Synthesis and biological activity of analogs of dynorphin-A(1-13) substituted in positions 2 and 4: design of [Ala2,Trp4]-Dyn-A(1-13) as a putative selective opioid antagonist

Mono- and di-substituted analogs of dynorphin-A(1-13) (Dyn-A(1-13)) were synthesized by the solid-phase procedure. The products were purified and analyzed for their ability to inhibit the electrically evoked contractions of the guinea pig ileum (GPI) and mouse vas deferens (MVD) and to compete with the binding of [3H]etorphine ([3H]ET) and [3H]ethylketocyclazocine ([3H]EKC) to homogenates of rat brain (mu-, delta-, kappa 2-receptors) and guinea pig cerebellum (kappa-receptor), respectively. Introduction of Ala in position 2 caused a drastic decrease in the activity of the peptide on the smooth muscle preparations (IC50 of 104 and 2.250 nM in the GPI and the MVD as compared with 0.7 and 21 nM for the parent peptide, respectively). Conversely, this analog retained much of the opioid binding activity of Dyn-A(1-13) (relative binding potencies of 15 and 72% for the displacement of [3H]ET and [3H]EKC, respectively). The replacement of Phe4 by Trp also caused drastic decreases in the activity of the peptide in the smooth muscle preparations (relative potencies of 0.8 and 8.8% on the GPI and MVD) while much of the binding potency to the opioid receptors was retained (31 and 67% for the displacement of [3H]ET and [3H]EKC, respectively). [Ala2,Trp4]-Dyn-A(1-13) was the least potent peptide tested in the smooth muscle assays (relative potencies: 0.1 and 0.6%). However, this latter analog still retained some opioid binding activity in the displacement of [3H]ET to rat brain homogenates (3%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of a myometrial oxytocin-receptor protein

The specific binding of [3H]oxytoxin to uterine membrane preparations derived from different species at late pregnancy was examined. The highest receptor density (bmax value) was found in membranes derived from the myometria of guinea pigs between day 60 post-conception (bmax = 3.6 +/- 0.1 pmol/mg) and day 65 (bmax = 4.4 +/- 0.1 pmol/mg). The similarity of Kd values for oxytocin binding (Kd = 2.6 +/- 0.2 nM) and for vasopressin binding (Kd = 2.1 +/- 0.4 nM) to the same membranes derived from a guinea pig myometrium indicate a homogeneous population of high-affinity binding sites which do not discriminate between these two hormones. Competitive binding experiments with specific oxytocin agonists containing either sarcosine or N-methylalanine in the place of Pro7 demonstrated that these myometrial receptors have the pharmacological properties of oxytocin receptors. The analogue of 1-deamino-[8-lysine]vasopressin containing a photoreactive azidophenylamidino group at the sidechain of Lys8 retained roughly the same receptor affinity as oxytocin. In photoaffinity labelling experiments with the tritium-labelled analogue a membrane protein from guinea pig myometrium with an apparent relative molecular mass Mr of 78,000 +/- 5000 (n = 13) was preferentially labelled. The labelling of this protein was completely suppressed by a 100-fold molar excess of either oxytocin, or [Sar7]oxytocin or [Thr4, Sar7]oxytocin, but not by other peptide hormones. These results provide evidence that the labelled 78,000-Mr protein is a myometrial oxytocin-receptor protein.     

Biological activity of parathyroid hormone antagonists substituted at position 13.

Lysine occupies position 13 in the parathyroid hormone (PTH) antagonist, [Nle8,18,Tyr34]bPTH(7-34)NH2. Acylation of the epsilon-amino group in lysine 13 by a hydrophobic moiety is well tolerated in terms of bioactivity: the analog [Nle8,18, D-Trp12,Lys 13 (epsilon-3-phenylpropanoyl),Tyr34]bPTH(7-34)NH2 is equivalent to the parent peptide in its affinity for PTH receptors and its ability to inhibit PTH-stimulated adenylate cyclase in both kidney- and bone-based assays. Truncation of this peptide by deletion of phenylalanyl7 with concomitant removal of the amino-terminal alpha-amino group yielded the analog desamino[Nle8,18,D-Trp12,Lys13 (epsilon-3-phenylpropanoyl),Tyr34]bPTH(8-34)NH2, an antagonist of high potency in vitro (Kb = 4 and 9 nM, Ki = 73 and 3.5 nM in kidney- and bone-based assays, respectively). Also this analog is potentially stable to aminopeptidases present in many biological systems.     

