Biochemical pharmacology of total retro-inverso analogues of bradykinin and angiotensin II: Molecular recognition by G-protein-coupled receptors and angiotensin converting enzyme

Total retro-inverso (TRI) analogues of bradykinin (BK), the B₂a-selective kinin antagonist d-Arg⁰[Hyp₃,d-Phe⁷,Leu⁸]BK, angiotensin II (AT II) and the AT II antagonist Saralasin ([Sar¹,Val⁵,Ala⁸]AT II) were prepared by conventional solid-phase synthesis. Molecular recognition of TRI peptidomimetics by G-protein-coupled receptors was studied by competitive radioligand displacement experiments. TRI analogues of d-Arg⁰[Hyp³,d-Phe⁷,Leu⁸]BK specifically bound to the kidney medulla B₂a bradykinin receptor with affinities (K_d) ranging from 64 M to 4 M. Conversely, TRI analogues of BK, AT II and Saralasin did not bind to either the B_2a bradykinin receptor or the rat AT_1a AT II receptor, respectively. These studies indicate that the TRI strategy is more compatible with the synthesis of antagonists than agonists. Three TRI peptidomimetics of d-Arg⁰[Hyp³,d-Phe⁷,Leu⁸]BK were weak inhibitors of angiotensin converting enzyme. All other TRI peptidomimetics had no effect upon ACE activity. These data endorse the utility of the TRI strategy for the synthesis of protease-resistant antagonists of peptide hormones and neuropeptides.     

Proton magnetic resonance studies of bradykinin antagonists

Bradykinin (BK) is a peptide hormone with sequence Arg¹-Pro²-Pro³-Gly⁴-Phe⁵-Ser⁶-Pro⁷-Phe⁸-Arg⁹ and has been implicated in a multitude of pathophysiological processes such as the ability to lower systemic blood pressure and stimulate pain. BK analogues having bulky, β-branched D -aliphatic residues at position 7 combined with bulky L -aliphatic residues at position 8 have now been observed to be strong antagonists. Conformational studies based on two-dimensional nmr experiments in methanol/water (80/20 v/v) were carried out on several such active antagonists in a polar solvent. Included in this study were the very active antagonists, [D -Arg⁰, Hyp³, Thi⁵, D -Cpg⁷, Cpg⁸]-BK [Cpg: α-cyclo-pentyl-glycine; Hyp: trans-4-hydroxy-L -proline; Thi: β-(2-thienyl)-L -alanine] ( I ), [D -Arg⁰, Hyp³, D -Cpg⁷, Cpg⁸] -BK ( II ), as well as its variant with D -Cpg⁷ replaced by Cpg⁷, namely [D -Arg⁰, Hyp³, Cpg⁷, Cpg⁸]-BK ( III ). A turn-like structure, which coexists with the extended conformation, was observed between residues 2 and 5 for the most active antagonists I and II , in direct correlation with the peptide activities. No turn-like structure was found for residues 6–9. In peptide III , a turn-like structure was not identified. The existence of a turn at the C-terminal end of bradykinin and its analogues has been predicted by empirical calculations and supported by nmr measurements. But the present nmr study on the most active antagonists ( I , II ) does not support this hypothesis. Instead, the data suggest that a turn-like structure between residues 2 and 5 could be important for antagonist activity. Finally, one weak inhibitor [D -Cpg⁷]-BK ( IV ) showed no defined secondary structure. © 1993 John Wiley & Sons, Inc.     

A proton magnetic resonance study of two synthetic agonist–antagonist pairs of bradykinin analogues

