Octopus gonadotrophin-releasing hormone: a multifunctional peptide in the endocrine and nervous systems of the cephalopod.

The optic gland, which is analogous to the anterior pituitary in the context of gonadal maturation, is found on the upper posterior edge of the optic tract of the octopus Octopus vulgaris. In mature octopus, the optic glands enlarge and secrete a gonadotrophic hormone. A peptide with structural features similar to that of vertebrate gonadotrophin-releasing hormone (GnRH) was isolated from the brain of octopus and was named oct-GnRH. Oct-GnRH showed luteinising hormone-releasing activity in the anterior pituitary cells of the Japanese quail Coturnix coturnix. Oct-GnRH immunoreactive signals were observed in the glandular cells of the mature optic gland. Oct-GnRH stimulated the synthesis and release of sex steroids from the ovary and testis, and elicited contractions of the oviduct. Oct-GnRH receptor was expressed in the gonads and accessory organs, such as the oviduct and oviducal gland. These results suggest that oct-GnRH induces the gonadal maturation and oviposition by regulating sex steroidogenesis and a series of egg-laying behaviours via the oct-GnRH receptor. The distribution and expression of oct-GnRH in the central and peripheral nervous systems suggest that oct-GnRH acts as a multifunctional modulatory factor in feeding, memory processing, sensory, movement and autonomic functions.

Novel excitatory neuropeptides isolated from a prosobranch gastropod, Thais clavigera: The molluscan counterpart of the annelidan GGNG peptides.

The GGNG peptides are excitatory neuropeptides identified from earthworms, leeches and polychaeta. Two structurally related peptides were purified and characterized from a mollusk, Thais clavigera (prosobranch gastropod). The peptides designated as Thais excitatory peptide-1 (TEP-1) (KCSGKWAIHACWGGN-NH2) and TEP-2 (KCYGKWAMHACWGGN-NH2) are pentadecapeptides having one disulfide bond and C-terminal GGN-NH2 structures, which are shared by most GGNG peptides. TEP augmented the motilities of Thais esophagus and penial complex. TEP-like immunoreactivity is distributed in both the neurons of the central nervous system and nerve endings in the penial complex. Thus, the involvement of TEP in the contraction of the digestive and reproductive systems is suggested. Substitution of amino acids in TEP revealed that two tryptophan residues in TEP are important for maintaining bioactivity.

PRQFVamide, a novel pentapeptide identified from the CNS and gut of Aplysia.

We have purified a novel pentapeptide from the Aplysia nervous system using bioassay on gut contractions. The structure of the peptide is Pro-Arg-Gln-Phe-Val-amide (PRQFVa). The precursor for PRQFVa was found to code for 33 copies of PRQFVamide and four related pentapeptides. Peaks corresponding to the predicted masses of all five pentapeptides were detected in Aplysia neurons by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Northern analysis revealed that expression of the precursor is abundant in the abdominal ganglion, much less in the pedal and cerebral ganglia, and rarely seen in the buccal and pleural ganglia. PRQFVa-positive neurons, mapped by immunohistochemistry and in situ hybridization, were present in all the central ganglia. PRQFVa immunopositive processes were observed in the gut, particularly in association with the vasculature. Some arteries and other highly vascularized tissues, such as the gill and the kidney, also contain numerous PRQFVa immunopositive processes. Application of synthetic PRQFVa suppresses not only contractions of the gut but also contractions of vasculature. PRQFVa is expressed in some of the neurons within the feeding circuitry and application of synthetic PRQFVa was found to decrease the excitability of some (B4/5 and B31/32) but not all (B8) neurons of the buccal feeding circuit. Our findings suggest that PRQFVa may act as a modulator within the feeding system as well as in other systems of Aplysia.

Deamidase inactivates a D-amino acid-containing Aplysia neuropeptide.

