Structure-activity and possible mode of action of S-Iamide neuropeptides on identified central neurons of Helix aspersa.

Intracellular recordings were made from identified neurons from the suboesophageal ganglia of Helix aspersa. The inhibitory action of nine S-Iamide peptides was investigated. Structure-activity studies suggest that all act through a common receptor, which normally requires FVRIamide at the C terminal, with a preferred length of seven amino acids. Substitution at the N-terminal with alanine (A), threonine (T), proline (P) or leucine (L) results in little change in potency, suggesting the N-terminal requirements are relatively flexible. Ion substitution experiments suggest that potassium is the main ion involved in the inhibitory response to S-Iamide application. Studies using a range of compounds, which modify second messenger systems, would suggest that S-Iamide peptides may interact with adenylate cyclase. No evidence was found for an interaction with either guanylate cyclase or nitric oxide synthase.

Mytilus inhibitory peptides (MIP) in the central and peripheral nervous system of the pulmonate gastropods, Lymnaea stagnalis and Helix pomatia: distribution and physiological actions.

The distribution and neuroanatomy of Mytilus inhibitory peptides (MIP)-containing neurons in the central nervous system and their innervation pattern in the peripheral nervous system of the pulmonate snail species, Lymnaea stagnalis and Helix pomatia, have been investigated immunocytochemically, by applying an antibody raised to GSPMFVamide. A significant number of immunoreactive neurons occurs in the central nervous system of both species (Lymnaea: ca 600-700, Helix: ca 400-500), but their distribution is different. In Lymnaea, labeled neurons are found in all central ganglia where a number of large and giant neurons, previously identified physiologically, reveal MIP immunoreactivity. In Helix, most of the immunolabeled neurons are small (12-30 microm) and concentrated in the buccal and cerebral ganglia; the parietal ganglia are free of labeled cells. In both species, the ganglionic neuropils, peripheral nerves, connectives, and commissures are richly supplied with immunolabeled fibers. The MIP-immunoreactive innervation pattern in the heart, intestine, buccal mass and radula, and foot is similar in both species, with labeled axonal bundles and terminal-like arborizations (buccal mass, foot) or a network of varicose fibers (heart, intestine). Intrinsic neurons are not present in these tissues. The application of GSPYFVamide inhibits the spontaneous contractions of the esophageal longitudinal musculature in Helix, indicating the bioactivity of the peptide. An outside-out patch-clamp technique has demonstrated that GSPYFVamide opens the K+ channels in central nerve cells of Helix. Injection of GSPYFVamide into the body cavity inhibits the feeding of starved Helix. A wide modulatory role of MIP at central and peripheral levels is suggested in Lymnaea and Helix, including the participation in intercellular signalling processes and remote neurohormonal-like control effects.

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.

Mytilus inhibitory peptide (MIP) induces a Na+-activated K+-current in snail neurons.

Two microelectrode voltage-clamp and single-channel recordings were performed on D-cluster neurons of snail right parietal ganglion in order to study the properties of MIP-activated potassium current. It was found that the octapeptide member of the MIP-family, ASHIPRFVa elicits an outward current, which possesses all the properties characteristic for the hexapeptide(s) inward membrane response. The main component of the peptide elicited response is highly [K+]o dependent, however the response was attenuated in Na-free extracellular saline. The peptide elicited response was mimicked by raising the [Na+]i by pressure injection of Na+ into the cell. Single channel recordings indicated that MIP-induced outward K-current is Na-dependent. The probability to find a channel in open state increases with increasing intracellular Na+-concentration. Excised inside-out patches obtained from D-neurons contained I(K(Na)) channels could be activated by exposure of the cytoplasmic face of the patch membrane to 40 mM Na+, and 40 mM Li+, as well. The single channel current amplitude at -60 mV is 15 pA and the single channel conductance is 212 pS between -80 and 0 mV. It was concluded that MIP's activate a novel type of K+-current in the snail neurons. This current is the Na-activated K+-current. The single channel properties of the MIP activated channel is in concert with I(K(Na)) data obtained on different vertebrate and invertebrate preparations.

Development of an antagonist of molluscan neuropeptide APGWamide with a peptide library.

