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.

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.

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.

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.

Taurine in rat posterior pituitary: localization in astrocytes and selective release by hypoosmotic stimulation.

Taurine, a gamma-aminobutyric acid (GABA)-like acidic amino acid, has previously been shown to be prominently localized to astrocytes in the supraoptic nucleus, the neurons of which contain only small amounts, and to have inhibitory actions on supraoptic neuronal activity. In the present study, taurine distribution in the neurohypophysis was determined by using a well-characterized monoclonal antibody against taurine itself. Preembedding immunohistochemistry was performed at light and electron microscopic levels by using diaminobenzidine and gold-substituted silver-intensified peroxidase (GSSP) methods. At the light microscopic level, the distribution pattern and cellular localization of taurine immunoreactivity corresponded to that of glial fibrillary acidic protein. Pituicyte cell bodies and processes displayed dense taurine immunoreactivity. Electron microscopic observations revealed strong taurine GSSP reactions in these neural lobe astrocytes, but weak taurine reactivity was seen within only some neurosecretory axons. High-performance liquid chromatography analyses demonstrated that in vitro hypoosmotic stimulation (reduction of 40 mOsm/kg) of isolated posterior pituitaries resulted in preferential increases in taurine release into the bathing medium without increased release of other amino acids. Conversely, tissue concentrations of taurine significantly decreased with hypoosmotic perfusion, while glutamate, glutamine, and GABA concentrations were not reduced. These results indicate that taurine is mainly concentrated in neurohypophysial astrocytes, which are known to engulf the neurosecretory axonal processes and terminals. Taurine released from pituicytes under basal and hypoosmotic conditions may act to suppress axon terminal depolarization and thereby depress release of neurohypophysial peptides.

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.

NdWFamide: a novel excitatory peptide involved in cardiovascular regulation of Aplysia.

Although diverse peptides are known to affect invertebrate cardiac activity, the peptidergic regulation of the cardiovascular system of Aplysia is still poorly understood. Asn-D-Trp-Phe-NH(2) (NdWFamide) is a recently purified cardioactive peptide in Aplysia. Pharmacological experiments showed that NdWFamide was one of the most potent cardioexcitatory peptides among the known endogenous cardioactive peptides in Aplysia. NdWFamide-immunopositive neuronal processes were abundant in the cardiovascular region of Aplysia, and many of them originated from neurosecretory cells in the abdominal ganglion (R3-R13 cells). The data suggest that NdWFamide is a cardioexcitatory peptide utilized by R3-R13 cells of Aplysia.

A novel gut tetradecapeptide isolated from the earthworm, Eisenia foetida.

A novel bioactive tetradecapeptide, GFKDGAADRISHGFamide, was isolated from the gut of the oligochaete annelid, Eisenia foetida, using the isolated anterior gut (crop-gizzard) as a bioassay system. A highly homologous peptide, GFRDGSADRISHGFamide, was also purified from the whole body of another species of earthworm, Pheretima vittata. These peptides were termed Eisenia tetradecapeptide (ETP) and Pheretima tetradecapeptide (PTP), respectively. Both the peptides showed a potent excitatory action on spontaneous contractions of the anterior gut with a threshold as low as 10(-10)-10(-9) M. These peptides were significantly homologous to molluscan tetradecapeptides and, to a lesser extent, to arthropodan tridecapeptides that have been reported to date. All these peptides seem to be evolutionally related to each other.

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.

Identification of a vasopressin-like immunoreactive substance in hydra.

Vasopressin (VP)-like immunoreactivity has long been known in the hydra nervous system, but has not yet been structurally identified. In this study, using HPLC fractionation and an immunological assay, we have purified two peptides, FPQSFLPRGamide and SFLPRGamide, from Hydra magnipapillata. Both the peptides shared the same C-terminal structure, -PRGamide, with Arg-VP. The nonapeptide proved to be Hym-355, a peptide that stimulates neuronal differentiation in hydra. Detailed evaluation by competitive enzyme-linked immunosorbent assay (ELISA) and double immunostaining using anti-VP and anti-Hym-355 antibodies enabled us to conclude that the two peptides account for a major part of the VP-like immunoreactivity in hydra nerve cells.

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.

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.