Membrane-catalyzed degradation of the cardioexcitatory peptide, Asn-D-Trp-Phe-NH(2) (N(d)WF-NH(2)), which was previously isolated from Aplysia, was investigated in relation to its inactivation mechanism. The principal degradation was deamidation of the C-terminal amide, producing biologically inert Asn-D-Trp-Phe-OH (N(d)WF-OH). Among membrane fractions prepared from different organs, the fraction from the ganglia showed the highest specific activity of the deamidation reaction. The deamidase activity was inhibited by Ebelactone B and the serine protease inhibitor, phenylmethanesulfonyl fluoride (PMSF), while the degradation of the synthetic stereoisomer, Asn-Trp-Phe-NH(2) (N(l)WF-NH(2)), was sensitive to the divalent cation-chelating agent, o-phenanthroline, and aminopeptidase inhibitors, amastatin and bestatin. The presence of D-Trp residue in the second position of N(d)WF-NH(2) endows this peptide not only with stereospecific bioactivity but also peptidase stability. The deamidation reaction seems to be the major inactivation mechanism for this peptide.

Distribution and function of an Aplysia cardioexcitatory peptide, NdWFamide, in pulmonate snails.

The distribution and function of an Aplysia cardioexcitatory peptide, NdWFamide, were examined in the nervous system of pulmonate snails. We chemically identified the authentic NdWFamide from a land snail (Euhadra congenita) and a freshwater snail (Lymnaea stagnalis). NdWFamide potentiated the heartbeat of those snails. Immunohistochemistry using anti-NdWFamide antibody demonstrated the distribution of NdWFamide-containing neurons and fibers in the central nervous system, as well as peripheral tissues, such as the cardiovascular region and accessory sex organs. These results suggest that NdWFamide is a neuropeptide mediating the neural regulation of the activity of the cardiovascular and reproductive systems of snails.

A novel GGNG-related neuropeptide from the polychaete Perinereis vancaurica.

The GGNG peptides are myoactive peptides so far identified from earthworms and leeches, which are the earthworm excitatory peptides (EEP) and the leech excitatory peptide (LEP), respectively. A novel GGNG peptide was isolated and structurally determined from a marine polychaete, Perinereis vancaurica, using a combination of immunological assay and high performance liquid chromatography (HPLC). The peptide was a pentadecapeptide whose amino acid sequence was similar to that of EEP and LEP, and showed myoactivity on isolated esophagus of P. vancaurica with a threshold concentration of 10(-10)M. The peptide was designated as polychaete excitatory peptide (PEP). Amidation of the alpha-carboxyl group of C-terminal residue occurred in PEP. This is the case for LEP, but not for EEP. The cDNA cloning revealed that the structure of the PEP precursor is more similar to the EEP precursor than to the LEP precursor. Immunohistochemical staining showed the presence of PEP in several neurons of central nervous system (CNS) as somata and neuropile structure, epithelial cells of the pharynx and epidermal cells throughout the body wall. Altogether these results support the physiological significance of PEP in regulation of the CNS neural activity and the peripheral myoactivity.

Potent antagonistic action of synthetic analogues of APGWGNamide, an antagonist of molluscan neuropeptide APGWamide.

Fifty-five kinds of analogues of APGWGNamide (Ala-Pro-Gly-Trp-Gly-Asn-NH2), which is an antagonist of molluscan neuropeptide APGWamide, were synthesized and their antagonistic activities were examined on two molluscan smooth muscles. Among all the analogues tested, on spontaneous contraction of the crop of the land snail, Euhadra congenita, APGWG(L-biphenylalanine, Bip)amide showed the most potent antagonistic activity and its potency was 50-100 times higher than that of APGWGNamide. Likewise, on phasic contraction of the anterior byssus retractor muscle (ABRM) of the sea mussel, Mytilus edulis, the effect of APGWG(D-homophenylalanine, dHfe) was the most potent and showed 5-10 times stronger activity than that of APGWGNamide. In the tolerance test to known exo- and endopeptidases or the crop tissue homogenate, APGWGNamide was not only easily degraded by a proline-specific endopeptidase but also by the homogenate. Two kinds of potent antagonists were thus developed: APGWG(Bip)amide and APGWG(dHfe)amide, which will be useful tools for investigation of the function of APGWamide in the snail and the mussel, respectively.