Fifty-seven kinds of APGWamide-related peptides and a peptide library consisting of 38 peptide mixtures, each of which contained 19 kinds of APGWamide-related peptides, were synthesized with a multipeptide synthesizer, and their APGWamide-agonistic or -antagonistic effects were examined on the anterior byssus retractor muscle of the bivalve Mytilus edulis and the crop of the land snail Euhadra congenita. The peptide mixtures having agonistic or antagonistic effects were subjected to HPLC purification to isolate the active peptides using the muscles as bioassay systems. Many peptides having agonistic or antagonistic effects were obtained. Of the antagonists, APGWGNamide, isolated from the peptide mixture of APGWGXamide, was the most potent. At 10(-4) M, APGWGNamide almost completely blocked the actions of 10(-6) M APGWamide on the anterior byssus retractor muscle of M. edulis and the crop of E. congenita.

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 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.

Ionic mechanism mediating Mytilus inhibitory peptides elicited membrane currents in identified Helix neurons.

Effects of seven, pressure applied MIP (Mytilus inhibitory peptides) had been studied on D-neurons of the CNS of Helix pomatia in voltage-clamp experiments. In physiological saline, the peptides produced a hyperpolarization usually coupled with the cessation of any spontaneous spiking activity. Clamped at the resting potential ( approximately -60 mV), peptide applications elicited an outward current, which increased its amplitude by shifting the holding potential towards depolarisation. The response was concentration-dependent and accompanied by an increased membrane conductance. Reversal potentials obtained at different [K+]o were plotted with a slope of 52 mV per ten-fold change in [K+]o showing that the peptide-elicited current was mainly due to the increased K+-conductance(s). The peptide-induced outward current could partially be blocked by Ba2+ (5 mM), CdCl2 (1 mM), TEACl (10 mM) or apamin (2.5x10(-5) M) or furosemide (10 mg/ml) and decreased either in Na+-free or Cl--free solutions. 4-Aminopyridine at 5 mM concentration completely blocked the peptide-induced current. In the presence of high [K+]o, the peptide(s) was still found to induce an outward current at membrane potentials beyond K+-reversal potential. This component was not present in Cl--free saline, suggesting that the current was due to the inward flow of Cl- ions. Our results show that the MIPs have at least two (three) independent actions, each associated with different voltage-, concentration-dependence and ionic mechanisms. It is suggested, that the peptide-induced currents are carried by K+, and Cl- ions. According to our present finding, the observed effects are mediated by the same receptor, activating different second messenger systems, inducing multiple conductance changes in the membrane of neurons of the snail ganglia.

Evidence for the involvement of nitric oxide in the inhibitory effect of GSPYFVamide on Helix aspersa central neurones.

Intracellular recordings were made from neurones E-8, E-16 and E-13a in the visceral ganglion of Helix aspersa. GSPYFVamide inhibits the activity of these neurones and the role of a second messenger system in this inhibition was investigated. 8-Bromo-cGMP, 100 microM was found to potentiate this inhibition while ODQ, 100 microM, an inhibitor of guanylyl cyclase, almost completely blocked GSPYFVamide-induced inhibition. Four NO donors sodium nitroprusside, 100 microM, sodium nitrite, 1 mM, SNOG, 50 microM, and SNAP, 10-50 microM, all potentiated the GSPYFVamide-induced inhibition. L-NAME, 100-1000 microM, a competitive inhibitor of NOS, blocked the GSPYFVamide-induced inhibition. In some cases recovery was only partial. The possible role of NO in modulating the inhibitory response to GSPYFVamide is discussed.

Peptides controlling stifness of connective tissue in sea cucumbers.

We present the first evidence of a system of four bioactive peptides that affect the stiffness of sea cucumber dermis. The body wall dermis of sea cucumbers consists of catch connective tissue that is characterized by quick and drastic stiffness changes under nervous control. The peptides were isolated from the body wall, their amino acid sequences determined, and identical peptides synthesized. Two peptides, which we named holokinins, are homologous with bradykinin. We tested the effect of the peptides on the mechanical properties of sea cucumber dermis. Both of the holokinins softened the dermis, and a pentapeptide that we designated as NGIWYamide stiffened it. Both effects were reversibly suppressed by anesthesia with menthol. We called the fourth peptide stichopin; it had no direct effect on the stiffness of the dermis but suppressed action of the neurotransmitter acetylcholine reversibly. The results suggest that the peptides are neuropeptides and are part of a sophisticated system of neurotransmitters and neuromodulators that controls the connective tissue stiffness of sea cucumber dermis.