The conformation of two agonist–antagonist pairs of bradykinin (Arg¹-Pro²-Pro³-Gly⁴-Phe⁵-Ser⁶-Pro⁷-Phe⁸-Arg⁹) analogues were studied in CD₃OH/H₂O solution by ¹H-nmr techniques. The first agonist peptide studied, D -Arg⁰-Arg¹-Pro²-Hyp³-Gly⁴-Thi⁵-Ser⁶-Pro⁷-Thi⁸-Arg⁹, differs from the bradykinin sequence by the addition of D -Arg⁰, the replacement of the Phe moieties in positions 5 and 8 by Thi (Thi = β-(2-thienyl)-L -alanine), and Hyp³ (Hyp = L -4-hydroxy-L -proline) in position 3. In the corresponding antagonist sequence, Pro⁷ is replaced by D -Phe⁷. The second agonist–antagonist pair studied does not contain the D -Arg⁰ residue, which is present only to slow down the rate of metabolism. Based on complete resonance assignments from two-dimensional total correlation spectroscopy and rotating frame nuclear Overhauser effect spectroscopy spectra at 500 MHz, the peptides were analyzed in terms of intraresidue, sequential, and medium-range nuclear Overhauser effects, amide proton temperature coefficients, and vicinal coupling constants. Both agonist peptides show clear evidence for the existence of a type I β-turn comprising the C-terminal residues Ser⁶-Pro⁷-Thi⁸-Arg⁹ in fast conformational equilibrium with extended structures throughout. Although the conformational space is dominated by extended structures, the presence of the β-turn is spectroscopically clearly discernible. The two antagonist peptides, on the other hand, do not show evidence of turn formation but rather the presence of an extended conformation with some irregularities in the N-terminal region of the peptide. While the existence of a turn at the C-terminal end of bradykinin and its analogues with agonist activity has been predicted by empirical calculations and measurements in very apolar solvents, this study, for the first time, provides evidence based on physical data in a polar solvent environment that the turn is present, that it is type I and that it is essential for agonist activity. In the particular solvent used in these studies, the Pro⁷ to D -Phe⁷ substitution precluded the formation of the turn for the C-terminal residues of the antagonist. © 1993 John Wiley & Sons, Inc.     

Structural elucidation of Leuprolide and its analogues in solution: insight into their bioactive conformation

Leuprolide [dLeu⁶, NHEt¹⁰]GnRH, a potent gonadotropin-releasing hormone (GnRH) agonist, is used in a wide variety of hormone-related diseases like cancer and endometriosis. In this report, the conformational behaviour of Leuprolide and its linear synthetic analogues, namely [Tyr⁵(OMe), dLeu⁶, Aze⁹, NHEt¹⁰]GnRH (1) and [Tyr⁵(OMe), dLeu⁶, NHEt¹⁰]GnRH (2) have been studied in DMSO and H₂O solutions by means of 2D nuclear magnetic resonance (NMR) experiments and detailed molecular dynamics (MD) simulations. The aim was to identify the conformational requirements of GnRH analogues for agonistic activity. This approach is of value as no crystallographic data are available for the GnRH receptor (G protein-coupled receptor, GPCR). The NOE data indicate the existence of a β-turn type I in the 2–5 segments of Leuprolide and its linear analogues in the case of using DMSO-d₆ as solvent, whereas a β-turn type II in the 3–6 segments is indicated using D₂O as solvent. The final structures fulfil the conformational requirements that are known, in the literature, to play a significant role in receptor recognition and activation. Finally, the linear analogues (1) and (2) are biologically active when tested against the human breast cancer cell line, MCF-7.     

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 Nucleotides on [3H]Bradykinin Binding in Guinea Pig: Further Evidence for Multiple B2 Receptor Subtypes

