Potent analgesic activity of the enkephalin-like tetrapeptide H-Tyr-D-Ala-Gly-Phe-NH2.

The N-terminal tetrapeptide amide analog of enkephalin (H-Tyr-D-Ala-Gly-Phe-NH₂) is approximately equipotent with highly active pentapeptide analogs of enkephalin (and with morphine) in producing analgesia after either intracerebroventricular or intravenous administration. Smaller fragments exhibited diminished potency, but even the dipeptide (H-Tyr-D-Ala-NH₂) retained naloxone-reversible analgesic activity at high intraventricular doses. These findings suggest that while the dipeptide has full intrinsic activity, the tetrapeptide sequence may be a minimum structural requirement for potent analgesia.     

Down regulation of enkephalin (δ) receptors Demonstration in membrane-bound and solubilized receptors

The pentapeptide leucine enkephalin induced down-regulation of enkephalin receptors in neuroblastoma-glioma NG108-15 hybrid cells in a reversible fashion, whereas the stable enkephalin analogue, d-Ala²-Met-enkephalinamide (AMEA), and the potent opiate alkaloid, etorphine, had a prolonged effect. The opiate alkaloid, morphine, which has low affinity to δ-type enkephalin receptors of these cells did not induce down-regulation, whereas AMEA decreased the binding of both opiate agonists and antagonists but had no effect on the binding of the α₂-adrenergic ligand, [³H]yohimbine. From several experiments that were designed to remove the tightly bound AMEA, and from experiments with solubilized receptor we ruled out the possibility that the decreased binding capacity of enkephalin-treated cells reflects only receptor masking. The study suggests that down-regulation of enkephalin receptors that may also occur in vivo can account for some of the abnormal physiological responses of subjects treated chromically with opiates. However, since opiates from the morphine type can induce opiate tolerance in vivo, but not down-regulation of enkephalin receptors in the cultured cells, we suggest that down-regulation of δ-type opiate receptors may not be prerequisite for the development of the physiological tolerance/dependence on these alkaloids.     

Interaction of iodinated enkephalin analogues with opiate receptors.

Radioiodinated derivatives of the metabolically stable enkephalin analogues, [DAla²,Leu⁵]- and [DAla²,DLeu⁵]-enkephalin, have been prepared. Such derivatives show sterospecific binding to receptors in brain homogenates and some neuroblastoma cell lines such as NG108-15 and N4TG1. The relative effects of levorphanol and dextrorphan and Na⁺ and Mn⁺⁺ ions on enkephalin binding in brain and cells indicate that the iodinated derivatives are interacting with opiate receptors. Levorphanol is considerably more potent in displacing specifically bound enkephalin than dextrorphan. Sodium ions at physiological concentrations decrease enkephalin binding whereas manganese ions enhance it. Unlabelled monoiodo derivatives retain high potency in the guinea-pig ileum, mouse vas deferens and receptor binding assays. Unlabelled diiodo derivatives show far lower potency in these assays. It is concluded that radio-iodinated derivatives containing one iodine per molecule retain high affinity for the opiate receptor but diiodo derivatives do not.     

Activation of the l,d‐transpeptidation peptidoglycan cross‐linking pathway by a metallo‐d,d‐carboxypeptidase in Enterococcus faecium

Bypass of the penicillin‐binding proteins by an l ,d ‐transpeptidase (Ldt_fm) confers cross‐resistance to β‐lactam and glycopeptide antibiotics in mutants of Enterococcus faecium selected in vitro. Ldt_fm is produced by the parental strain D344S although it insignificantly contributes to peptidoglycan cross‐linking as pentapeptide stems cannot be used as acyl donors by this enzyme. Here we show that production of the tetrapeptide substrate of Ldt_fm is controlled by a two‐component regulatory system (DdcRS) and a metallo‐d ,d ‐carboxypeptidase (DdcY). The locus was silent in D344S and its activation was due to amino acid substitutions in DdcS or DdcR that led to production of DdcY and hydrolysis of the C‐terminal d ‐Ala residue of the cytoplasmic peptidoglycan precursor UDP‐MurNAc‐pentapeptide. The T¹⁶¹A and T¹⁶¹M substitutions affected a position of DdcS known to be essential for the phosphatase activity of related sensor kinases. Complete elimination of UDP‐MurNAc‐pentapeptide, which was required specifically for resistance to glycopeptides, involved substitutions in DdcY that increased the catalytic efficiency of the enzyme (E¹²⁷K) and affected its interaction with the cell envelope (I¹⁴N). The ddc locus displays striking similarities with portions of the van vancomycin resistance gene clusters, suggesting possible routes of emergence of cross‐resistance to glycopeptides and β‐lactams in natural conditions.     