Structural requirements and ionic mechanism of the Mytilus inhibitory peptides (MIPs) on Helix central neurons.

1. The structural and ionic requirements for potent FVamide-induced inhibition were investigated using Helix aspersa central neurons under current or voltage clamp. 2. For potent FVamide inhibition the full hexapeptide sequence, GSPYFVamide, was required. The rank order of decreasing potency was GSPYFVamide >> SPYFVamide> PYFVamide > YFVamide. 3. GSPYFVamide inhibition involved an increase in conductance to both potassium and chloride ions as demonstrated by ion substitution experiments and addition of 4-aminopyridine, tetraethylammonium, 4,4-di-isothiocyanatostilbene-2,2'-disulfonic acid, 4,4-dinitrostilbene-2,2'-disulfonic acid disodium salt and furosemide to the solution bathing the preparation.

Isolation and characterization of a novel bioactive peptide, Carassius RFamide (C-RFa), from the brain of the Japanese crucian carp.

A novel bioactive peptide with the C-terminal RFamide was isolated from the brain of the Japanese crucian carp, Carassius auratus langsdorfii, by using the intestine of the fish as the bioassay system. The primary structure of the peptide was determined to be SPEIDPFWYVGRGVRPIGRFamide and it was designated Carassius RFamide (C-RFa). The sequence of C-RFa was found to be significantly homologous to the molluscan neuropeptide termed Achatina cardioexcitatory peptide 1 (ACEP-1). Both peptides have RPXGRFamide structure in their C-terminal moieties. C-RFa was found to have an excitatory effect on visceral muscle tissues of fish, newt, quail, and rat. In the stomach of the fish, Zacco temminckii, IGRFamide (C-RFa 17-20) was shown to be the minimal structure required for the excitatory effect and PIGRFamide (C-RFa 16-20) to be almost equipotent with the parent peptide C-RFa.

A novel D-amino-acid-containing peptide isolated from Aplysia heart.

A novel cardio-excitatory peptide was purified from the hearts of Aplysia kurodai. The peptide was a tripeptide containing a D-amino acid residue in the second position, Asn-D-Trp-Phe-NH2 (NdWFamide). NdWFamide increased the amplitude of contractions of the perfused Aplysia heart with little effect on beating frequency, showing a threshold of approximately 10(-11) M. The peptide also potentiated spontaneous contractions of the anterior aorta. The synthetic peptide having L-Trp instead of D-Trp was about 1,000 times less potent than the native one. NdWFamide seems to play an important role in regulation of cardiac activity in Aplysia.

Isolation of bioactive compounds from Helix aspersa nerve tissue and the effect of pQPPLPRYamide on heart, esophagus and central neurons of H. aspersa and rectum of Anodonta woodiana.

1. Both acetone and methanol extraction was used to isolate bioactive compounds from 1000 Helix aspersa brains. 2. Seven compounds were isolated of which four were identified as follows: Ha-1, 5-hydroxytryptamine; Ha-3, GSPYFVamide; Ha-4, pQPPLPRYamide; Ha-5, SGYLAFPRMamide. There was insufficient material to identify Ha-2, Ha-6 and Ha-7. 3. Ha-4, pQPPLPRYamide, was found to excite the heart of H. aspersa, relax the esophagus and both excite (mainly) and inhibit central neurons. In addition, this peptide contracted the rectum of Anodonta woodiana. 4. It is concluded that pQPPLPRYamide is an example of a new molluscan peptide family, designated as PRYamide.

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.

Systematic isolation of peptide signal molecules regulating development in hydra: LWamide and PW families.