The enterins: a novel family of neuropeptides isolated from the enteric nervous system and CNS of Aplysia.

To identify neuropeptides that have a broad spectrum of actions on the feeding system of Aplysia, we searched for bioactive peptides that are present in both the gut and the CNS. We identified a family of structurally related nonapeptides and decapeptides (enterins) that are present in the gut and CNS of Aplysia, and most of which share the HSFVamide sequence at the C terminus. The structure of the enterin precursor deduced from cDNA cloning predicts 35 copies of 20 different enterins. Northern analysis, in situ hybridization, and immunocytochemistry show that the enterins are abundantly present in the CNS and the gut of Aplysia. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry we characterized the enterin-precursor processing, demonstrated that all of the precursor-predicted enterins are present, and determined post-translational modifications of various enterins. Enterin-positive neuronal somata and processes were found in the gut, and enterins inhibited contractions of the gut. In the CNS, the cerebral and buccal ganglia, which control feeding, contained the enterins. Enterin was also present in the nerve that connects these two ganglia. Enterins reduced the firing of interneurons B4/5 during feeding motor programs. Such enterin-induced reduction of firing also occurred when excitability of B4/5 was tested directly. Because reduction of B4/5 activity corresponds to a switch from egestive to ingestive behaviors, enterin may contribute to such program switching. Furthermore, because enterins are present throughout the nervous system, they may also play a regulatory role in nonfeeding behaviors of Aplysia.

Characterization of a cDNA encoding a novel avian hypothalamic neuropeptide exerting an inhibitory effect on gonadotropin release.

We previously isolated a novel dodecapeptide containing a C-terminal -Arg-Phe-NH(2) sequence, SIKPSAYLPLRF-NH(2) (RFamide peptide), from the quail brain. This quail RFamide peptide was shown to decrease gonadotropin release from the cultured anterior pituitary and to be located at least in the quail hypothalamo-hypophysial system. We therefore designated this RFamide peptide gonadotropin inhibitory hormone (GnIH). In the present study we characterized the GnIH cDNA from the quail brain by a combination of 3' and 5' rapid amplification of cDNA ends ('RACE'). The deduced GnIH precursor consisted of 173 amino acid residues, encoding one GnIH and two putative gene-related peptide (GnIH-RP-1 and GnIH-RP-2) sequences that included -LPXRF (X=L or Q) at their C-termini. All these peptide sequences were flanked by a glycine C-terminal amidation signal and a single basic amino acid on each end as an endoproteolytic site. Southern blotting analysis of reverse-transcriptase-mediated PCR products demonstrated a specific expression of the gene encoding GnIH in the diencephalon including the hypothalamus. Furthermore, mass spectrometric analyses detected the mass numbers for matured GnIH and GnIH-RP-2, revealing that both peptides are produced from the precursor in the diencephalon as an endogenous ligand. Taken together, these results lead to the conclusion that GnIH is a hypothalamic factor responsible for the negative regulation of gonadotropin secretion. Furthermore, the presence of a novel RFamide peptide family containing a C-terminal -LPXRF-NH(2) sequence has been revealed.

Conformational comparison of mu-selective endomorphin-2 with its C-terminal free acid in DMSO solution, by 1H NMR spectroscopy and molecular modeling calculation.