Enkephalin dimers: Regulation of cyclic AMP levels in NG108-15 cells

Intracellular cyclic AMP levels were determined for dimeric and monomeric enkephalins interacting with PGE₁-stimulated NG108-15 cells. The dimeric pentapeptide enkephalin (DPE₂), [D-Ala², Leu⁵ -NH-CH₂]₂, displaying very high affinity (K = 4.2 ± 0.3 nM^?1) for the δ-opiate receptor, inhibited cyclic AMP production by 70%. Its IC₅₀-value was between 0.1 and 0.2 nM, similar to that of the potent δ-agonist [D-Ala², D-Leu⁵] enkephalin (DADLE) with K = 1.0 ± 0.1 nM^?1. [D-Ala², Leu⁵] enkephalin amide (DALEA), which is the monomer of DPE₂, showed an IC₅₀ = 4 nM. The dimeric tetrapeptide enkephalin (DTE₁₂), [D-Ala², des-Leu⁵-NH-(CH₂)₆]₂ and its monomer [D-Ala², desLeu⁵] enkephalin amide (DAPEA) showed IC₅₀ = 2 and 20 nM, respectively. These results indicate that the DPE₂ and DTE₁₂ enkephalin dimers are potent δ-agonists.     

Dimeric pentapeptide enkephalin: a novel probe of delta opiate receptors

A dimeric pentapeptide enkephalin (DPE2) consisting of two molecules of [D-Ala 2, Leu 5] enkephalin linked at C-terminal leucine with ethylenediamine, (H-Tyr-D-Ala-Gly-Phe-Leu-NH-Ch2)2 is a bivalent ligand for the delta enkephalin receptors of rat brain and neuroblastoma-glioma hybrid (NG108-15) cells. This new enkephalin analog shows dramatically increased affinity in radioligand assays using whole brain membranes when delta but not mu specific radioligands are employed. When membranes from NG108-15 cells are used, the dimer shows greatly increased activity irrespective of the mu or delta specificity of the tracer. The dimer DPE2 shows a four-fold, "sodium shift" in its IC50 for competition with [3H]naloxone, suggestive of agonist behavior. Agonist activity was confirmed by demonstrating that DPE2 inhibits cyclic AMP production in prostaglandin E1 stimulated NG108-15 cells, and by demonstrating very high potency in the mouse vas deferens bioassay. DPE2 binds to the same delta sites as the delta-selective monomer [D-Ala2, D-Leu5] enkephalin, since the two ligands show complete crossdisplacement. Radiolabeled 3H-DPE2 shows a five-fold higher affinity constant, a 2.5-fold higher association rate constant, and a two-fold lower dissociation rate than the monomer. These results are consistent with the hypothesis that the dimeric pentapeptide enkephalin can bridge two delta receptors. This enkephalin dimer provides a valuable new probe of opiate receptors and their organization in cell membranes.     

The effect of modification of the carboxyl group in short D-arginine 2-containing enkephalin analogs on their affinity and selectivity for opiate receptors

Radioreceptor binding assay using a membrane fraction from the rat brain was applied to study [D-Arg2, Leu5] enkephalin and two series of its analogues truncated at the C-terminus with a free or modified carboxyl group: tetra- and tripeptide amides and ethyl esters. The affinity to mu-specific opiate receptor subtype of the N-terminal [D-Arg2] tetrapeptide ethyl ester was 44 times as high as that of the tripeptide with a free carboxyl, and thus the ester retained up to 10% of leucine-enkephalin binding potency. However, a comparable esterification of the carboxyl group in the N-terminal [D-Arg2] tripeptide led to a 6-fold reduction in its affinity to mu-receptors. Consequently, identical modifications of the C-terminal carboxyl group in enkephalin analogues of various length can have completely different effects. Substitution of the natural glycine residue by D-arginine residue in position 2 of the enkephalin molecule truncated at the C-terminus increased the mu-receptor binding potency of the tetrapeptide, whereas its delta receptor binding potency declined by more than one order of magnitude. Simultaneous replacement of glycine2 by D-arginine2 and carboxyl amidation resulted in the short enkephalin analogue Tyr--D--Arg--Gly--Phe--NH2, whose affinity to mu receptors was four times as high as that of leucine--enkephalin, the tetrapeptide being 284 times more selective for the mu vs. delta opiate receptors.     