Carassius RFamide, a novel FMRFa-related peptide, is produced within the retina and involved in retinal information processing in cyprinid fish.

Carassius RFamide (C-RFa) is a novel peptide, isolated originally from the brain of the Japanese crucian carp and sharing homologies with mammalian prolactin-releasing peptide (PrRP). It has been demonstrated previously that C-RFa mRNA is abundant in the proximal half (fundus) of the Japanese crucian carp eye. In the present work, we localized C-RFa by immunohistochemistry mainly to perikarya, in the proximal half of the inner nuclear layer (amacrine cell layer). This distribution is different from that of FMRFamide, which is confined to axon terminals of terminal nerve efferent fibers in the inner plexiform layer. Electrophysiological recording revealed that C-RFa depolarized some amacrine cells and hyperpolarized L-type horizontal cells in the carp. These results suggest that C-RFa is produced within the cyprinid retina and functions as a transmitter or neuromodulator in retinal image processing.

Not only osmotic stress but also repeated restraint stress causes structural plasticity in the supraoptic nucleus of the rat hypothalamus.

Magnocellular neuroendocrine cells (MNCs) in the supraoptic nucleus (SON) of the hypothalamus have been known to undergo dramatic structural changes during chronic stimulation such as osmotic stress. In the present study, we examined whether this anatomical neural plasticity is associated with an another stress, such as restraint. Rats were chronically stimulated by either dehydration with 2% saline drinking instead of water or daily restraint with leg immobilization. The structural reorganizations of MNCs in the SON were analyzed morphometrically with use of light and electron microscopy. The results were compared to control animals that had free access to water and food. In restraint rats, the soma size of both oxytocin (OXT) and arginine vasopressin (AVP) neurons was enlarged, and the percent of soma-somatic/dendritic membrane contact (juxtaposition) was elevated significantly. The number of total synapses per 100 microns soma membrane was not changed, although soma profiles were enlarged. However, the number of multiple synapses (which contacted with more than one postsynaptic element) per 100 microns soma membrane was significantly increased. Similar structural changes were observed in dehydrated animals, and the degree of morphological changes was stronger than the restraint one. These findings indicate that NMCs undergo structural plasticity during not only osmotic stress but also restraint stress.

Immunohistochemical Localization of C-RFamide, a FMRF-related Peptide, in the Brain of the Goldfish, Carassius auratus.

Carassius RFamide (C-RFa) is a novel peptide found in the brain of the Japanese crucian carp. It has been demonstrated that mRNA of C-RFa is present in the telencephalon, optic tectum, medulla oblongata, and proximal half of the eyeball in abundance. Immunohistochemical methods were employed to elucidate the distribution of the peptide in the brain of the goldfish (Carassius auratus) in detail. C-RFaimmunoreactive perikarya were observed in the olfactory bulb, the area ventralis telencephali pars dorsalis and lateralis, nucleus preopticus, nucleus preopticus periventricularis, nucleus lateralis tuberis pars posterioris, nucleus posterioris periventricularis, nucleus ventromedialis thalami, nucleus posterioris thalami, nucleus anterior tuberis, the oculomotor nucleus, nucleus reticularis superior and inferior, facial lobe, and vagal lobe. C-RFa immunoreactive fibers and nerve endings were present in the olfactory bulb, olfactory tract, area dorsalis telencephali pars centralis and medialis, area ventralis telencephali, midbrain tegmentum, diencephalon, medulla oblongata and pituitary. However, in the optic tectum the immunopositive perikarya and fibers were less abundant. Based on these results, some possible functions of C-RFa in the nervous system were discussed.

A myomodulin-CARP-related peptide isolated from a polychaete annelid, Perinereis vancaurica.