Evidence that the potential antipsychotic agent rimcazole (BW 234U) is a specific, competitive antagonist of sigma sites in brain

Rimcazole (BW 234U) is a potential antipsychotic agent which in open-clinical trials appears to be effective in acute schizophrenic patients. In the present study, rimcazole was found to block the specific binding of [3H]-(+)-SKF 10,047 to sigma sites in rat and guinea pig brain (IC50 = 5.0 X 10(-7) M). The compound was 100 times weaker as a blocker of phencyclidine sites (IC50 = 4.3 X 10(-5) M). At 1 X 10(-5) M, rimcazole had only weak effects on mu, delta, kappa and epsilon opioid receptors. Scatchard analysis of the binding data from guinea pig brain revealed an apparent KD for [3H]-(+)-SKF 10,047 of 85 +/- 5 nM and a Bmax of 824 +/- 27 fmole/mg protein. In the presence of 5 X 10(-7) M BW 234U, the apparent KD was 165 +/- 35 nM, but the Bmax (892 +/- 146 fmoles/mg protein) was not affected. This suggests that rimcazole is a competitive inhibitor of sigma sites. The agent was also capable of blocking sigma sites in vivo (ID50 = 6 mg/kg i.p., mice) as judged by an in vivo sigma receptor binding assay. Thus, if the antipsychotic activity of rimcazole is confirmed in double-blind, placebo-controlled trials, it would be the first compound whose mechanism of antipsychotic activity may best be explained by a direct blockade of sigma sites and not by a direct blockade of dopamine (D2) receptors in brain.     

Identification of a VIP-specific receptor in guinea pig tenia coli

Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) interact with VPAC(2) receptors in rabbit and guinea pig (GP) gastric muscle but with functionally distinct VIP and PACAP receptors in GP tenia coli. This study examined whether selectivity for VIP was determined by two residues (40, 41) in the extracellular domain that differ in the VIP receptors of GP gastric and tenial muscle. A mutant rat VPAC(2) receptor (L40F, L41F), and two chimeric receptors in which the NH(2)-terminal domain of rat VPAC(2) receptor was replaced with that of GP gastric (chimeric-G) or tenia coli (chimeric-T) VIP receptors, were constructed and expressed in COS-1 cells. VIP and PACAP bound with equal affinity to wild-type and mutant rat VPAC(2) receptors and to chimeric-G receptor (IC(50): VIP 0.3 +/- 0.1 to 1.5 +/- 0.4 nM, PACAP 0.4 +/- 0.1 to 2.5 +/- 0.1 nM) and stimulated cAMP with equal potency (EC(50): VIP 13 +/- 5 to 48 +/- 8 nM, PACAP 8 +/- 3 to 31 +/- 14 nM). VIP bound with high affinity also to chimeric-T receptor (IC(50): 0.5 +/- 0.1 nM) and stimulated cAMP with high potency (EC(50): 3 +/- 1 nM). In contrast, PACAP exhibited >1,000-fold less affinity for binding or potency for stimulating cAMP. We conclude that GP tenia coli express a VIP-specific receptor and that selectivity is determined by a pair of extracellular phenylalanine residues.     

Identity of a membrane-bound vasoactive intestinal peptide-binding protein with calmodulin.

A vasoactive intestinal peptide (VIP)-binding protein purified from guinea pig lung membranes (p18) was digested with trypsin, and the amino acid sequence of the peptide fragments was determined. The sequence of six tryptic fragments of p18 was identical with subsequences present in mammalian calmodulin. Authentic porcine brain calmodulin and p18 co-migrated on an sodium dodecyl sulfate-electrophoresis gel and displayed identical chromatographic behavior on a reverse phase high performance liquid chromatography column. The VIP-binding properties of p18 and calmodulin were indistinguishable. Both proteins displayed saturable and apparent high affinity binding of VIP, evidenced by potent inhibition of complexation with [Tyr10-125I]VIP by unlabeled VIP (IC50 = 6.0-8.1 nM). Rat growth hormone releasing factor and a C terminally extended form of VIP ([Leu17]VIP-GKR) also displayed potent inhibition of the binding (IC50 = 6.4 and 4 nM, respectively). These neuropeptides are potential modulators of calmodulin function.     