Enkephalin analogs containing 4,4-difluoro-2-aminobutyric acid: Synthesis and fluorine effect on the biological activity

Analogs of Met-enkephalin and [d -Pen², d -Pen⁵]enkephalin (DPDPE) containing the partially fluorinated amino acid 4,4-difluoro-2-aminobutyric acid (DFAB) in the 2- or 3-position of the peptide sequence were synthesized and their opioid activities and receptor selectivities were determined in vitro. The linear fluorinated [d -DFAB², Met⁵-NH₂]enkephalin showed μ and δ agonist potencies comparable to those of natural [Leu⁵]enkephalin. The partially fluorinated DPDPE analogs behaved differently as compared with their non-fluorinated correlates. While l -amino acid substitution in position 3 of DPDPE usually resulted in higher δ agonist potency than d -amino acid substitution, [d -DFAB³]DPDPE turned out to be a more potent δ agonist than [l -DFAB³]DPDPE. Furthermore, [d -DFAB³]DPDPE showed over 100-fold higher δ agonist potency than [d -Abu³]DPDPE (Abu=2-aminobutyric acid), indicating that the fluorine substituents interact favorably with a δ opioid receptor subsite. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Demonstration and Characterization of Opiate Inhibition of the Striatal Adenylate Cyclase

Abstract: The conditions in which Leu⁵-enkephalin inhibition of striatal adenylate cyclase was observed were defined. It was determined that enkephalin inhibition was dependent on GTP. The apparent K_m for GTP in opiate inhibition was determined to be 0.5 and 2 μM when 0.1 mM- and 0.5 mM-ATP were used as substrate. ITP, but not CTP or UTP, could substitute for GTP in the reaction. Though the addition of monovalent cations—Na⁺,K⁺, Li⁺, Cs⁺, and choline⁺—stimulated striatal adenylate cyclase activity, enkephalin inhibition of striatal adenylate cyclase did not require Na⁺ when theophylline was used as the phosphodiesterase inhibitor. Under optimal conditions, i.e., 20 μM-GTP and 100 mM-Na⁺, Leu⁵-enkephalin inhibited the striatal adenylate cyclase activity by 23–27%. When the enkephalin regulation of the cyclase activity was further characterized, it was observed that Leu⁵-enkephalin inhibited the rate of the enzymatic reaction. Kinetic analysis revealed that the opioid peptide decreases V_max values but not the K_m values for the substrates Mg²⁺ and Mg-ATP. Agents such as MnCl₂, NaF, and guanyl-5′-ylimido-diphosphate, which directly activated the adenylate cyclase, antagonized the opiate inhibition. Levorphanol and (–)naloxone were more potent than dextrorphan and (+)naloxone in inhibiting adenylate cyclase and in reversing the enkephalin inhibition, respectively. There were differences in the potencies of various opiate peptides in their inhibition of striatal adenylate cyclase activity, with Met⁵- > Leu⁵-enkephalin > β-endorphin. The opiate receptor through which the enkephalin inhibition was observed is most likely δ in nature, since in the presence of either Na⁺ or K⁺, the magnitude of the alkaloid inhibition was reduced, whereas the peptide inhibition was either potentiated or not affected.     

Purification and characterization of l,(l/d)-aminopeptidase from Guinea pig serum

