Yojiro Muneoka

THE LEECH EXCITATORY PEPTIDE, A MEMBER OF THE GGNG PEPTIDE FAMILY : ISOLATION AND COMPARISON WITH THE EARTHWORM GGNG PEPTIDES

A member of the GGNG peptide family was isolated from Hirudo nipponia (leech). GGNG peptides had only been isolated previously from earthworms. The C‐terminus structure of the leech peptide, LEP (leech excitatory peptide), was –Gly–Gly–Asn–amide, while that of the earthworm peptides, EEP (earthworm excitatory peptide), was –Gly–Gly–Asn–Gly. LEP exerted 1000‐fold more potent activities on leech gut than did EEP‐2. On the other hand, EEP‐2 was 1000‐fold more potent than LEP on the crop‐gizzard of the earthworm. Analog peptides of LEP and EEP‐2 were synthesized, and the myoactive potency of each analog on the leech and earthworm tissues was compared.

Distribution of catch-relaxing peptide (CARP)-like immunoreactive neurons in the central and peripheral nervous system of Helix pomatia

Immunocytochemistry was performed on the nervous system of Helix by the use of an antibody raised against a myotropic neuropeptide, the catch-relaxing peptide (CARP), isolated from Mytilus edulis. In each ganglion of the central nervous system of Helix pomatia, numerous CARP-immunoreactive cell bodies and a dense immunoreactive fiber system could be observed with a dominancy in the cerebral and pedal ganglia. The majority of the immunoreactive neurons are unipolar, although multipolar neurons also occur. In the neuropil areas, CARP-immunoreactive fibers show extensive arborization, which may indicate a central role of CARP. CARP-immunoreactive elements could be observed in each investigated peripheral nerve and peripheral areas, namely in the intestine, heart, aorta, buccal mass, lips, and foot. However, CARP-immunoreactive cell bodies could only be demonstrated in the intestine and the foot musculature. Thin varicose CARP-immunoreactive fibers were observed over both muscle and gland cells in the different peripheral organs, suggesting a peripheral role of CARP. In vivo CARP injection into the body cavity (10-3, 10-4, 10-5 M) altered the general behavioral state of the animals and induced the relaxation of the musculature of the whole body wall indicating that CARP has a significant role in the regulation of muscle contraction.

Inhibitory pentapeptides isolated from the gut of the earthworm, Eisenia foetida☆

Abstract The isolated anterior gut (crop-gizzard) of the oligochaete annelid, Eisenia foetida, was used as a bioassay system to identify the bioactive peptides that regulate rhythmic spontaneous contractions of the gut. Three pentapeptides were purified from the extract of the gut (crop, gizzard and intestine) of the earthworm. These peptides showed inhibitory actions on gut motility and were termed Eisenia inhibitory pentapeptides (EIPPs). EIPPs had the most remarkable effect on the crop among the various parts of the gut with a threshold concentration as low as 10−10–10−9 M. From the pharmacological results, the inhibitory actions of EIPPs on the gut motility seem to be different from those of FMRFamide. The amino acid sequences of EIPPs were very similar to certain molluscan and echiuroid peptides previously identified. This is the first isolation of annelid peptides inhibiting gut motility except for FMRFamide.

Isolation and characterization of bioactive peptides from the sea cucumber, Stichopus japonicus

In the previous study, we isolated a number of bioactive peptides from the body wall of the sea cucumber, Stichopus japonicus, and determined the primary structures of four species of the isolated peptides [1]. In the present study, we made further identification of bioactive peptides of the sea cucumber using newly prepared body-wall extract and determined the structures of 14 peptides including the four found in the previous study. Here, we report the primary structures and the actions of the 14 peptides.

Demonstration, localization, and development of galanin receptors in the quail oviduct