Localization of biotinylated gonadotropin releasing hormone on pituitary monolayer cells with avidin-biotin-peroxidase complexes

The new avidin-biotin-peroxidase complex (ABC) technique was used to localize the [D-Lys6] analog of gonadotropin releasing hormone (GnRH), labeled with biotin, on pituitary monolayer cultures from female rats. Staining was diffuse, or in patches, on the surface of 10-17% of the cells 30 sec-3 min after the addition of 10(-10)-10(-12) M biotin-labeled GnRH. In parallel studies, double stains for gonadotropins showed label on 16.3 +/- 2% of the monolayers. Capping was evident by 3 min after exposure and the stain appeared in dense patches, vesicles, or granules 10-30 min after exposure. The stain was abolished by the addition of a 10- to 100-fold excess of unlabeled [D-Lys6] GnRH. Biotinylated GnRH released luteinizing hormone (LH) and follicle stimulating hormone (FSH) and was either equipotent or 10 times more potent than the unlabeled analog in multiple dose-response tests. The ED50 of the 4 hr release was 0.075 nM for LH and 0.02 nM for FSH. Competitive binding assays showed that the binding affinity of the biotinylated GnRH was within the range found for the unlabeled analog (0.7 nM-IC50). This report describes the localization of biotinylated GnRH on the surfaces of cells exposed to low concentrations of the analog with a technique that requires minimal manipulation of the cells, and is performed in less than one day.     

ONO-4057, a novel, orally active leukotriene B4 antagonist: effects on LTB4-induced neutrophil functions.

ONO-4057(5-[2-(2-Carboxyethyl)-3-[6-(4-methoxyphenyl)-5E- hexenyl]oxyphenoxy]valeric acid), an orally active leukotriene B4(LTB4) antagonist, displaced the binding of [3H] LTB4 to the LTB4 receptor in human neutrophil (Ki = 3.7 +/- 0.9 nM). ONO-4057 inhibited the LTB4-induced rise in cytosolic free calcium (the concentration causing 50% inhibition (IC50) = 0.7 +/- 0.3 microM) and inhibited human neutrophil aggregation, chemotaxis or degranulation induced by LTB4 (IC50 = 3.0 +/- 0.1, 0.9 +/- 0.1 and 1.6 +/- 0.1 microM) without showing any agonist activity at concentration up to 30 microM. ONO-4057 did not inhibit fMLP or C5a-induced neutrophil activation at concentrations up to 30 microM. In the in vivo study, ONO-4057 given orally, prevented LTB4-induced transient neutropenia or intradermal neutrophil migration in guinea pig (the dose causing 50% efficacy (ED50) = 25.6mg/kg or 5.3mg/kg). Furthermore, ONO-4057 given topically, suppressed phorbol-12-myristate-13-acetate (PMA)-induced neutrophil infiltration in guinea pig ear (the effective dose = 1 mg/ear). These results indicate that ONO-4057 is a selective and orally active LTB4 antagonist and may be a potential candidate for the treatment of various inflammatory diseases.     

Synthesis and biological activity of a biotinylated vasopressin analog

To study vasopressin receptor-mediated endocytosis using electronmicroscopy methods and to develop avidin affinity columns for receptor purification, we synthesized and tested the biological properties of a biotinylated vasopressin (VP) analog [1-(2-mercapto) propionic acid] 8-[lysine-N6-biotin] VP (B-MLVP). B-MLVP was prepared by coupling biotin to the epsilon amine of the lysine residue in [1-(2-mercapto) propionic acid] 8-(lysine) VP (MLVP). The structure of HPLC purified B-MLVP was confirmed by fast atom bombardment mass spectrometry. B-MLVP effectively competed for arginine vasopressin (AVP) binding sites in canine renal plasma membranes on the surface of LLC-PK1 kidney cells. Dissociation constants of 15 nM and 202 nM were calculated from the results of competition binding assays conducted with membranes and cells, respectively. B-MLVP stimulated adenylate cyclase activity and elevated cellular 3',5',cyclic-AMP (cAMP) content in a manner similar to AVP, indicating it is an agonist of VP action in renal tissue. These observations indicate that B-MLVP is an agonist of VP action and may be used to study renal VP receptors by employing avidin coupled to various reporter groups.     

Biological activity of leukotriene B4 analogs: inhibition of guinea pig eosinophil migration in vitro by the 2,6-disubstituted pyridine analogs U-75,302 and U-75,485.