Abstract: We have suggested recently the existence of three subtypes of B₂ bradykinin receptors in tissues of guinea pigs. We have classified these B₂ bradykinin receptors into B_2a, B_2b, and B_2c subtypes depending on their affinity for various bradykinin antagonists. Because the actions of bradykinin in different cell systems appear to be both dependent on and independent of G proteins, we sought to determine whether the binding of [³H]bradykinin to the B₂ subtypes is sensitive to guanine nucleotides and, therefore, possibly coupled to G proteins. In the ileum, where we have demonstrated B_2a and B_2b subtypes, specific [³H]bradykinin binding was reduced with GDP (100 μM) and the nonmetabolized analogue of GTP, guanyl-5′-yl-imidodiphosphate (GppNHp; 100 μM). Competition studies with bradykinin and with [Hyp³]-bradykinin, which shows approximately 20-fold greater selectivity for the B_2a subtype than bradykinin, were performed in the presence or absence of GppNHp (100 μM). The competition experiments demonstrated that binding to the B_2a subtype, which has higher affinity for [Hyp³]-bradykinin and bradykinin than the B_2b subtype, was lost in the presence of GppNHp, whereas binding to the B_2b subtype was unaffected. In contrast, GppNHp (100 μM) and GDP (100 μM) failed to alter specific [³H]bradykinin binding to B_2b and B_2c subtypes in lung. [³H]Bradykinin binding was unaffected by AMP, ADP, ATP, and GMP (100 μM each). Based on this evidence, we suggest that the B_2a bradykinin subtype is coupled to G proteins. The B_2b and B_2c subtypes are either not coupled to G proteins, or may be coupled to the G^o-type GTP binding proteins, which have been suggested to be less sensitive to guanine nucleotides. These data provide further evidence for three subtypes of B₂-type bradykinin receptors in guinea pig.     

Conformational Studies of Two Bradykinin Antagonists by Using Two-dimensional NMR Techniques and Molecular Dynamics Simulations

Abstract

The solution conformations of two potent antagonists of bradykinin (Arg¹-Pro²-Pro³-Gly⁴- Phe⁵-Ser⁶-Pro⁷-Phe⁸-Arg⁹), [Aca-¹, DArg⁰, Hyp³, Thi⁵, DPhe⁷,(N-Bzl)Gly⁸]BK (1) and [Aaa- ¹, DArg⁰, Hyp³, Thi⁵,(2-DNal)⁷, Thi⁸]BK (2), were studied by using 2D NMR spectroscopy in DMSO-dg and molecular dynamics simulations. The NMR spectra of peptide 1 reveals the existence of at least two isomers arising from isomerization across the DPhe⁷-(N-Bzl)Gly⁸peptide bond. The more populated isomer possesses the cis peptide bond at this position. The ratio of cis/trans isomers amounted to 7:3. With both antagonists, the NMR data indicate a β-turn structure for the Hyp³-Gly⁴ residues. In addition, for peptide 2, position 2,3 is likely to be occupied by turn-like structures. The cis peptide bond between DPhe⁷ and (N- Bzl)Gly⁸ in analogue 1 suggests type VI β-turn at position 7,8. The molecular dynamics runs were performed on both peptides in DMSO solution. The results indicate that the structure of peptide 1 is characterized by type VIb β-turn comprising residues Ser-Arg⁹ and the βI or βII-turn involving the Pro²-Thi⁵ fragment, whereas peptide 2 shows the tendency towards the formation of type I β-turn at position 2,3. The structures of both antagonists are stabilized by a salt bridge between the guanidine moiety of Arg¹ and the carboxyl group of Arg⁹. Moreover, the side chain of DArg⁰ is apart of the rest of molecule and is not involved in structural elements except for a few calculated structures.     

Characterization of rat brain opioid receptors by [Tyr-3,5-3H]1,d-Ala2, Leu5-enkephalin binding

[Tyr-3,5-³H]¹,d-Ala², Leu⁵-enkephalin ([³H]DALA) was used for labeling the opioid receptors of rat brain plasma membranes. The labeled ligand was prepared from [Tyr-3,5-diiodo]¹,d-Ala², Leu⁵-enkephalin by catalytic reductive dehalogenation in the presence of Pd catalyst. The resulting [Tyr-3,5-³H]¹,d-Ala², Leu⁵-enkephalin had a specific activity of 37.3 Ci/mmol. In the binding experiments steady-state level was reached at 24°C within 45 min. The pseudo first order association rate constant was 0.1 min^–1. The dissociation of the receptor-ligand complex was biphasic with k_–1-s of 0.009 and 0.025 min^–1. The existence of two binding sites was proved by equilibrium studies. The high affinity site showed aK _D=0.7 nM andB _max=60 fmol/mg protein; the low affinity site had aK _D=5 nM andB _max=160 fmol/mg protein. A series of opioid peptides inhibited [³H]DALA binding more efficiently than morphine-like drugs suggesting that labeled ligand binds preferentially to the subtype of opioid receptors. Modification of the original peptides either at the C or N terminal ends of the molecules resulted in a decrease in their affinity.     