Mammalian sera contain enzymes that catalyze the hydrolytic degradation of peptidoglycans and molecules of related structure and are relevant for the metabolism of peptidoglycans. We now report on a novel L,(L/D)-aminopeptidase found in human and mammalian sera. The enzyme hydrolyses the pentapeptide L-Ala-D-iso-Gln-meso-DAP(omegaNH(2))-D-Ala-D-Ala yielding the free L-alanine and the respective tetrapeptide (K(M) 18 mM). L,(L/D)-aminopeptidase from guinea pig serum was highly purified in four chromatographic steps, up to 700-fold. Molecular weight of the enzyme was estimated by HPLC to be approximately 175,000. The configuration of alanine obtained by hydrolysis of the pentapeptide was determined by oxidation with L-amino acid oxidase. The amino acids sequence in the respective tetrapeptide was deduced from the results of mass spectrometry. The novel L,(L/D)-aminopeptidase also hydrolyzed alanine-4-nitroanilide (K(M)=0.6 mM) and several peptides comprising L-amino acids. Peptides containing D-amino acid at the amino end and L-Asp-L-Asp were not the substrates for this enzyme. The purified enzyme also exhibited enkephalin degrading activity, hydrolyzing enkephalins comprising L,L- and L,D-peptide bonds. The enzyme was inhibited strongly by metal chelating agents, bestatin and amastatin.     

Structural requirements for microvascular permeability-increasing ability of peptides: Studies on analogues of a fibrinogen pentapeptide fragment

A pentapeptide, Ala-Arg-Pro-Ala-Lys, liberated from fibrinogen during plasmin-mediated fibrinolysis, was shown earlier to increase microvascular permeability in rat and human skin. Eighteen new analogues have now been synthesized in addition to the 15 previously prepared and examined for their effect on permeability. The old concept that a tetrapeptide with basic amino acids at both ends and a proline residue adjacent to the N-terminal amino acid is essential for high activity on permeability, has now been challenged. The results obtained with several of the new analogues strengthen this concept. More interestingly, however, the third amino acid, which was found in earlier studies to be less sensitive to exchange, has now been deleted as well as duplicated with only a modest loss of activity of the peptide. The chirality of the C-terminal amino acid, most surprisingly, does not seem to be crucial for peptide activity. Slightly superpotent analogues were obtained on amidation of the C-terminus. In addition, a few naturally occurring peptides, namely tuftsin, substance P, neurotensin and bradykinin, the amino acid sequences of which all exhibit characteristic features of some of our active peptide analogues were investigated in the same test system. Tuftsin displayed a potency equal to that of the pentapeptide. The other three peptides were all highly superpotent in this assay system.     

Dermorphin tetrapeptide analogs as potent and long-lasting analgesics with pharmacological profiles distinct from morphine

Dermorphin (Tyr-d-Ala-Phe-Gly-Tyr-Pro-Ser-NH₂) is a heptapeptide isolated from amphibian skin. With a very high affinity and selectivity for μ-opioid receptors, dermorphin shows an extremely potent antinociceptive effect. The structure-activity relationship studies of dermorphin analogs clearly suggest that the N-terminal tetrapeptide is the minimal sequence for agonistic activity at μ-opioid receptors, and that the replacement of the d-Ala² residue with d-Arg² makes the tetrapeptides resistant to enzymatic metabolism. At present, only a handful of dermorphin N-terminal tetrapeptide analogs containing d-Arg² have been developed. The analogs show potent antinociceptive activity that is greater than that of morphine with various injection routes, and retain high affinity and selectivity for μ-opioid receptors. Interestingly, some analogs show pharmacological profiles that are distinct from the traditional μ-opioid receptor agonists morphine and [d-Ala²,NMePhe⁴,Gly-ol⁵]enkephalin (DAMGO). These analogs stimulate the release of dynorphins through the activation of μ-opioid receptors. The activation of κ-opioid receptors by dynorphins is suggested to reduce the side effects of μ-opioid receptor agonists, e.g., dependence or antinociceptive tolerance. The dermorphin N-terminal tetrapeptide analogs containing d-Arg² may provide a new target molecule for developing novel analgesics that have fewer side effects.     

Opiates and enkephalins: a common binding site mediates their analgesic actions in rats

³H-Labelled opiate and enkephalin ligands appear to bind with highest affinity to a single site responsible for their analgesic properties. Administered $\text{in vivo}$, naloxazone, an irreversible opiate, selectively inhibits for over 24 hours the high affinity binding of ³H-labelled mu, and kappa opiates and enkephalins. This inhibition of binding gradually resolves over 3 days, perhaps correlating with receptor turnover. Naloxazone treatment also abolishes morphine, D-ala²-met⁵-enkephalinamide and betah-endorphin analgesia. Although morphine and D-ala²-met⁵-enkephalinamide bind with similar potencies to the high affinity site, morphine's potency for the low affinity D-ala²-met⁵-enkephalinamide site is far less than the enkephalin analog. These results imply that all ³H-ligands examined bind with highest affinity to a mu-like receptor while low affinity D-ala²-met⁵-enkephalinamide binding, with a K_D of 6 nM, represents a delta-like receptor.     