A "late phase" antigen-induced bronchoalveolar eosinophilia has been demonstrated in ovalbumin sensitized guinea pigs (1,2). This in vivo response to antigen inhalation can be inhibited by a 2,6-disubstituted pyridine analog of LTB4, U-75,302(2) (3). In the present study, the mechanism of the drug action was studied by assessing the activity of U-75,302 and a second analog, U-75,485 to displace [3H]-leukotriene B4 binding at the guinea pig eosinophil membrane, as well as their action as chemoattractants or inhibitors of the directional migration of guinea pig eosinophils in vitro. Radioligand competition experiments demonstrated that both analogs interacted strongly with the high affinity LTB4 binding sites on guinea pig eosinophil membrane. Both analogs are powerful chemoattractants for guinea pig eosinophils since they induced directional migration of guinea pig eosinophils when administered alone. In addition, when the cells were treated with either analog and their chemotaxis response was measured in response to a natural chemoattractant, both U-75,302 and U-75,485 at concentrations of 0.1 to 100 microM dose dependently inhibited the LTB4 induced chemotaxis response. The EC50s obtained for U-75,302 and U-75,485 as inhibitors of LTB4 induced guinea pig eosinophil chemotaxis were estimated to be 11.5 +/- 5.5 microM and 5.4 +/- 2.5 microM respectively. Under the same conditions, they had no significant effect upon eosinophil migration induced by zymosan activated plasma at concentrations below 100 microM. We suggest that the inhibition of antigen-induced eosinophil infiltration in guinea pig airway in vivo by U-75,302 or U-75,485 may be a result of partial antagonism or desensitization at the LTB4 receptor level of guinea pig eosinophils.     

Cloning of bradykinin precursor cDNAs from skin of Bombina maxima reveals novel bombinakinin M antagonists and a bradykinin potential peptide

Bombinakinin M (DLPKINRKGP-bradykinin) is a bradykinin-related peptide purified from skin secretions of the frog Bombina maxima. As previously reported, its biosynthesis is characterized by a tandem repeats with various copy numbers of the peptide and sometimes co-expressed with other structure-function distinguishable peptides. At present study, two novel cDNAs encoding bombinakinin M and its variants were cloned from a cDNA library from the skin of the frog. The encoded two precursor proteins are common in that each contains three repeats of a novel 16-amino acid peptide unit and one copy of kinestatin at their N- and C-terminal parts, respectively. They differ in that the first precursor contains two copies of bombinakinin M and the second one contains one copy of a novel bombinakinin M variant. Bombinakinin M was found to elicit concentration-dependent contractile effects on guinea pig ileum, with an EC50 value of 4 nM that is four times higher than that of bradykinin (1 nM). Interestingly, the synthetic peptide (DYTIRTRLH-amide), as deduced from the 16-amino acid peptide repeats in the newly cloned cDNAs, possessed weak inhibitory activity on the contractile effects of bombinakinin M, but not on that of bradykinin. Furthermore, the newly identified bombinakinin M variant (DLSKMSFLHG-Ile1-bradykinin), did not show contractile activity on guinea pig ileum, but showed potentiation effect on the myotropic activity of bradykinin. In a molar ratio of 1:58, it augmented the activity of bradykinin up to two-fold.     

Probing peptide backbone function in bombesin. A reduced peptide bond analogue with potent and specific receptor antagonist activity

Each peptide bond CONH group in the most important COOH-terminal octapeptide region of [Leu14]bombesin was replaced by a CH2NH group using recently developed rapid solid-phase methods. The resulting analogues were then examined for amylase releasing activity in guinea pig pancreatic acini and for their ability to inhibit binding of [125I-Tyr4]bombesin to acinar cells. Replacement of the Trp8-Ala9, Gly11-His12, and His12-Leu13 peptide bonds resulted in about 1000-, 200-, and 300-fold losses in both amylase releasing activity and binding affinity. The Val10-Gly11 replacement, however, retained 30% potency relative to the parent peptide. Ala9-Val10 and Leu13-Leu14 bond replacement analogues exhibited no detectable amylase releasing activity but were still able to bind to acini with Kd values of 1060 and 60 nM, respectively (compared to 15 nM for [Leu14]bombesin itself). Subsequently, both analogues were demonstrated to be competitive inhibitors of bombesin-stimulated amylase release with IC50 values of 937 and 35 nM, respectively. [Leu14-psi-CH2NH-Leu13]Bombesin exhibits a 100-fold improvement in binding affinity compared to previously reported bombesin receptor antagonists and showed no affinity for substance P receptors. It was also a potent inhibitor of bombesin-stimulated growth of murine Swiss 3T3 cells with an IC50 of 18 nM. In terms of a bombesin receptor-binding conformation, these results may aid in the delineation of intramolecular hydrogen-bonding points and the eventual design of improved, conformationally restricted analogues.     

