻⁷ M ANG II) and by increases of [Ca²⁺]_i in the lower range (at 10⁻¹² M ANG II) and 2) inhibition of the exchanger at high [Ca²⁺]_i levels (at 10⁻⁹ – 10⁻⁷ M ANG II) through cytochrome P450 epoxygenase-dependent metabolites of the arachidonic acid signaling pathway.     

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 approximately 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 less than 5, while substitution of Phe for Tyr gave pA2 values of 7.4, 6.7, and less than 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.     

Angiotensin-Induced Changes in the Apparent Size of Rat Liver Angiotensin Receptors

Abstract

Angiotensin receptors from rat liver were labeled using four different ligands : (Sar¹-(³H)Tyr⁴)-Angiotensin II ((³H)SarAII); (Sar¹-(³H)Tyr⁴-IIe⁸) -Angiotensin II ((³H)SarIIeAII); (Sar¹-(¹²⁵I)Tyr⁴-(4′-N₃)Phe⁸)-Anglotensin II (IN₃AII); (Sar¹-(¹²⁵I)Tyr⁴-(4′N₃D-Phe)⁸)-Angiotensin II (IN₃DPheAII) (³H) SarAII and IN₃AII behaved like agonists and (³H) SarlleAII and IN₃DPheAII like antagonists. All four ligands labeled the same population of sites. The azido derivatives allowed covalent labeling of receptors with a high yield (about 40%). Membranes were solubilized by Triton X-100 under experimental conditions which ensured complete solubilization of the liganded receptors in a stable form (less than 40% dissociation after 20 h). The apparent size of liganded angiotensin receptors was determined by gel filtration on Ultrogel ACA-34 columns and by SDS gel electrophoresis (in the case of covalent labeling). The apparent Stokes radius of solubilized angiotensin receptors was different wether the receptor was labeled with an agonist (Stokes radius = 6.2 ± 0. 1 nm (6) after labeling with (³H) SarAII) or with an antagonist (Stokes radii of 5. 5 ± 0. 1 (7), and 5.6 ± 0.1 nm (4) after labeling with (³H) SarIIeAII and IN₃DPheAII respectively). After covalent labeling with IN₃All anglotensin receptors were eluted as a mixture of light and heavy forms. SDS gel electrophoresis revealed only one molecular entity of M_r 64,000. It is concluded that binding of an agonist to liver angiotensin receptors triggers or stabilizes an interaction with another membrane component Involved in the coupling of the receptor to its primary effector.     

Oscillatory Cl current induced by angiotensin II in rat pulmonary arterial myocytes: Ca dependence and physiological implication

We have previously reported that angiotensin II (ANG II) induces oscillations in the cytoplasmic calcium concentration ([Ca²⁺]_i) of pulmonary vascular myocytes. The present work was undertaken to investigate the effect of ANG II in comparison with ATP and caffeine on membrane currents and to explore the relation between these membrane currents and [Ca²⁺]_i. In cells clamped at −60 mV, ANG II (10 μM) or ATP (100 μM) induced an oscillatory inward current. Caffeine (5 μM) induced only one transient inward current. In control conditions, the reversal potential (E_rev) of these currents was close to the equilibrium potential for Cl⁻ ions (E_Cl = −2.1 mV) and was shifted towards more positive values in low-Cl⁻ solutions. Niflumic acid (10–50 μM) and DIDS (0.25-1 mM) inhibited this inward current. Combined recordings of membrane current and [Ca²⁺]_i by Indo-1 microspectrofluorimetry revealed that ANG II- and ATP-induced currents occurred simultaneously with oscillations in [Ca²⁺]_i, whereas the caffeine-induced current was accompanied by only one transient increase in [Ca²⁺]_i Niflumic acid (25 μM) had no effect on agonist-induced [Ca²⁺]_i responses, whereas thapsigargin (1 μM) abolished both membrane current and the [Ca²⁺]_i response. Heparin (5 mg/ml in the pipette solution) inhibited both [Ca²⁺]_i responses and membrane currents induced by ANG II and ATP, but not by caffeine. In pulmonary arterial strips, ANG II-induced contraction was inhibited by niflumic acid (25 μM) or nifedipine (1 μM) to the same extent and the two substances did not have an additive effect. This study demonstrates that, in pulmonary vascular smooth muscle, ANG II, as well as ATP, activate an oscillatory calcium dependent chloride current which is triggered by cyclic increases in [Ca²⁺]_i and that both oscillatory phenomena are primarily IP₃ mediated. It is suggested that ANG II-induced oscillatory chloride current could depolarise the cell membrane leading to activation of voltage-operated Ca²⁺ channels. The resulting Ca²⁺ influx contributes to the component of ANG II-induced contraction that is equally sensitive to chloride or calcium channel blockade.     