Photoaffinity labeling of opioid receptor of rat brain membranes with 125+I(d-Ala2, p-N3-Phe4-Met5)enkephalin

A photoreactive (d-Ala², p-N₃-Phe⁴-Met⁵)enkephalin derivative was prepared, iodinated with carrier-free ¹²⁵I, and then purified by high-performance liquid chromatography. The purified radioactive photoprobe was monoiodinated at the amino terminal tyrosine residue. This radioactive photoprobe was used to photoaffinity label membranes prepared from the rat brain (minus cerebellum) and the spinal cord. The photolabeled membranes were analyzed by sodium dodecyl sulfate gel electrophoresis. A 46,000-Da protein was specifically photolabeled in these membrane preparations. The photolabeling of this protein was inhibited by peptides related to enkephalin but not by unrelated substance P or gastrin tetrapeptide. A concentration-dependent inhibition of the photolabeling of the 46,000-Da protein was observed in the presence of competing ligands specific for the μ-, δ-, and κ-opioid receptors. These data demonstrate that the radioactive photoprobe labels the μ-, δ-, and κ-opioid receptors. Although there is no evidence available to show that the 46,000-Da protein is identical in all the cases, our data strongly suggest that it is a binding protein common to all of the opioid receptor subtypes.     

Chemical synthesis of [des(tetrapeptide B27--30), Tyr(NH2)26-B] and [des(pentapeptide B26--30), Phe(NH2)25-B] bovine insulins.

Two analogs of bovine insulin, [des(tetrapeptide B27--30), Tyr(NH2)26-B] and [des(pentapeptide B26--30), Phe(NH2)25-B] insulin, which differ from the parent molecule in that the C-terminal tetrapeptide and pentapeptide sequences, respectively, from the B chain have been eliminated and the newly exposed residues are amidated, have been synthesized. The [des(tetrapeptide B27--30), Tyr(NH2)26-B] insulin shows potencies of 16.8 IU/mg by the mouse convulsion assay method and 10.8 IU/mg by the radioimmunoassay method. The [des(pentapeptide B26--30), Phe(NH2)25-B] insulin possesses a potency of 10.5 IU/mg when assayed by the mouse convulsion method and 14 IU/mg by the radioimmunoassay technique. The potencies of these analogs are higher than the potencies of the respective non-amidated derivatives (Katsoyannis et al., 1973, 1974). It is speculated that the gradual decline of biological activity observed as amino acid residues are eliminated from the C-terminal region of the B chain of insulin is due to the proximity of a hydrophilic carboxyl group to the hydrophobic core of the protein molecule.     

The flexible termini of conantokin G define its interactions with NMDA receptors

Summary Converging lines of evidence suggest that theN-methyl-d-aspartic acid (NMDA) antagonist properties of conantokin G (ConG) are mediated through a novel polyamine-associated site. Moreover, structural modification of the heptadecapeptide yields peptides that can either mimic the NMDA antagonist properties of the parent peptide or produce polyamine-like actions at NMDA receptors. We synthesized a panel of ConG fragments and evaluated their effects using a neurochemical assay that predicts pharmacological actions at NMDA receptors. While the C-terminal tetrapeptide elicited a polyamine-like activation of [³H]MK-801 binding with a potency comparable to spermine, the N-terminal pentapeptide produced a marginal inhibition of spermine-enhanced [³H]MK-801 binding. These observations suggest that the parent peptide interacts with two distinct sites on NMDA receptors. In contrast, amino acid replacements in the middle region of ConG resulted in analogues that were of comparable or greater potency than the parent peptide. The Ala⁷, Tyr¹⁰ derivative is of particular interest since it is a potent inhibitor (IC₅₀≈80 nM) of spermine-enhanced [³H]MK-801 binding, and may thus serve as a precursor for studies designed to¹²⁵I-label putative ConG binding sites. Our observations are also consistent with the hypothesis that the termini of ConG are essential for an interaction with NMDA receptors, while the middle region of this peptide serves as a spacer unit. This hypothesis is consonant with spectroscopic evidence that ConG possesses a central rigid helical backbone with flexible N- and C-terminal regions. Nonetheless, ConG variants in which the termini were connected with conformationally stabilized α-or 3₁₀-helical spacers grew progressively less potent as NMDA antagonists as the structural stability of these peptides increased. Thus, the middle region of ConG appears to possess functions other than providing conformational stability. These newly synthesized ConG derivatives may serve as a basis for the design of novel peptide or peptidomimetic agents.     