Neosurugatoxin, a Specific Antagonist of Nicotinic Acetylcholine Receptors

Neosurugatoxin (NSTX) (3 nM-30 nM), recently isolated from the Japanese ivory mollusc (Babylonia japonica) exerted a potent antinicotinic action in the isolated guinea pig ileum. Specific [3H]nicotine binding to rat forebrain membranes was saturable, reversible, and of high affinity. Nicotinic cholinergic agonists exhibited a markedly greater affinity for [3H]nicotine binding sites than a muscarinic agonist, oxotremorine. Although alpha-bungarotoxin had no effect on [3H]nicotine binding, low concentrations (1 nM-1 microM) of NSTX inhibited [3H]nicotine binding in the forebrain membranes and its IC50 value was 69 +/- 6 nM. On the other hand, NSTX did not affect muscarinic receptor binding in the brain. These data indicate that NSTX may be of appreciable interest as a neurotoxin with a selective affinity for ganglionic nicotinic receptors.     

Stereoselectivity for the (R)-Enantiomer of HA-966 (l-Hydroxy-3-Aminopyrrolidone-2) at the Glycine Site of the N-Methyl-d-Aspartate Receptor Complex

HA-966 (1-hydroxy-3-aminopyrrolidone-2) is an antagonist at the glycine allosteric site of the N-methyl-D-aspartate receptor ionophore complex. Unlike presently known glycine antagonists, HA-966 is chiral. We report stereoselectivity for the (R)-enantiomer at the glycine antagonist site. In [3H]glycine binding, the (R)-enantiomer has an IC50 of 4.1 +/- 0.6 microM. The racemic mixture has an IC50 of 11.2 +/- 0.5 microM, whereas (S)-HA-966 has an IC50 greater than 900 microM. In glycine-stimulated [3H]1-[1-(2- thienyl)cyclohexyl]piperidine binding, the (R)-enantiomer inhibits with an IC50 of 121 +/- 61 microM, whereas the racemic mixture has an IC50 of 216 +/- 113 microM and (S)-HA-966 is inactive. The inhibition by (R)-HA-966 can be prevented by the addition of glycine. (R)-HA-966 and racemic HA-966, but not (S)-HA-966, also prevent N-methyl-D-aspartate cytotoxicity in cortical cultures. The (R)-enantiomer and, less potently, the (S)-enantiomer inhibit N-methyl-D-aspartate-evoked [3H]norepinephrine release from rat hippocampal slices (IC50 values of about 0.3 mM and 1.6 mM, respectively), but only the inhibition by (R)-HA-966 is reversed by added glycine. In glutamate-evoked contractions of the guinea pig ileum, (R)-HA-966 causes a glycine-reversible inhibition (IC50 of about 150 microM), whereas (S)-HA-966 is much less potent (IC50 of greater than 1 mM). These results demonstrate stereoselectivity of the glycine antagonist site of the N-methyl-D-aspartate receptor complex in a variety of tissues and assays. The stereoselectivity also confirms the specificity of N-methyl-D-aspartate receptors in glutamate-evoked contractions of the guinea pig ileum, and supports their similarity to central N-methyl-D-aspartate receptors.     

Synthesis and biological activity of dynorphin-(1-13) and analogs substituted in positions 8 and 10