Influence of polyfluorination of the phenylalanine ring of angiotensin II on conformation and biological activity

[Phe(F5)8]angiotensin II was synthesized by the solid phase method and purified by reverse-phase HPLC. In rat uterus and rabbit aorta bioassays the analogue had 10 and 50%, respectively, of the contractile activity of angiotensin II and demonstrated antagonist properties. These findings illustrate that inversion of the Phe8 ring quadrupole moment in angiotensin II decreases agonist activity and invokes antagonist properties. 1H-NMR studies at 400 MHz in DMSO-d6 demonstrated the presence of cis and trans isomers in the ratio 1:3 due to restricted rotation of the His-Pro bond. Downfield shifts of the His C2 and C4 protons in [Phe(F5)]ANG II compared to ANG II suggest that the Phe(F5) residue may be involved in a parallel-plate ring pairing interaction with the imidazole group. However heteronuclear NOE studies, carried out by measuring the proton difference spectrum before and after saturation of the fluorine resonances, showed the absence of any NOE enhancement illustrating that electrostatic influences of the Phe(F5) ring occur at relatively long range.     

Stabilization of hydrolytically labile iron(II)–cysteine peptide thiolate complexes in aqueous triton X-100 micelle solution: Spectroscopic properties mimicking of reduced rubredoxin

The absorption, CD, and ¹H- and ¹⁹F-nmr spectroscopic features of Fe(II) complexes with a series of cysteine-containing oligopeptides were investigated in aqueous (H₂O or D₂O) 10% Triton X-100 micelle solution. The complexes with distal aromatic rings, [Fe(Z-cys-Pro-Leu-cys-Gly-X)₂]²⁻ (Z = benzyloxycarbonyl; X = NH-C₆H₄-p-F, NH-CH₂-CH₂-C₆H₄-p-F, and Phe-OMe), were found to be quite stable in such aqueous micelle solution. The coordination of cysteine–peptide ligands to the Fe(II) ion is revealed by isotropically shifted ¹H-nmr signals due to the Cys C_βH₂ protons occurring at 120 ∼ 250 ppm in a D₂O Triton X-100 micelle solution (10%) at 60°C that are very similar to those reported for native reduced rubredoxin. The high stability of these cysteine peptide–Fe(II) complexes in aqueous micellar system was explained by the combined contributions from NH—S hydrogen bonds and the effect of the proximity of aromatic groups. The existence of such NH—S hydrogen bonds and interactions between aromatic ring and sulfur atom was confirmed by ¹⁹F-nmr spectral and ¹⁹F spin–lattice relaxation times (T₁) measurements. © 1998 John Wiley & Sons, Inc. Biopoly 46: 1–10, 1998     

Conformationally Restricted 2-Substituted Wyosine Derivatives. 1H, 13C,and 15N NMR Study

Abstract

It was found by ¹H, ¹³C and ¹⁵N NMR study that substitution of 4,9-dihydro-4, 6-dimethyl-9-oxo-3-(2′,3′,5′-tri-O-acetyl-β-D-ribofuranosyl) imidazo [1,2-a]purine (wyosine triacetate, 1) at C2 position with electronegative groups CH₃O and C₆H₅CH₂O results in a noticeable electron distribution disturbance in the “left-hand” imidazole ring and a significant increase in the North conformer population of the sugar moiety.     

SR 140333, a Novel, Selective, and Potent Nonpeptide Antagonist of the NK1 Tachykinin Receptor: Characterization on the U373MG Cell Line