Rat brain and kidney metalloendopeptidase: Enkephalin heptapeptide conversion to form a cardioactive neuropeptide,Phe-Met-Arg-Phe-Amide

Rat brain or kidney metalloendopeptidase purified from particulates cleaved Met-enkephalin-Arg⁶-Phe⁷ and its amide at the Gly³-Phe⁴ bond to release Phe-Met-Arg-Phe or the tetrapeptide amide. The latter, a neuropeptide with cardioactive properties, was relatively stable upon further incubation. The metallo-nature of the enzyme was established by inhibition with chelating agents (EDTA, o-phenanthroline) and its endopeptidase nature by cleavage at the Gly³-Phe⁴ bond of pentapeptide enkephalins or precursors such as the heptapeptide, or analogs bearing N- or C-terminal protective groups. Presence of C-terminal amides decreased the rate of hydrolysis. Thiorphan, (DL-3-mercapto-2-benzylpropanoyl)-glycine, competitively inhibited cleavage at the Gly³-Phe⁴ bond of enkephalin (Ki 10 nM). The thiorphan sensitive metalloendopeptidase provides a pathway for conversion of an enkephalin precursor to form a non-opioid peptide of biological interest.     

The effect of asteltoxin and citreomontanine, two polyenic alpha-pyrone mycotoxins, on Escherichia coli adenosine triphosphate

Conformational similarities between morphine and the enkephalin analogue Tyr-D-Ala-Gly-Phe which interact preferentially with opiate μ-receptors were investigated using a constrained energy minimization procedure. This method takes into account several structural features of morphine-like substances including enkephalin analogues and uses them to search for conformations of peptides exhibiting low energies and good similarity with the μ-opiate pharmacophore. This latter involves as critical components the A-ring, the N-atom of D-ring and the D₆-O₂ bond is morphine which correspond to the N-terminal tyrosine moiety and the Gly³-CO group in Tyr-D-Ala-Gly-Phe respectively. Several low energy conformers present a good similarity with rigid opiates and are consistent with activity of sterically constrained enkephalins. Conformational changes of peptides from solid or solvated states to μ-receptors bound state involve a transconformational binding process.     

Synthesis,biological evaluation and structural analysis of novel peripherally active morphiceptin analogs

Morphiceptin (Tyr-Pro-Phe-Pro-NH₂), a tetrapeptide amide, is a selective ligand of the μ-opioid receptor (MOR). This study reports the synthesis and biological evaluation of a series of novel morphiceptin analogs modified in positions 2 or/and 4 by introduction of 4,4-difluoroproline (F₂Pro) in l or d configuration. Depending on the fluorinated amino acid configuration and its position in the sequence, new analogs behaved as selective full MOR agonists showing high, moderate, or relatively low potency. The most potent analog, Tyr-F₂Pro-Phe-d-F₂Pro-NH₂, was also able to activate the κ-opioid receptor (KOR), although with low potency. Docking studies and the comparison of results with the high resolution crystallographic structure of a MOR-agonist complex revealed possible structure–activity relationships of this compound family.     

Morphine-like activity of the enkephalin analog Tyr-D-Ala-Gly-Phe-NH2

The analgetic activity of the tetrapeptide enkephalin analog, its influence on the interneuronal transmission of excitation in various areas of the central nervous system and on opiate receptors of vas deferens were studied. The tetrapeptide was found to have a marked analgetic effect during intravenous injection to mice but to be less active than morphine. The tetrapeptide as well as morphine inhibited the impulse summation in rabbits and both spontaneous and bradykinin-induced neuronal activity in the rat sensory motor cortex. The tetrapeptide inhibited the contractions of isolated vas deferens in mice. The opiate antagonist naloxone eliminated both analgetic effect of the tetrapeptide and its inhibitory effect on the impulse summation, neuronal activity and contractions of vas deferens.