In order to make clear the structural role of the C-terminal amide group of endomorphin-2 (EM2, H-Tyr-Pro-Phe-Phe-NH2), an endogenous mu-receptor ligand, in the biological function, the solution conformations of endomorphin-2 and its C-terminal free acid (EM2OH, H-Tyr-Pro-Phe-Phe-OH), studied using two-dimensional 1H NMR measurements and molecular modeling calculations, were compared. Both peptides were in equilibrium between the cis and trans isomers around the Tyr-Pro omega bond in a population ratio of approximately/= 1:2. The lack of significant temperature and concentration dependence of NH protons suggested that the NMR spectra reflected the conformational features of the respective molecules themselves. Fifty possible 3D structures for the each isomer were generated by the dynamical simulated annealing method under the proton-proton distance constraints derived from the ROE cross-peaks. These energy-minimized conformers, which were all in the phi torsion angles estimated from J(NHCalphaH) coupling constants within +/- 30 degrees, were then classified in groups one or two according to the folding backbone structures. All trans and cis EM2 conformers adopt an open conformation in which their extended backbone structures are twisted at the Pro2-Phe3 moiety. In contrast, the trans and cis conformers of EM2OH show conformational variation between the 'bow'-shaped extended and folded backbone structures, although the cis conformers of its zwitterionic form are refined into the folded structure of the close disposition of C- and N-terminal groups. These results indicate clearly that the substitution of carboxyl group for C-terminal amide group makes the peptide flexible. The conformational requirement for mu-receptor activation has been discussed based on the active form proposed for endomorphin-1 and by comparing conformational features of EM2 and EM2OH.

Comparative aspects of invertebrate neuropeptides.

1. We searched for bioactive peptides, most of which were considered to be neuropeptides, in various animals of several phyla. These peptides were compared with each other and with peptides identified by many other investigators. Consequently, we found that structures of neuropeptides are generally conserved in each phylum. 2. We also found some exceptional interesting aspects. First, there are a number of peptide groups whose members are distributed among several phyla. Second, there are many structural similarities between molluscan and annelidan peptides as if molluscs and annelids were the animals in a phylum. Third, certain toxic peptides of invertebrates are closely related to vertebrate neuropeptides. 3. In addition to the above phylogenetic aspects, we found some other interesting aspects. A wide structural variety of members of a peptide group is generally found in invertebrate species. Invertebrate muscles seem to be generally regulated not only by some or several classical non-peptidic neuromediators but also by various peptidic neuromediators. Peptides containing a D-amino acid residue are not rare.

Fulicin regulates the female reproductive organs of the snail, Achatina fulica.

Fulicin is a D-amino acid-containing neuropeptide that has been thought to control male copulatory behavior in the land snail, Achatina fulica. In the present study, we demonstrated that the vagina and the oviduct of Achatina were densely innervated by fulicin-like immunoreactive neuronal fibers. We confirmed that fulicin was actually present in the vagina by mass spectrometry. Furthermore, fulicin showed a profound excitatory effect on contractions of the vagina and the oviduct. These results suggest that fulicin controls female egg-laying behavior as an excitatory neuropeptide regulating the female reproductive organs of the snail.

Cardioactive peptides isolated from the brain of a Japanese octopus, Octopus minor.

Octopus cardioactive peptides (Ocp-1: Gly-D-Phe-Gly-Asp, and Ocp-3: Gly-Ser-Trp-Asp) were isolated from brain extracts of the octopus, Octopus minor, using the isolated systemic heart as a bioassay. These peptides showed both positive chronotropic and inotropic effects on the heart. The stereoisomers at position 2 were also isolated, but their activities were only 1/10(3)-1/10(4) those of the corresponding isomers. The presence of the peptides in the systemic heart was confirmed by time-of-flight mass spectrometry (MS) and tandem MS analysis. The results suggested that Ocp-1 and Ocp-3 might be involved in excitatory control of the octopus cardiovascular system as neuropeptides and/or neurohormones.

Poly-L-glutamine forms cation channels: relevance to the pathogenesis of the polyglutamine diseases.

