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.

Dopamine stimulates snail albumen gland glycoprotein secretion through the activation of a D1-like receptor.

The catecholamine dopamine is present in both the central nervous system and in the peripheral tissues of molluscs, where it is involved in regulating reproduction. Application of exogenous dopamine to the isolated albumen gland of the freshwater pulmonate snail Helisoma duryi (Wetherby) induces the secretion (release) of perivitelline fluid. The major protein component of the perivitelline fluid of Helisoma duryi is a native 288 kDa glycoprotein that is secreted around individual eggs and serves as an important source of nutrients for the developing embryos. The secretion of glycoprotein by the albumen gland is a highly regulated event that must be coordinated with the arrival of the fertilized ovum at the carrefour (the region where the eggs receive albumen gland secretory products). In order to elucidate the intracellular signalling pathway(s) mediating dopamine-induced glycoprotein secretion, albumen gland cAMP production and glycoprotein secretion were measured in the presence/absence of selected dopamine receptor agonists and antagonists. Dopamine D1-selective agonists dihydrexidine, 6,7-ADTN and SKF81297 stimulated cAMP production and glycoprotein secretion from isolated albumen glands whereas D1-selective antagonists SCH23390 and SKF83566 suppressed dopamine-stimulated cAMP production. Dopamine D2-selective agonists and antagonists generally had no effect on cAMP production or protein secretion. Based on the effects of these compounds, a pharmacological profile was obtained that strongly suggests the presence of a dopamine D1-like receptor in the albumen gland of Helisoma duryi. In addition, secretion of albumen gland glycoprotein was not inhibited by protein kinase A inhibitors, suggesting that dopamine-stimulated protein secretion might occur through a protein kinase A-independent pathway.

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 putative hyperglycemic factor from the cerebral ganglia of Otala lactea (Mollusca: Pulmonata).

Mantle tissue pieces from adult Otala lactea continuously synthesized glycogen over a 72-h incubation period. Acid-saline extract of the cerebral ganglia inhibited glycogen synthesis by mantle tissue in vitro. This effect was dose-dependent. The glycogen reduction factor from the cerebral ganglia was heat stable, protease sensitive, and relatively hydrophobic. The cerebral ganglia extract also stimulated mantle glycogen phosphorylase in vitro in a dose-dependent manner. The results suggest the presence of a hyperglycemic factor in the cerebral ganglia of Otala. The molecular weight of this factor, estimated by size-exclusion chromatography, was approximately 10,000. Mammalian glucagon had no significant effect on glycogen synthesis by the mantle pieces.

Vacuolar-type ATPase in the accessory boring organ of Nucella lamellosa (Gmelin) (Mollusca : Gastropoda): role in shell penetration.

The structure and function of the accessory boring organ (ABO) of muricid gastropods has been described in numerous studies, and the ABO of Nucella lamellosa was found to be similar to those of other muricid species. The active cap region of the ABO is composed of tall, mitochondria-rich cells with distinct brush borders at their apicies, surrounding a hemolymph-containing central sinus. Using antibodies specific for vacuolar-type ATPase (V-ATPase), enzyme immunoreactivity was found to be limited to the brush border of the epithelial cells. Electron immunohistochemistry revealed that V-ATPase immunoreactivity resides in the plasma membranes of the microvilli. Immunodot blotting using yeast V-ATPase as a positive control confirmed the specificity of the reactions. SDS-PAGE of membrane suspensions from the ABO revealed protein bands of the requisite molecular weight for V-ATPase subunits. Western blots suggest that antibodies raised against mammalian V-ATPase subunits recognize subunits of the molluscan V-ATPase. The molecular weights of these identified subunits are similar to those in mammals. The V-ATPase-specific inhibitor bafilomycin A1 inhibited ATPase activity in samples of ABO homogenate by about 10% relative to control, providing further evidence for the presence of V-ATPase. Specific V-ATPase activity was about 67 picomoles of inorganic phosphate per microgram of protein per minute in the homogenate. Collectively this evidence strongly suggests that a vacuolar-type proton transporting ATPase is present in the brush border of the accessory boring organ of Nucella lamellosa, and is responsible for acidifying secretions from this gland. Similarities between the ABO, osteoclasts, and the mantle of freshwater bivalves also suggest that the mechanism for decalcification of calcareous substrates is conserved.

Osmoregulation and FMRFamide-related peptides in the salt marsh snail Melampus bidentatus (Say) (Mollusca: Pulmonata).