We recently isolated an oviposition-inducing peptide that was identified as avian galanin from the oviducts of the Japanese quail. Avian galanin was localized in neural fibers distributed in muscle layers in the uterine and vaginal oviduct regions, and potentiated spontaneous contractions of the uterus and vagina. To elucidate whether an oviposition-inducing effect of avian galanin is due to the direct action on the oviduct, therefore, a specific binding site for avian galanin was determined in the functional quail oviduct in this study. The binding of [125I]iodoavian galanin was primarily located in the oviduct as well as the brain. The galanin binding was specifically inhibited as a function of the concentrations of both avian and rat galanins. The specific binding of avian galanin to the quail oviduct was temperature dependent and reached the maximum level for 1 h at 20 degrees C. In several regions of the oviduct, a higher level of specific galanin binding was observed only in the uterus and vagina. In contrast, the specific binding was low in the isthmus and negligible in the magnum. A similar localization was evident in the functional chicken oviduct. The Scatchard plot analysis of the binding of avian galanin to the uterine preparation revealed that the dissociation constant (Kd) was 0.249 (95% confidence interval, 0.192-0.356) nM, and the number of binding sites was 1.13 (0.99-1.36) fmol per mg tissue, respectively. During development, the galanin-binding sites were apparent in the quail oviduct at 3 weeks of age and the number of binding sites markedly increased between 3 weeks and 3 months of age. However, there was no significant change in the Kd value in the developing quail oviduct. This is the first demonstration of the presence of galanin receptors in the reproductive tract, such as the uterine and vaginal oviduct. The present results suggest that the number of galanin receptors in the oviduct increases during development and that galanin acts directly on the mature uterus and vagina to induce their contractions. This mechanism may be essential to the avian oviposition.

ISOLATION AND CHARACTERIZATION OF FOUR NOVEL BIOACTIVE PEPTIDES FROM A POLYCHAETE ANNELID, PERINEREIS VANCAURICA

Four bioactive peptides were purified from an extract of the polycheate annelid Perinereis vancaurica using an HPLC system for fractionation of the extract and the esophagus of the worm as bioassay system. The sequences of the peptides were determined to be H-Trp-Val-Val-Gly-Asp-Val-Gln-OH, H-Ala-Thr-Trp-Leu-Asp-Thr-OH, H-Trp-Met-Val-Gly-Asp-Val-Gln-OH and H-Phe-Tyr-Glu-Gly-Asp-Val-Pro-Tyr-OH. These peptides showed an excitatory activity on the esophagus of P. vancaurica. The excitatory effect of the second and fourth peptides was marked. They may be neuropeptides involved in regulation of the esophagus.

Characterization of binding of the annelidan myoactive peptides, GGNG peptides, to tissues of the earthworm, Eisenia foetida

The GGNG peptides are myoactive peptides which we have isolated from several species of annelids. Two types of peptides have been isolated as GGNG peptides: earthworm excitatory peptides (EEP) and leech excitatory peptide (LEP). The EEP and LEP are highly homologous with each other but act specifically on earthworms and leeches, respectively. To approach the mechanism of the specific action, we attempted to characterize the receptors for EEP in the earthworm Eisenia foetida, from which EEP have been isolated, by a radioreceptor assay. To prepare a radiolabeled ligand, we first coupled EEP to Bolton-Hunter reagent and then labeled it with 125I ([125I] BH-EEP). Binding of the [125I] BH-EEP to the membrane preparation of the earthworm tissue (crop-gizzard) was reversible, saturable, and specific with a Kd of 4.9 ± 1.2 nM and a Bmax of 15.9 ± 2.0 fmol/mg wet tissue. For the earthworm membrane preparation, EEP showed a much greater potency than LEP in displacing [125I] BH-EEP, suggesting that the earthworm tissues contain EEP-specific receptors. This result would account for the specific biological activity of EEP and LEP on the earthworm and leech tissues, respectively. The binding capacity was high in the anterior part of digestive tract including the esophagus, crop and gizzard, and the nephridia. From these results, together with the observed myotropic activity of EEP on gut tissues, it is assumed that EEP is physiologically involved in regulation of the gut motility in the earthworm E. foetida.J. Exp. Zool. 279:562–570, 1997.© 1997 Wiley-Liss, Inc.

Identification of a novel analgesic peptide including an unnatural amino acid from peptide libraries synthesized with a multipeptide synthesizer

Combinatorial peptide libraries are powerful resources for developing pharmacologically active compounds such as, for example, potent agonists and antagonists for opioid receptors [1,2]. In a previous study, tetraor pentapeptide libraries which contain Trp at the C-terminal position were found to show inhibitory activity on the rectum of a quail. Two types of peptides, YGHW–NH2 and TFVAW–NH 2, showed strong activity in the assay [3]. These peptides also showed relaxant activity on the guinea pig ileum (GPI). In this study, penta–, tetra–, tri–, and di-peptide libraries which contain a Trp amide (or carboxyl) group at C terminus were synthesized with a multipeptide synthesizer in order to obtain more effective peptides for the GPI.