Dynorphin-(1-13) (Dyn-(1-13)) and various analogs substituted in positions 8 and 10 were synthesized by the solid-phase technique and analyzed for their ability to inhibit the electrically evoked contraction of the guinea pig ileum (GPI) and to compete with the binding of [3H]-ethylketocyclazocine (EKC, kappa ligand), [3H]-[D-Ala2, MePhe4-Gly-ol5]-enkephalin (DAGO, mu ligand) and [3H]-[D-Ser2, Thr6]-Leu-enkephalin (DSLET, delta ligand) to membrane preparations of the guinea pig cerebellum or rat brain. Introduction of Ala in position 8 decreased the activity of the peptide on the GPI by 50% but induced a 2.22-fold increase in its affinity for the kappa receptor ([3H]-EKC binding displacement from guinea pig cerebellum; Ki of 0.05 nM as compared with 0.11 nM for Dyn-(1-13)). On the other hand, the ability of [Ala8] Dyn-(1-13) to displace the binding of [3H]-DSLET from rat brain membranes was decreased by a factor of 1.7 while its affinity for the mu receptor was not greatly affected ([3H]-DAGO displacement; Ki of 0.44 nM as compared with 0.50 nM for Dyn-(1-13)). Replacement of position 8 by D-Ala caused similar changes in the activity of the peptide but the increase in its affinity for the kappa site was somewhat smaller (Ki of 0.08 nM as compared with 0.11 nM). [D-Pro10]-Dyn-(1-13) was equipotent to [Ala8]-Dyn-(1-13) in the GPI but its affinity for the mu binding site was decreased by a factor of 2.7 as compared with Dyn-(1-13).(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis of fully active biotinylated analogues of parathyroid hormone and parathyroid hormone-related protein as tools for the characterization of parathyroid hormone receptors.

The synthesis, purification, and characterization of biotinylated analogues of parathyroid hormone (PTH) and PTH-related protein (PTHrP) are described. A novel methodology was developed which allowed the selective biotinylation during solid-phase synthesis of either the Lys13 or Lys26 residue in PTH/PTHrP sequences. Incorporation of orthogonally protected N alpha-Boc-Lys(N epsilon-Fmoc) at a selected position in the sequence, followed by selective side-chain deprotection and biotinylation of the epsilon-amino group, permitted modification of the specific lysine only. Biotinylated analogues of [Nle8,18,Tyr34]bPTH(1-34)NH2 (analogue 1a) were prepared by modification of Lys13 with a biotinyl group (analogue 1) or a biotinyl-epsilon-aminohexanoyl group (analogue 2) or at Lys26 with a biotinyl-epsilon-aminohexanoyl group (analogue 3). A biotinylated PTHrP antagonist [Leu11,D-Trp12,Lys13(N epsilon-(biotinyl-beta-Ala))]PTHrP(7-34)NH2 (analogue 5), was also prepared. In a different synthetic approach, selective modification of the thiol group of [Cys35]PTHrP(1-35)NH2, in solution, with N-biotinyl-N'-(6-maleimidohexanoyl)hydrazide, resulted in analogue 4. The high affinities of the biotinylated analogues for PTH receptors present in human osteosarcoma B-10 cells or in porcine renal cortical membranes (PRCM), were comparable to those of the underivatized parent peptides. The analogues were also highly potent in stimulation of cAMP formation (analogues 1-4) or inhibition of PTH-stimulated adenylyl cyclase (analogue 5) in B-10 cells. The most potent analogue (analogue 1) had potencies in B-10 cells (Kb = 1.5 nM, Km = 0.35 nM) and in porcine renal membranes (Kb = 0.70 nM) identical or similar to those of its parent peptide, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Streptokinase binds preferentially to the extended conformation of plasminogen through lysine binding site and catalytic domain interactions

Binding of streptokinase (SK) to plasminogen (Pg) activates the zymogen conformationally and initiates its conversion into the fibrinolytic proteinase, plasmin (Pm). Equilibrium binding studies of SK interactions with a homologous series of catalytic site-labeled fluorescent Pg and Pm analogues were performed to resolve the contributions of lysine binding site interactions, associated changes between extended and compact conformations of Pg, and activation of the proteinase domain to the affinity for SK. SK bound to fluorescein-labeled [Glu]Pg(1) and [Lys]Pg(1) with dissociation constants of 624 +/- 112 and 38 +/- 5 nM, respectively, whereas labeled [Lys]Pm(1) bound with a 57000-fold tighter dissociation constant of 11 +/- 2 pM. Saturation of lysine binding sites with 6-aminohexanoic acid had no effect on SK binding to labeled [Glu]Pg(1), but weakened binding to labeled [Lys]Pg(1) and [Lys]Pm(1) 31- and 20-fold, respectively. At low Cl(-) concentrations, where [Glu]Pg assumes the extended conformation without occupation of lysine binding sites, a 23-fold increase in the affinity of SK for labeled [Glu]Pg(1) was observed, which was quantitatively accounted for by expression of new lysine binding site interactions. The results support the conclusion that the SK affinity for the fluorescent Pg and Pm analogues is enhanced 13-16-fold by conversion of labeled [Glu]Pg to the extended conformation of the [Lys]Pg derivative as a result of lysine binding site interactions, and is enhanced 3100-3500-fold further by the increased affinity of SK for the activated proteinase domain. The results imply that binding of SK to [Glu]Pg results in transition of [Glu]Pg to an extended conformation in an early event in the SK activation mechanism.     