The pulmonate snail Melampus bidentatus occupies the high intertidal zone of salt marshes in a nearly terrestrial environment. The hemolymph osmolarity of the snails collected in the field paralleled that of the adjacent water and was affected by the tides and precipitation. The snails initially gained or lost weight when submerged in hypo- or hyperosmotic media, respectively, but returned to their original weight after 24 h. The content of their immunoreactive (IR)-FMRFamide-Related Peptides (FaRPs) was measured in various tissues by radioimmunoassay, and IR-FaRPs were found in every tissue analyzed. The subesophageal part of the central nervous system (CNS) contained more IR-FaRPs than the supraesophageal part, and the kidney and the tissues of the reproductive tract contained more than other peripheral tissues. The levels of IR-FaRPs in the CNS, kidney, and hemolymph were higher in snails that were immersed in higher concentrations of seawater. Many IR neurons are present in all ganglia of the CNS except the pleural ganglia, and IR neurites are extensively distributed within the CNS and its connective tissue sheath. The visceral nerve from the visceral ganglion is immunoreactive and could be seen to innervate the kidney, which contains IR-varicosities. An osmoregulatory role for the FaRPs is suggested.

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

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.

FMRFamide-related peptides, partial serotonin depletion, and osmoregulation in Helisoma duryi (Mollusca: Pulmonata).

Serotonergic neurons were studied by specific histological methods, and neurons containing Phe-Met-Arg-Phe-NH2 (FMRFamide)-related heptapeptides were identified with an antiserum specific for these substances in the central nervous system of the freshwater snail Helisoma duryi. Serotonergic neurons and their axons are present in all of the ganglia (paired buccal, cerebral, pedal, pleural, parietal, and single visceral) and major nerves of the central nervous system. Large neurons containing FMRFamide-related peptide immunoreactivity are located in the left parietal and visceral ganglia, whereas a few small neurons are located in the cerebral and pedal ganglia. Both serotonergic and FMRFamide-related peptide-immunoreactive dendrites and varicosities were observed in the kidney. A second antiserum with high affinity for FMRFamide-related heptapeptides was used to measure the levels of the immunoreactive material in various tissues, and such material was found in every tissue analyzed. When snails were exposed to a medium isosmotic to their hemolymph, the levels of immunoreactive FMRFamide-related peptides increased in the hemolymph, central nervous system, mantle, and kidney. Injection of dihydroxytryptamine, which is known to deplete serotonin content in the snail, also reduced the levels of FMRFamide-related-immunoreactive material in the above tissues. Therefore, serotonin may influence the levels of FMRFamide-related peptides in tissues by regulating the rate of their synthesis, axonal transport, or release. Both serotonin and FMRFamide-related peptides could be involved in osmoregulation.

An electron microscopic study of the endocrine dorsal bodies in reproductively active and inactive Siphonaria pectinata (Pulmonata: Mollusca).

The fine structure of the dorsal bodies of the pulmonate limpet Siphonaria pectinata is described in the context of female reproduction involving egg production. In reproductively-active (egg-laying) animals, the ciliated dorsal body cells are filled with lipid droplets and mitochondria. Gap junctions are commonly seen between the cells. The Golgi complexes and the smooth endoplasmic reticulum constitute the other prominent cell organelles. In reproductively-inactive (non-egg-laying) animals, there is a significant reduction in the number of lipid droplets and evidence of reduced synthetic activity in the dorsal bodies. About 12 dorsal body cells are present immediately underneath the perineurium of each cerebral ganglion of the central nervous system. These internal cells are structurally similar to those outside the central nervous system. Cell processes of some of these cells exit the central nervous system at a minimum of three locations on each side and they come in close proximity to the dorsal body cells outside the cerebral ganglia. Like the external cells, the internal cells also communicate via gap junctions and exhibit structural differences according to whether or not the animals are reproductively active. The dorsal body cells, inside and outside the central nervous system, appear to be innervated by neurosecretory axons suggesting neuronal control of dorsal body activity.

Cell junctions in the endocrine dorsal body cells of Helisoma duryi (Mollusca: Pulmonata): an in vitro study.

The endocrine dorsal bodies of gastropod molluscs regulate reproduction and are closely associated with the central nervous system. Previous studies on Helisoma duryi have shown that the dorsal body cells of reproducing snails contain more gap junctions than those of non-reproducing virgin snails. More dorsal body cells were isolated from virgin snails than reproducing snails in Ca(2+) and Mg(2+) free experimental saline. The isolated spherical cells attached to the culture dish, spread and formed filopodium-like processes within a few hours in culture medium containing Ca(2+) and Mg(2+). Many isolated cells reaggregated after 4-6 h in culture forming septate junction-like and gap junction-like cell contacts, as revealed by thin section and freeze-fracture studies. Following 10 min incubation in carboxyfluorescein diacetate the isolated cells fluoresced and, after aggregation, these cells transferred fluorescent dye to unlabelled cells. Cell aggregation was inhibited by cytochalasin D. Staining by NBD-phallacidin revealed the presence of actin in the filopodium-like processes of spread cells and in the perinuclear cytoplasm. It is likely that the septate junction-like contacts provide sites of cell attachment between aggregating cells; gap junctions are involved in intercellular communication, and actin is required in this process.

Neuronal pathways of three neurosecretory cells from the lateral lobes in Helisoma (Mollusca): innervation of the dorsal body.

