Fumihiro Morishita

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.

Regulatory actions of neuropeptides and peptide hormones on the reproduction of molluscs.

Reproductive success of individual animals is essential for the survival of any species. Molluscs have adapted to a wide variety of environments (freshwater, brackish water, seawater, and terrestrial habits) and have evolved unique tactics for reproduction. Both of these features attract the academic interests of scientists. Because neuropeptides and peptide hormones play critical roles in neural and neurohormonal regulation of physiological functions and behaviors in this animal group, the regulatory actions of these messengers in reproduction have been extensively investigated. In this review, we will briefly summarize how peptidergic messengers are involved in various aspects of reproduction, using some peptides such as egg-laying hormone, caudo-dorsal cell hormone, APGWamide, and gonadotropin-releasing hormone as typical examples.

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

Abstract 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.

Release of proteins and polysaccharides from the albumen gland of the freshwater snail Helisoma duryi: effect of cAMP and brain extracts.

Abstract The albumen gland is a compound tubular exocrine gland found in the female reproductive tract of freshwater pulmonate snails such as Helisoma duryi. It secretes a perivitelline fluid, composed of protein and polysaccharide complexes, and coats each fertilized egg. A 288-kDa native glycoprotein, composed of several 66-kDa subunits, was identified in soluble extracts of albumen gland. Forskolin stimulates the release of secretory granules, containing both proteins and polysaccharides, from the cytoplasm of the glandular cells. An acid extract of the central nervous system or the adenosine-3′,5′-cyclic monophosphate (cAMP) analogue 8-bromo cAMP, stimulates protein secretion from the gland. Pretreatment of the albumen gland with cAMP antagonist (Rp isomer of cAMP) inhibits the stimulatory effect of a brain extract. Digestion of brain extract with proteolytic enzymes abolishes its activity, suggesting the factor from the brain is peptidergic. The neuroactive agents serotonin, Phe-Met-Arg-Phe-amide, Tyr-Gly-Gly-Phe-Met-Arg-Phe-amide, small cardioactive peptide B, and caudodorsal cell hormone were also tested for potential secretion-promoting ability. Brain extracts were partially purified with a Sep-Pak C18 reverse-phase cartridge and indicate the peptide is relatively hydrophobic. These results suggest that a brain peptide promotes the secretion of perivitelline fluid, and this is mediated by the adenylate cyclase/cAMP signal transduction pathway.

Subtypes of beta adrenergic receptors mediating pigment dispersion in chromatophores of the medaka, oryzias latipes

Actions of the adrenergic beta-2 agonists, salbutamol and terbutaline, and the beta-1 antagonists, metoprolol and atenolol, were examined on denervated melanophores and leucophores of a teleost, Oryzias latipes. Beta-2 agonists depressed the pigment-aggregation response of melanophores to norepinephrine, while beta-1 antagonists inhibited the dispersion response of leucophores to isoproterenol but not the melanophore response. These findings suggest that adrenergic receptors mediating pigment dispersion in melanophores are beta-2 and those of leucophores are beta-1. The possible relations between receptor mechanisms and the responses of chromatophores are discussed.

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.

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.

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.

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.

Distribution of the Aplysia cardioexcitatory peptide, NdWFamide, in the central and peripheral nervous systems of Aplysia

NdWFamide is an Aplysia cardioexcitatory tri-peptide containing D-tryptophan. To investigate the roles of this peptide, we examined the immunohistochemical distribution of NdWFamide-positive neurons in Aplysia tissues. All the ganglia of the central nervous system (CNS) contained NdWFamide-positive neurons. In particular, two left upper quadrant cells in the abdominal ganglion, and the anterior cells in the pleural ganglion showed extensive positive signals. NdWFamide-positive processes were observed in peripheral tissues, such as those of the cardio-vascular system, digestive tract, and sex-accessory organs, and in the connectives or neuropils in the CNS. NdWFamide-positive neurons were abundant in peripheral plexuses, such as the stomatogastric ring. To examine the NdWFamide contents of tissues, we fractionated peptidic extracts from the respective tissues by reversed-phase high-pressure liquid chromatography and then assayed the fractions by competitive enzyme-linked immunosorbent assay. A fraction corresponding to the retention time of synthetic NdWFamide contained the most immunoreactivity, indicating that the tissues contained NdWFamide. The prevalence of the NdWFamide content was roughly in the order: abdominal ganglion >heart >gill >blood vessels >digestive tract. In most of the tissues containing NdWFamide-positive nerves, NdWFamide modulated the motile activities of the tissues. Thus, NdWFamide seems to be a versatile neurotransmitter/modulator of Aplysia and probably regulates the physiological activities of this animal.