We report that long-chain poly-L-glutamine forms cation-selective channels when incorporated into artificial planar lipid bilayer membranes. The channel was permeable to alkali cations and H(+) ions and virtually impermeable to anions; the selectivity sequence based on the single-channel conductance was H(+) >> Cs(+) > K(+) > Na(+). The cation channel was characterized by long-lived open states (often lasting for several minutes to tens of minutes) interrupted by brief closings. The appearance of the channel depended critically on the length of polyglutamine chains; ion channels were observed with 40-residue stretches, whereas no significant conductance changes were detected with 29-residue tracts. The channel-forming threshold length of poly-L-glutamine was thus between 29 and 40 residues. A molecular mechanics calculation suggests a mu-helix (. Biophys. J. 69:1130-1141) as a candidate molecular structure of the channel. The channel-forming nature of long-chain poly-L-glutamine may provide a clue to the elucidation of the pathogenetic mechanism of the polyglutamine diseases, a group of inherited neurodegenerative disorders including Huntington's disease.

Identification of multiple urechistachykinin peptides, gene expression, pharmacological activity, and detection using mass spectrometric analyses.

Urechistachykinin I and II (Uru-TK I and II) are invertebrate tachykinin-related peptides (TRPs), which have been isolated from echiuroid worms. The cDNA sequence encoding the Uru-TK I and II revealed that the precursor also encoded five TRP-like peptides. Here, we report the characterization of these Uru-TK-like peptides named as Uru-TK III-VII. Northern and Southern blot analyses demonstrated that Uru-TK mRNA is localized in nerve tissue. In addition, the presence of the Uru-TK-like peptides as matured forms in the nerve tissue was detected by mass spectrometric analysis, and identified these peptides were shown to exhibit a contractile activity on cockroach hindgut that was as potent as that of Uru-TK II. Furthermore, synthetic Uru-TK-like peptide analogs which contained Met-NH2 instead of Arg-NH2 at their C-termini were shown to possess a potential to bind to a mammalian tachykinin receptor, indicating that Uru-TK-like peptides are likely to correspond to vertebrate tachykinins, except for the difference at the C-terminal residue. These findings show that Uru-TK-like peptides are essentially equivalent to Uru-TK I and II, leading to the proposal that Uru-TK-like peptides play an essential role as invertebrate tachykinin neuropeptides.

Characterization of cDNA and expression of mRNA encoding an Achatina cardioexcitatory RFamide peptide.

Achatina cardioexcitatory peptide-1 (ACEP-1) is an RFamide family peptide isolated from the atria of the African giant snail, Achatina fulica. In this report, we describe an identification of the ACEP-1 cDNA sequence and localizations of the ACEP-1 mRNA. Southern blot analysis revealed that the ACEP-1 mRNA was present in the atrium as well as in the central nervous system. Furthermore, in situ hybridization revealed the localizations of the ACEP-1 mRNA in small neurons of the cerebral and pedal ganglia and a few large neurons of the right parietal and visceral ganglia.

The importance of C-terminal residues of vertebrate and invertebrate tachykinins for their contractile activities in gut tissues.

The C-terminal residues of mammalian tachykinins and urechistachykinins (Uru-TKs), tachykinin-related peptides of echiuroid worm origin, were substituted for each other. Their contractile effects were assayed on the cockroach hindgut and the guinea pig ileum. [Met(10)] substitution of Uru-TKs caused a 1000 times lower activity on the hindgut, but a 1000 times higher activity on the ileum. In contrast, [Arg(11)]substance P (SP) was 100 times more and 400 times less potent than SP on the hindgut and ileum, respectively. A SP antagonist blocked these Uru-TK activities on the hindgut. These results demonstrated that the C-terminal Met-NH(2) is necessary for ileum contraction and the Arg-NH(2) is required for hindgut contraction, which was caused by binding to the cockroach's neurokinin-like receptor.