Modulation of membrane K+ conductance in T-lymphocytes by substance P via a GTP-binding protein

Summary Modulation of the voltage-gated K⁺ conductance in T-lymphocytes by substance P was examined. Whole-cell recordings from Jurkat E6-1 human T-lymphocytes revealed two components of substance P action on the outward K⁺ current: (i) dose- and time-dependent reduction of current peak amplitude; and (ii) acceleration of the current inactivation rate. This action was blocked by substituting Cs⁺ for K⁺ in the recording pipette and by the substance P antagonist, [d-Arg¹,d-Phe⁵,d-Trp^7.9, Leu¹¹]-substance P. As indicated by conductance-voltage relationship, the reduction in current peak amplitude as a result of substance P application was not due to a shift of the voltage dependence of the channel. Raising intracellular free calcium concentration from 2 to 200nm reversed the reduction, induced by substance P, in current peak amplitude and disclosed an apparent desensitization towards the neuropeptide action. The treatment, however, did not reverse substance P-induced acceleration of the rate of current decay. Intracellular administration of hydrolysis-resistant guanosine triphosphate (to persistently activate GTP-binding protein) and guanosine diphosphate (to competitively inhibit GTP—binding proteins) analogues mimicked and inhibited substance P-induced reduction of K⁺ conductance, respectively. The data demonstrate a modulation of T-lymphocyte K⁺ channels by substance P and substantiate a possible role for GTP-binding proteins in this modulation.     

Synthesis and biological activity of oxytocin analogues containing unnatural amino acids in position 9: structure activity study

We report the solid phase synthesis and some pharmacological properties of 24 oxytocin (OT) analogues. Basic modifications at position 9 (introduction of l- or d-β-(2-thienyl)-alanine [L- or D-Thi], or l- or d-3-Pyridylalanine [l- or d-3-Pal]) were combined with d-tyrosine(OEthyl) [d-Tyr(Et)] or d-1-naphthylalanine [d-1-Nal] in position 2 and β-mercaptopropionic acid (Mpa) in position 1 modifications in altogether 14 analogues. Additionally, 8 analogues having α-aminoisobutyric acid [Aib] or d-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (d-Tic) or diethylglycine (Deg) in position 9 and d-Tyr(Et) or d-1-Nal or d-Tic in position 2 and Mpa or Pen (ββ-dimethylcysteine) in position 1 were prepared. Two of these analogues have one more modification in position 6, i.e. Pen. Furthermore, two analogues having Mpa in position 1 and d-Tyr(Et) or d-1-Nal in position 2 were prepared for comparison purposes. The analogues were tested for rat uterotonic activity in vitro, in the rat pressor assay and for binding affinity to human OT receptor. The analogue having the highest anti-oxytocic activity was [Mpa¹, d-Tyr(Et)², Deg⁹]OT (pA₂ = 8.68 ± 0.26); this analogue was also selective.     

Autoradiographic Localization of [125I-Tyr0]Bradykinin Binding Sites in Brains of Wistar-Kyoto and Spontaneously Hypertensive Rats