To isolate new peptide signal molecules involved in regulating developmental processes in hydra, a novel screening project was developed. Peptides extracted from the tissue of Hydra magnipapillata were systematically purified to homogeneity using HPLC. A fraction of each purified peptide was examined by differential display-PCR for its ability to affect gene expression in hydra. Another fraction was used to determine the tentative structure using an amino acid sequence analyzer and/or a mass spectrometer. Based on the results, peptides of potential interest were selected for chemical synthesis, followed by confirmation of the identity of the synthetic with the native peptides using HPLC. Using this approach, 286 peptides have been isolated, tentative amino acid sequences have been determined for 95 of them, and 19 synthetic peptides identical to native ones were produced. The 19 synthetic peptides were active in a variety of biological tests. For example, Hym-54 stimulated muscle contraction in adult polyps of hydra and sea anemone, Anthopleura fuscoviridis, and induced metamorphosis of planula, the larval stage, into polyps in a marine hydrozoan species, Hydractinia serrata. Another peptide, Hym-33H, inhibited nerve cell differentiation in hydra and induced tissue contraction in planula of Hydractinia serrata. The evidence obtained so far suggests that hydra contains a large number (>350) of peptide signal molecules involved in regulating developmental or other processes in cnidaria. These peptides can be isolated and their functions examined systematically with the new approach developed in this study.

Time course of appearance of GABA and GABA receptors during retinal regeneration in the adult newt.

Appearance and maturation of the GABA (gamma-aminobutyric acid) system during newt retinal regeneration were studied by electrophysiological, immunohistochemical, and biochemical techniques. (1) Responses to GABA appeared in neurons dissociated from regenerating retinae before the segregation of the plexiform layers; whereas (2) GABA immunoreactivity appeared at sites of the presumptive horizontal cell and amacrine cell layers at the beginning of the segregation of these layers. During subsequent regeneration, GABA-immunoreactive cells at the amacrine cell layer increased in number and extended lateral processes, forming a GABA-immunoreactive inner plexiform layer. Also GABA immunoreactivity increased in the region of the outer plexiform layer, but not their somata which showed decreased GABA immunoreactivity. (3) GABA synthesis in the retina increased significantly at the beginning of the segregation of the plexiform layers. These results suggest that the increase of GABA synthesis during retinal regeneration correlates well with the development of GABA-immunoreactive cells and that functional GABA receptors appear earlier than increased GABA synthesis.

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.

An oviposition-inducing peptide: isolation, localization, and function of avian galanin in the quail oviduct.

It is well established that avian oviposition is regulated, at least partly, by a neurohypophysial hormone, arginine vasotocin, and ovarian hormones, prostaglandins, are regulated through mechanisms of the induction of uterine contractions. Although abundant nerves are terminated in the musculature in the uterine and vaginal oviduct regions, limited information is available on the neuronal control mechanism of avian oviposition. To identify the oviduct factor that plays an important role in the induction of oviposition as a neurotransmitter or a neuromodulator, a study was conducted to isolate the bioactive substance involved in the vaginal and uterine contractions from mature oviducts of the Japanese quail. Acetic acid extracts of 200 quail oviducts were forced through disposable C-18 reversed-phase cartridges, and then the retained material was subjected to the reversed-phase and cation-exchange HPLC purifications. A purified bioactive substance showed a single peak on the reversed-phase HPLC and was further subjected to amino acid sequence analysis and molecular weight presumption. The substance was identified as avian galanin, which was previously isolated from chicken intestine. The synthetic peptide enhanced contractions of both the vagina and the uterus in a manner similar to the native peptide, and the threshold concentrations were 10(-9) - 10(-8) M in the vagina and 10(-10) - 10(-9) M in the uterus. An intraperitoneal injection of the synthetic peptide significantly evoked quail oviposition during 5 min after injection. Immunohistochemical analysis with the anti-galanin serum revealed that abundant immunoreactive fibers were distributed in muscle layers of the vagina and the uterus. The immunoreaction examined in these regions was completely inhibited by preincubation of the antibody with synthetic peptide. These results suggest that avian galanin in the oviduct evokes oviposition through mechanisms of the induction of uterine and vaginal contractions. This peptide may contribute as a neurotransmitter or a neuromodulator to avian oviposition.

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.