Characterization of a novel cDNA sequence encoding invertebrate tachykinin-related peptides isolated from the echiuroid worm, Urechis unicinctus.

Tachykinin is one of the most well-known bioactive peptides found in vertebrates, and tachykinin-related peptides have also been isolated from various invertebrate species. Urechistachykinin I (Leu-Arg-Gln-Ser-Gln-Phe-Val-Gly-Ser-Arg-NH(2)) and II (Ala-Ala-Gly-Met-Gly-Phe-Phe-Gly-Ala-Arg-NH(2)) were purified from the ventral nerve cords of echiuroid worm, Urechis unicinctus. In the present study, we described the characterization of a novel cDNA encoding the urechistachykinin precursor. Amino acid sequence analysis of the deduced polypeptide revealed that the urechistachykinin precursor included seven structurally related peptides, unlike mammalian tachykinin precursors which encode only one or two tachykinin peptides. This is the first identification of an invertebrate tachykinin-related peptide cDNA.

The Aplysia mytilus inhibitory peptide-related peptides: identification, cloning, processing, distribution, and action.

Neuropeptides are a ubiquitous class of signaling molecules. In our attempt to understand the generation of feeding behavior in Aplysia, we have sought to identify and fully characterize the neuropeptides operating in this system. Preliminary evidence indicated that Mytilus inhibitory peptide (MIP)-like peptides are present and operating in the circuitry that generates feeding in Aplysia. MIPs were originally isolated from the bivalve mollusc Mytilus edulis, and related peptides have been identified in other invertebrate species, but no precursor has been identified. In this study, we describe the isolation and characterization of novel Aplysia MIP-related peptides (AMRPs) and their precursor. Several AMRPs appear to have some structural and functional features similar to vertebrate opioid peptides. We use matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to confirm that all 14 AMRPs predicted by the precursor are processed in isolated neurons. Northern analysis, whole-mount in situ hybridization, and immunohistochemistry are used to map the abundant expression of these peptides in the CNS and peripheral tissues such as the digestive tract, vasculature, and the reproductive organs. Physiological studies demonstrate that the rank order of the inhibitory actions of these peptides is different for three target muscles. These results underscore the importance of using a multidisciplinary approach to identifying and characterizing the actions of neuropeptides in an effort to gain understanding of their role in systems of interest. The widespread distribution of the AMRPs indicates that they may be operating in many different systems of Aplysia.

Possible functions of oxytocin/vasopressin-superfamily peptides in annelids with special reference to reproduction and osmoregulation.

Annetocin is an earthworm oxytocin-related peptide that we previously isolated from the whole body of a lumbricid earthworm Eisenia foetida. We have reported that annetocin induces egg-laying-like behaviors in E. foetida and a gnathobdellid leech, Whitmania pigra, when it is injected into the respective animals. The present study was undertaken to probe physiological functions of invertebrate oxytocin-vasopressin-superfamily peptides with special reference to reproductive and osmoregulatory events in which vertebrate peptides of this superfamily are involved. Annetocin, Lys-conopressin (a leech vasopressin-related peptide) and two analog peptides, [Tyr(3)]-annetocin ((3)Y-annetocin) and [Phe(3)]-annetocin ((3)F-annetocin), were compared for their activities to induce egg-laying-like behavior and to change body weight as a measure of water balance in the leech W. pigra. Injection of annetocin, Lys-conopressin, and (3)F-annetocin caused both egg-laying-like behavior and reduction of body weight in the animals, but (3)Y-annetocin induced neither. Furthermore, leeches in the non-breeding season responded to peptides less conspicuously than those in the breeding season. Such a concomitant induction of egg-laying-like behavior and body-weight reduction suggests that these two phenomena are unitary and might be accounted for by the fact that egg-laying in leeches and earthworms is accompanied by secretion of a large quantity of mucus, which should significantly contribute to body-weight loss. J. Exp. Zool. 284:401-406, 1999.