Myomodulin-CARP-family peptides have been isolated only from molluscs. In the present study, a heptapeptide, Ala-Met-Gly-Met-Leu-Arg-Met-NH2, termed Pev-myomodulin, was isolated from a polychaete annelid, Perinereis vancaurica using the esophagus of the animal as the bioassay system. The sequence of the annelid peptide is highly homologous with those of the myomodulin-CARP-family peptides found in molluscs. The annelid peptide is regarded as a member of the myomodulin-CARP family, though all the molluscan peptides have a Leu-NH2 at their C-termini. The annelid peptide showed a potnet contractile action on the esophagus of the annelid. The peptide may be an excitatory neuromediator involved in the regulation of the esophagus. Among various myomodulin-CARP-family peptides and their analogues, the annelid peptide showed the most potent contractile action on the esophagus. Replacement of the C-terminal Met-NH2 of the annelid peptide with a Leu-NH2 decreased its contractile potency, while replacement of the C-terminal Leu-NH2 of myomodulin and CARP with a Met-NH2 increased their potency. The C-terminal Met-NH2 of the annelid peptide seems to be important, but not essential, for exhibiting its contractile activity on the esophagus. On the anterior byssus retractor muscle of the bivalve mollusc Mytilus edulis, the annelid peptide showed catch-relaxing and contraction-modulating effects qualitatively similar to those of the authentic peptide CARP, though the annelid peptide was less potent than CARP.

Immunohistochemical Localization of Annetocin, an Earthworm Oxytocin-Related Peptide, and Identification and Ultrastructural Characteristics of the Annetocin-Secretory Cells in the Oligochaete Earthworm Eisenia foetida.

Annetocin is an egg-laying-inducing oxytocin-related peptide which we have previously isolated from the earthworm, Eisenia foetida. Here we report the results of immunohistochemical and ultrastructural studies on annetocin-secretory cells in the earthworm. Annetocin-immunoreactive (IR) cell-somata were located mainly at the ventro-lateral side of the subesophageal ganglion. Only four annetocin-IR cells were seen in the cerebral ganglion. Some annetocin-IR cells displayed unipolar-like structure with a process directing to the core region (the neuropile) of the ganglion. Annetocin-IR fibers were also observed in the neuropile of the ventral ganglia and the ventral nerve cord between the 4th and the 30th segments including the clitellum, but not in the posterior segments (31-55th). The number of annetocin-IR fibers decreased from the 4th to the 30th segment. The annetocin-secretory cells were identified by the immunogold staining, and filled with gold-labeled vesicles, 200-250 nm in diameter, which included moderately electron dense material. The annetocin-secretory cells possessed a euchromatic nucleus, well-developed rough endoplasmic reticulum and Golgi apparatus. Some of the annetocin-secretory cells were found to form a neurohemal-like structure, where somata or fibers with loose glial investment came in contact with the coelomic space at the ventral side of the subesophageal ganglion. The results suggest that annetocin is a neuropeptide produced and secreted by the neuron in the cerebral and subesophageal ganglia of the earthworm.

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.

Distribution of Eisenia tetradecapeptide immunoreactive neurons in the nervous system of earthworms.

A detailed mapping of Eisenia-tetradecapeptide-immunoreactive neurons in the central and peripheral nervous system combined with quantitative morphological measurements was performed in Eisenia fetida and Lumbricus terrestris. In Eisenia, most labelled neurons were observed in the ganglia of the ventral cord (20.38% of the total cell number of the ganglion) and 15.67% immunoreactive cells occurred in the brain, while 6% of the neurons could be shown in the subesophageal ganglion. In the case of Lumbricus, most immunoreactive cells were found in the subesophageal ganglion (16.17%) and in the ventral ganglia (12.54%). The brain contained 122 ETP-immunoreactive cells (5.6%). The size of the immunoreactive cells varied between 35-75 microm. A small number of Eisenia-tetradecapeptide immunoreactive fibres were seen to leave the ventral ganglia via segmental nerves, and labelled processes could also be observed in the stomatogastric system and the body wall. Labelled axon branches originating from the segmental nerves formed an immunoreactive plexus both between the circular and longitudinal muscle layer and on the inner surface of the longitudinal muscle layer. This inner plexus was especially rich in the setal sac. Among the superficial epithelial cells the body wall contained a significant number of immunoreactive cells. Only a few Eisenia-tetradecapeptide immunoreactive neurons and fibres occurred in the stomatogastric ganglia. In the enteric plexus the number of immunoreactive neurons and fibres decreased along the cranio-caudal axis of the alimentary tract. Eisenia-tetradecapeptide immunoreactive cells were also present among the epithelial cells in the alimentary canal. Some of these cells resembled sensory neurons in the foregut, while others showed typical secretory cell morphology in the midgut and hindgut.

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.