1. The present study was undertaken to localize and characterize bradykinin (BK) binding sites in brains from Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR).2. Serial sections of brains were cut from adult WKY and SHR and specific [¹²⁵I-Tyr⁰]bradykinin ([¹²⁵I-Tyr⁰]BK) binding was determined using in vitro quantitative receptor autoradiographic techniques.3. Specific binding of [¹²⁵I Tyr⁰]BK was localized in the medulla oblongata to the regions of the nucleus of the solitary tract (NTS), area postrema (AP), dorsal motor nucleus of the vagus (X), and caudal subnucleus of the spinal trigeminal nucleus in both strains of rat. The specific binding (85–90% of total binding) was of high affinity and saturable with K _D values in the range of 100 pM and a B _max of 0.75 fmol per mg tissue equivalent in the NTS–X–AP complex of both the WKY and SHR. In competition studies, the rank order of potencies was similar in both strains with BK = Lys-BK > icatibant >>> DesArg⁹-BK. The B₂ receptor antagonist icatibant inhibited [¹²⁵I-Tyr⁰]BK binding with a K _i value of 0.63 ± 0.19 nM in WKY and 0.91 ± 0.73 nM in SHR, while K _i values for the B> ₁ receptor agonist DesArg⁹-BK were 1475 ± 1055 and 806 ± 362 nM in WKY and SHR, respectively.4. Our finding of specific high-affinity [¹²⁵I-Tyr⁰]BK B₂ binding sites in the NTS, AP, and the X of WKY and SHR is important because these brain areas are associated with central cardiovascular regulation. However, alterations in BK B₂ receptors in the medulla that could contribute to the hypertensive state in the SHR were not detected.     

Bradykinin-induced biphasic response in the rat isolated stomach fundus: Functional evidence for a novel bradykinin receptor

The responses of the rat isolated stomach fundus to bradykinin (BK) and des-Arg⁹-BK (DA-BK) have been examined. In rat isolated stomach fundus pre-contracted with BaCl₂ (0.5-1 mM), BK caused concentration-dependent biphasic responses characterized by relaxation followed by contraction. DA-BK also caused marked relaxations, but, unlike BK, induced only small contractions. Removal of the mucosal layer initially abolished the relaxant responses to BK and both responses to DA-BK without affecting BK-induced contractions, but repeated challenges with BK or DA-BK revealed a time-depeendent reappearance of the relaxant responses, suggesting “de novo” synthesis of BK receptors. Pretreatment of rat stomach fundus with tetrodotoxin (1 μM), atropine (1 μM), captopril (3 μM), prazosin (1 μM) or glibenclamide (1 μM) did not significantly modify the biphasic responses to BK (300 nM). The biphasic responses to DA-BK were antagonized selectivley by the B₁ receptor antagonist des-Arg⁹-[Leu ⁸]-BK (DAL- BK) (1 μM). In contrast, the biphasic responses to BK were unaffected by DAL-BK or by several selective peptide antagonists of B₂ receptors including NPC 431 (Thi^5,8, D-Phe⁷)-BK, NPC 349 (D-Arg Hyp³, Thi^5,8, D-Phe⁷)-BK, NPC 567 (D-Arg -Hyp³, D-Phe⁷)-BK and NPC 361 (D-Phe⁷)-BK (3 to 10 μM). These results are consistent with the view that the biphasic responses of the rat isolated stomach fundus to BK appear to be mediated by a novel BK receptor which is insensitive to blockade by B₁ and B₂ selective BK receptor antagonists.     

B2 receptor-mediated dual effect of bradykinin on proximal tubule Na-ATPase: Sequential activation of the phosphoinositide-specific phospholipase Cβ/protein kinase C and Ca-independent phospholipase A2 pathways

In a previous paper we showed that bradykinin (BK), interacting with its B₂ receptor, inhibits proximal tubule Na⁺-ATPase activity but does not change (Na⁺ + K⁺)ATPase activity. The aim of this paper was to investigate the molecular mechanisms involved in B₂-mediated modulation of proximal tubule Na⁺-ATPase by BK. To abolish B₁ receptor-mediated effects, all experiments were carried out in the presence of (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Leu), des-Arg⁹-[Leu⁸]-BK (DALBK), a specific antagonist of B₁ receptor. A dual effect on the Na⁺-ATPase activity through the B₂ receptor was found: short incubation times (1-10 min) stimulate the enzyme activity; long incubation times (10-60 min) inhibit it. The stimulatory effect of BK is mediated by activation of phosphoinositide-specific phospholipase C β (PI-PLCβ)/protein kinase C (PKC); its inhibitory action is mediated by Ca²⁺-independent phospholipase A₂ (iPLA₂). Prior activation of the PI-PLCβ/PKC pathway is required to activate the iPLA₂-mediated inhibitory phase. These results reveal a new mechanism by which BK can modulate renal sodium excretion: coupling between B₂ receptor and activation of membrane-associated iPLA₂.     