Isolation, structural characterization and pharmacological activity of dog neuromedin U

The neuromedin U-like immunoreactivity in an extract of dog small intestine was resolved by reversed-phase HPLC into two molecular forms. The primary structure of the larger form (NMU-25) was established as: Phe-Arg-Leu-Asp-Glu-Glu-Phe-Gln-Gly-Pro10-Ile-Ala-Ser-Gln-Val-Arg- Arg-Gln-Phe- Leu20-Phe-Arg-Pro-Arg-Asn-NH2. This sequence shows five substitutions relative to pig neuromedin U-25. The primary structure of the second peptide (NMU-8) was established as: pGlu-Phe-Leu-Phe-Arg-Pro-Arg-Asn-NH2. The sequence contains the substitution pGlu for Tyr1 compared with pig neuromedin U-8. The potency of synthetic dog NMU-8 in contracting smooth muscle from the rat uterus (EC50 10 +/- 2 nM; mean +/- S.E., n = 6) was not significantly different from the corresponding potency of pig NMU-8 (EC50 16 +/- 5 nM) but the maximum response produced by the dog peptide was greater (58%; p less than 0.05) than that produced by pig NMU-8.     

Structure-activity studies of new melanocortin peptides containing an aromatic amino acid at the N-terminal position

Cyclic melanotropin peptides, designed with an aromatic amino acid substitution at the N-terminal position of the MT-II-type scaffold, were prepared by solid-phase peptide synthesis and evaluated for their ability to bind to and activate human melanocortin-1, -3, -4, and -5 receptors. The structure-activity studies of these MT-II analogues have identified a selective antagonist at the hMC4R (H-Phe-c[Asp-Pro-d-Nal(2')-Arg-Trp-Gly-Lys]-NH(2), pA(2)=8.7), a selective partial agonist at the hMC4R (H-d-Nal(2')-c[Asp-Pro-d-Phe-Arg-Trp-Gly-Lys]-NH(2), IC(50)=11nM, EC(50)=56nM), and a selective partial agonist at the hMC3R (H-d-Phe-c[Asp-Pro-d-Phe-Arg-Trp-Lys]-NH(2), IC(50)=3.7nM, EC(50)=4.9nM). Aromatic amino acid substitution at the N-terminus in conjuction with the expansion of the 23-membered cyclic lactam MT-II scaffold to a 26-membered scaffold by addition of a Gly residue in position 10 leads to melanotropin peptides with enhanced receptor selectivity.     

Existence of Three Subtypes of Bradykinin B2 Receptors in Guinea Pig

We describe the binding of [3H]bradykinin to homogenates of guinea pig brain, lung, and ileum. Analysis of [3H]bradykinin binding kinetics in guinea pig brain, lung, and ileum suggests the existence of two binding sites in each tissue. The finding of two binding sites for [3H]bradykinin in ileum, lung, and brain was further supported by Scatchard analysis of equilibrium binding in each tissue. [3H]Bradykinin binds to a high-affinity site in brain, lung, and ileum (KD = 70-200 pM), which constitutes approximately 20% of the bradykinin binding, and to a second, lower-affinity site (0.63-0.95 nM), which constitutes the remaining 80% of binding. Displacement studies with various bradykinin analogues led us to subdivide the high- and lower-affinity sites in each tissue and to suggest the existence of three subtypes of B2 receptors in the guinea pig, which we classify as B2a, B2b, and B2c. Binding of [3H]bradykinin is largely to a B2b receptor subtype, which constitutes the majority of binding in brain, lung, and ileum and represents the lower-affinity site in our binding studies. Receptor subtype B2c constitutes approximately 20% of binding sites in the brain and lung and is equivalent to the high-affinity site in brain and lung. We suggest that a third subtype of B2 receptor (high-affinity site in ileum), B2a, is found only in the ileum. All three subtypes of B2 receptors display a high affinity for bradykinin, whereas they show different affinities for various bradykinin analogues displaying agonist or antagonist activities.(ABSTRACT TRUNCATED AT 250 WORDS)