The effects of a novel nonpeptide NK1 tachy-kinin receptor antagonist, SR 140333, on the functional consequences of NK1 receptor activation in a human astrocytoma cell line, U373MG, were investigated. Radioligand binding conducted with ¹²⁵l-Bolton-Hunter substance P revealed a competitive inhibition by SR 140333 and its R enantiomer SR 140603 with K_i values of 0.74 and 7.40 nM, respectively. The NK1-selective agonist, [Sar⁹,Met(O₂)¹¹]-substance P, stimulated the formation of inositol phosphates with an EC₅₀ of 3.8 × 10^?9M. SR 140333 blocked the stimulatory effect of this agonist (10^?7M) with an IC₅₀ of 1.6 × 10^?9M,whereas the effect of another NK1 agonist, septide (EC₅₀= 1.5 × 10^?8M)was antagonized with an IC₅₀ of 2.1 × 10^?10M.Enhancement of [³H]taurine release by [Sar⁹,Met(O₂)¹¹]-substance P (EC₅₀= 7.4 × 10^?9M) was also inhibited by SR 140333 with an IC₅₀ of 1.8 × 10^?9 M. SR 140603 was 10-fold less potent than SR 140333 in inhibiting inositol monophosphate formation and [³H]taurine release. The calcium mobilization induced by [Sar⁹,Met(O₂)¹¹]-substance P (10^?8M) was totally prevented by 10^?8MSR 140333. Patchclamp experiments showed that SR 140333 depressed the outward current evoked by 5 × 10^?8M [Sar⁹, Met(O₂)¹¹]-substance P with an IC₅₀ of 1.3 × 10^?9M. The expression of c-fos was stimulated by [Sar⁹,Met(O₂)¹¹]-substance P with an EC₅₀ of 2.5 × 10^?10M, an effect that was also inhibited by SR 140333 with an IC₅₀ of 1.1 × 10^?9M. The present results illustrate the sequential events of the response elicited by NK1 agonists, which were antagonized by SR 140333, demonstrating its powerful NK1 antagonist activity on a functional basis.     

1H-NMR studies of [Sar1]angiotensin II conformation by nuclear Overhauser effect spectroscopy in the rotating frame (ROESY): clustering of the aromatic rings in dimethylsulfoxide

The conformational properties of the octapeptide [Sar1]ANG II in dimethylsulfoxide-d6 were investigated by rotating frame nuclear Overhauser effect spectroscopy (ROESY). Interresidue ROESY interactions were observed between Tyr ortho and Phe ring protons, between Phe ring and Pro C gamma protons, and also between His C alpha and Pro C delta protons. A weak connectivity was also observed between the Sar N-CH3 protons and a Tyr ortho proton. Intraresidue interactions between alpha and beta protons in Tyr, His and Phe indicated restricted rotation for the side-chains of the three aromatic residues. These findings suggest that [Sar1]ANG II takes up a folded conformation in DMSO in which the three aromatic rings form a cluster. Connectivities between the His C alpha proton and the two Pro C delta protons illustrated a preferred conformation for angiotensin II in DMSO in which the His-Pro bond exists as the trans isomer. The NMR spectroscopic evidence is consistent with the presence of a Tyr charge relay system in the biologically active conformation of angiotensin II and with the postulated role of the Tyr hydroxyl group in angiotensin II for receptor activation.     

Insect Trypsin Modulating Oostatic Factor (Neb-TMOF) and Its Analogs: Preliminary Structure/Biological Function Relationship Studies

Neb-TMOF, the trypsin modulating oostatic factor of gray fleshfly Neobellieria bullata, is a hexapeptide with the following sequence: H-Asn-Pro-Thr-Asn-Leu-His-OH. It has been isolated from vitellogenic ovaries in 1994. TMOF, the newly discovered insect peptide, inhibits trypsin biosynthesis in the gut, lowers yolk polypeptide concentration in the hemolymph and strongly inhibits ecdysone biosynthesis by larval ring glands. It is interesting that this short non-protected peptide contains in its molecule two Asn residues at positions 1 and 4 and His at its C-terminus. To obtain information about the role of the His-6 and Asn-4 residues we synthesised two series of Neb-TMOF analogs, modified: (1) in position 6 by D-His (I), His(Bzl) (II) and Phe(p-X) derivatives, where X = NH₂ (III), NO₂ (IV), OEt (V) and OH (VI) and (2) in position 4 by such amino acid residues as Ser (VII), Thr (VIII), Gly (IX), Asp (X), Glu (XI) and D-Asn (XII). The influence of these peptides on trypsin biosynthesis in N. bullata was determined in vivo. In preliminary investigations, we found that Neb-TMOF, [Phe(NH₂)⁶], and [Phe(NO₂)⁶]-Neb-TMOF inhibited trypsin biosynthesis, whereas [D-His)⁶]- and [D-His(Bzl)⁶]-Neb-TMOF were inactive. In further biological studies performed in vitro on heart of Tenebrio molitor we found that Neb-TMOF and [Phe(p-NH₂)⁶-Neb-TMOF showed weak cardioexcitatory activity, about 30% of the cardioexcitatory activity of proctolin, an insect neuromodulating peptide.