Opioid Peptides: Synthesis and Biological Activity of New Endomorphin Analogues

Summary Syntheses are described of new endomorphin 1 and 2 peptoid–peptide hybrids in which Tyr¹ and either one or both Phe³ and Phe⁴ have been replaced by N-substituted-glycine. The preparation is also described of two glycosylated Hyp²-endomorphin 2 analogues in which either 2,3,4,6-tetra-O-acetyl glucose or glucose are β-O-glycosidically linked to the hydroxyproline residue. The Hyp²-endomorphin sequences have also been elongate by adding a C-terminal β-alanine residue and several linear dimers have been prepared by coupling either the native peptides or the modified analogues. The cyclo endomorphin 2 has also been synthesized. Preliminary pharmacological experiments on isolated organ preparations showed that the agonist activities of both endomorphin 1 and 2 are not significantly affected by the Pro/Hyp substitution. Phe⁴/Nphe substitution in the endomorphin 1 reduced the potency on guinea pig ileum (GPI) by about 100 times and abolished the agonist activity on mouse vas deferens (MVD) preparation. The decrease of the agonist activity induced by modification of one phenylalanine residue only, either Phe³ or Phe⁴, is lower on endomorphin 2. Either modification of both Phe³ and Phe⁴ or glycosylation of the Hyp²-endomorphin 2 cancelled any agonist activity on both preparations. The linear peptide dimers [endomorphin 1]₂, [endomorphin 2]₂, [Hyp²-endomorphin 1]₂, [Hyp²-endomorphin 2]₂, [Hyp²-endomorphin 1-Hyp²-endomorphin 2]₂ or [Hyp²-endomorphin 2-Hyp²-endomorphin 1]₂, are 7–19 times less potent than endomorphin 1 on GPI and significantly less active than endomorphins 1 and 2 on MVD. The other afforded modifications significantly affected or abolished the agonist activity of the resulting endomorphin analogues on both GPI and MVD preparations.The α-amino acid residues are of the L-configuration. Standard abbreviations for amino acid derivatives and peptides are according to the suggestions of the IUPAC-IUB Commission on Biochemical Nomenclature (1984) Eur. J. Biochem., 138, 9–37. Abbreviations listed in the guide published in (2003) J. Peptide Sci., 9, 1–8 are used without explanation.     

Biomedical Applications of Synthetic Melanotropins

Alpha-melanotropin (alpha-melanocyte stimulating hormone, alpha-MSH) is a hormone produced by the pituitary gland of most vertebrate animals. This melanotropic peptide, Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2, regulates melanin pigmentation of the skin of some mammals. Although MSH may be absent from the human pituitary gland, this peptide can stimulate pigment formation in human skin. We have synthesized several analogues of alpha-MSH, which are superpotent, prolonged-acting, and resistant to inactivation by serum enzymes. One such analogue, [NLe⁴, D-Phe⁷]alpha-MSH, has proven particularly useful in a number of physiological studies. In addition, some [Nle⁴, D-Phe⁷]-substituted fragment analogues of MSH are even more active than the native hormone, alpha-MSH. For example, these analogues are 100–1,000 times more active than alpha-MSH in stimulating S-91 mouse melanoma tyrosinase activity in vitro. We have successfully labeled one such peptide to high specific activity; this melanotropin, [³H]-Ac-[Nle⁴, D-Phe⁷]alpha-MSH_4–11NH₂, has been shown by others to bind to B16 melanoma cells. We have also conjugated several ligands (fluorescein and biotin) to [Nle⁴, D-Phe⁷]alpha-MSH. These melanotropin conjugates might prove useful for melanotropin receptor studies and for the clinical localization of metastatic melanoma. We have demonstrated that [Nle⁴, D-Phe⁴]alpha-MSH can be topically applied and transdermally delivered across the skin of mice and humans in vitro, as determined by bioassay and RIA. Initial toxicologic studies indicate that the analogue is nontoxic to mice and is not mutagenic. Studies are underway to determine whether this analogue may prove useful as a “tanning hormone” for increasing the pigmentation of light-skinned individuals or possibly even for treating people with certain hypopigmentary disorders.     

Cyclic hexapeptide antagonists of the bradykinin B2 receptor: Receptor binding and solution backbone conformation

Summary Cyclic hexapeptide analogs of bradykinin, based on a folded receptor-bound model of bradykinin, were found to be able to antagonize the action of bradykinin at its B₂ bradykinin receptor. The best of these, cyclo(d-Lys(Arg)-Phe-Ser-d-Tic-Oic- Arg) [compound 17], has affinities at the human and rat B₂ bradykinin receptors of 230 and 8.5 nM, respectively. This potency is significant, since the analogs lack the C-terminal carboxylate group, residues 2–4 and the important interaction of Phe⁵. These constrained analogs may serve as tools for the determination of the receptor-bound conformation of antagonists at the bradykinin receptor and for the design of even smaller and more potent antagonist analogs.Abbreviations Arg(Me) N-methyl-l-arginine - Arg(Me)₂ N,N-dimethyl-l-arginine - Boc t-butoxycarbonyl - Oic (S,S,S)-octahydroindole-2-carboxylic acid - PAM phenylacetamidomethyl - PyBOP benzotriazole-l-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate - Thi -(2-thienyl)-l-alanine - Tic l-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid     

Nonselective Cation and BK Channels in Apical Membrane of Outer Sulcus Epithelial Cells

The outer sulcus epithelium was recently shown to absorb cations from the lumen of the gerbil cochlea. Patch clamp recordings of excised apical membrane were made to investigate ion channels that participate in this reabsorptive flux. Three types of channel were observed: (i) a nonselective cation (NSC) channel, (ii) a BK (large conductance, maxi K or K _Ca ) channel and (iii) a small K⁺ channel which could not be fully characterized. The NSC channel found in excised insideout patch recordings displayed a linear current-voltage (I-V) relationship (27 pS) and was equally conductive for Na⁺ and K⁺, but not permeable to Cl⁻ or N-methyl-d-glucamine. Channel activity required the presence of Ca²⁺ at the cytosolic face, but was detected at Ca²⁺ concentrations as low as 10⁻⁷ m (open probability (P _o ) = 0.11 ± 0.03, n= 8). Gadolinium decreased P _o of the NSC channel from both the external and cytosolic side (IC₅₀∼ 0.6 μm). NSC currents were decreased by amiloride (10 μm− 1 mm) and flufenamic acid (0.1 mm). The BK channel was also frequently (38%) observed in excised patches. In symmetrical 150 mm KCl conditions, the I-V relationship was linear with a conductance of 268 pS. The Goldman-Hodgkin-Katz equation for current carried solely by K⁺ could be fitted to the I-V relationship in asymmetrical K⁺ and Na⁺ solutions. The channel was impermeable to Cl⁻ and N-methyl-d-glucamine. P _o of the BK channel increased with depolarization of the membrane potential and with increasing cytosolic Ca²⁺. TEA (20 mm), charybdotoxin (100 nm) and Ba²⁺ (1 mm) but not amiloride (1 mm) reduced P _o from the extracellular side. In contrast, external flufenamic acid (100 μm) increased P _o and this effect was inhibited by charybdotoxin (100 nm). Flufenamic acid inhibited the inward short-circuit current measured by the vibrating probe and caused a transient outward current. We conclude that the NSC channel is Ca²⁺ activated, voltage-insensitive and involved in both constitutive K⁺ and Na⁺ reabsorption from endolymph while the BK channel might participate in the K⁺ pathway under stimulated conditions that produce an elevated intracellular Ca²⁺ or depolarized membrane potential. Received: 14 October 1999/Revised: 10 December 1999     

Functional and biochemical characteristics of a putative quisqualate-type receptor in rat striatum: Effect of brain lesions

Excitatory amino acids such asl-glutamate (Glu) and quisqualate (QUIS) markedly potentiated K⁺-evoked release of exogeneous [³H]dopamine (DA) from rat striatal slices. Intranstriatal kainic acid injections resulted in a total disappearance of the stimulatory effects of Glu on evoked-release of [³H]DA as well as in a parallel reduction in the maximal number (B_max) of ad-aspartate-insensitivel-[³H]Glu binding site in striatal particulate fractions. Following cortical ablation, the potentiating effect of Glu on [³H]DA release in decorticated striatal slices lasted longer, compared to normal slices, and occured during the 2nd min following K⁺-depolarization. However, the extent (%) of Glu stimulation on [³H]DA release remained the same in decorticated and normal striatal slices. Cortical ablation produced also a significant decrease in the B_max and in theK _d of thed-aspartateinsensitive binding site towardsl[³H]Glu. These results support the proposal that thed-aspartate-insensitive Glu binding site is somehow related to an amino acid receptor-mediated modulation of dopaminergic transmission in the rat corpus striatum.     

Interaction between antagonists of angiotensin II and antidiuretic hormone in isolated toad tissues

Responses of isolated aorta and toad skin from Bufo arenarum to angiotensin II (AT II) and antidiuretic hormone (ADH) were examined. Inhibitory effects on both responses were obtained either by AT II antagonist or ADH (ADHant). Contractile responses to AT II and AVT were inhibited in a similar way by both Leu⁸ AT II and ADHant. No blocking effect could be obtained against norepinephrine. Leu⁸ AT II, ADHant and an oxytocin antagonist were able to inhibit osmotic water permeability (P_osm) and short-circuit current (SCC) response in toad skin. The inhibitor not only blocked its own agonist response but also other peptide agonistics' responses. No antagonist affected P_osm response to isoproterenol (Isop). The striking similarities among ADH and AT II receptors in amphibian tissues suggest a common peptide hormone receptor.     

Nmr investigation of CYCLO7,10[C7,X9,C10,Nle12] analogues of the α-factor from saccharomyces cerevisiae

The cyclo^7,10[Cys⁷,Cys¹⁰,Nle¹²], cyclo^7,10[Cys⁷,D -Ala⁹,Cys¹⁰,Nle¹²], and cyclo^7,10[Cys⁷,L -Ala⁹,Cys¹⁰,Nle¹²] analogues of the α-factor mating pheromone (WHWLQLKPGQPMY) of the yeast Saccharomyces cerevisiae were studied in DMSO/water (80 : 20) and aqueous solution by nmr spectroscopy. In addition, the cyclo^7,10[Cys⁷,D -Val⁹,Cys¹⁰,Nle¹²] α-factor was examined in DMSO/water. Nuclear Overhauser effect (NOE) and NH dδ/dT data indicate that the cyclo^7,10[Cys⁷,D -Val⁹,Cys¹⁰,Nle¹²] α-factor adopts a type II β-turn in DMSO/water and that the cyclo^7,10[Cys⁷,D -Ala⁹,Cys¹⁰,Nle¹²] - and cyclo^7,10-[Cys⁷,L -Ala⁹,Cys¹⁰,Nle¹²] α-factor analogues adopt type II and type I/III β-turns, respectively, in both DMSO/water and aqueous solutions. In aqueous solution, residues 8 and 9 of the cyclo^7,10[Cys⁷,Cys¹⁰,Nle¹²] α-factor appear to adopt at least two distinct conformations, one of these being identified as a type I/III β-turn. In contrast, the cyclo^7,10[Cys⁷,Cys¹⁰,Nle¹²] α-factor appears to adopt predominately a type II β-turn in DMSO/water. Quantitative NOE measurements of the cyclo^7,10[Cys⁷,Cys¹⁰,Nle¹²]-, cyclo^7,10[Cys⁷,D -Val⁹,Cys¹⁰,Nle¹²]-, and cyclo^7,10[Cys⁷,L -Ala⁹,Cys¹⁰,Nle¹²] α-factors in DMSO/water were used to derive three-dimensional structures of the cyclo^7,10[Cys⁷,Pro⁸,X⁹Cys¹⁰] portion of these analogues. © 1994 John Wiley & Sons, Inc.