The neurite distribution of three large neurosecretory cells, namely one canopy cell and two lateral-lobe cells from each lateral lobe of the cerebral ganglia in Helisoma duryi were studied by thick and thin plastic serial sections. These cells from only the right lateral lobe innervate the dorsal body. Neurites from the canopy cell innervate the cell-bodies whereas those from the lateral-lobe cells innervate the cell-processes of the dorsal body. Neurosecretory granules from these neurites are released at their sites of innervation. The neurites of the optic nerve form synapses with lateral-lobe cell(1), and synapse-like contacts with lateral-lobe cell(2) while a neurite of each canopy cell is found within its collateral optic nerve. Based on the anatomy of the lateral-lobe nerve cells and the optic nerve, it is argued that the stimulatory effect of long days on the dorsal body and on egg production is mediated through the lateral-lobe cells and the canopy cell.

FMRFamide-related peptides from the kidney of the snail, Helisoma trivolvis.

Three FMRFamide-related peptides (FaRPs) were purified and characterized from the kidney of the snail, Helisoma trivolvis, by HPLC and detected using two radioimmunoassays (RIA) for FaRPs. Automated sequencing and mass spectrometry of the isolated peptides suggest the following sequences: Phe-Met-Arg-Phe-NH2 (FMRFamide), Phe-Leu-Arg-Phe-NH2 (FMRFamide), and Gly-Asp-Pro-Phe-Leu-Arg-Phe-NH2 (GDPFLRFamide). The FaRPs, predominantly the heptapeptides, were also detected by HPLC and RIA in other osmoregulatory tissues such as the skin, mantle, and the hemolymph. The level of FaRPs, detected by radioimmunoassay, appears to be lower in snails kept under hyposmotic stress than in snails kept under isosmotic stress. The FaRPs appear to be involved in osmoregulation in H. trivolvis.

Insulin-like peptide(s) in the central nervous system of the snail Helisoma duryi.

The central nervous system of the snail Helisoma duryi contains porcine insulin-immunoreactive cells. Most of these cells are known as neurosecretory mediodorsal cells (MDC), and are involved in regulation of growth. There are about 25-40 large and small MDC in each cerebral ganglion near the commissure. Besides the MDC, 6-8 insulin-immunoreactive cells are also seen in the left parietal ganglion and the visceral ganglion. Insulin-immunoreactive material can be traced from the MDC perikarya into their axons in the median lip nerve. The elementary granules of the MDC are 150-250 nm in diameter, and are insulin-immunoreactive. Insulin-like material is released by the CNS in vitro, and can be measured by radioimmunoassay using an antiserum against human insulin. High potassium and 4-aminopyridine increase such release in vitro. It is likely that insulin-like peptide is a growth hormone in H. duryi.

Immunocytochemical localization of FMRFamide in the central nervous system and the kidney of Helisoma duryi (Mollusca): its possible antidiuretic role.

The distribution of FMRFamide-immunoreactive neurons in the central nervous system of the freshwater pulmonate, Helisoma duryi is described. All parts of the central nervous system except the two pleural and the right parietal ganglia, contain immunoreactive neurons. By immunogold techniques, only one kind of neurosecretory FMRFamide-immunoreactive cell (previously identified as the type-3 cell) was localized in the visceral and left parietal ganglia. This cell type has been previously implicated in an antidiuretic role. FMRFamide-immunoreactive material is found in the whole mount of the kidney as well as in kidney sections. Electron microscopic examination shows that the axons innervating either the smooth muscles of the kidney or the kidney itself contain neurosecretory granules morphologically similar to type-3 cells of the visceral and left parietal ganglia. When incubated in saline containing nanogram quantities of FMRFamide, the wet weight of the kidney increased. It is suggested that FMRFamide-like substance may function as an antidiuretic factor and that the kidney is a target organ of this peptide for osmoregulation.

Changes in the fine structure of the endocrine dorsal body cells of Helisoma duryi (mollusca) induced by mating.

The morphology and the organization of the endocrine dorsal bodies (DB) of non-reproducing virgin and castrated, and reproducing mated Helisoma duryi have been examined using serial sectioning. The DB cells occur in two masses on the mid-dorsal side of the cerebral commissure, each of which has a cortical zone containing the cell bodies and a medulla where cell processes terminate. The cell bodies measure 10-15 µm in diameter, and are arranged in lobules of 6-12 cells. The complex cell processes are winding and terminate at various distances from their cell bodies in both reproducing and non-reproducing snails. Few 70-90-nm membrane-bound granules are found in the cell bodies and many are seen in the cell processes, which seem to penetrate the perineurium of the cerebral ganglia and make close contacts with neurosecretory cells. In reproducing snails the DB cells display a significantly larger amount of plasma membrane sproutings in the form of loops and circles compared to that in reproductively inactive virgin or castrated snails. Images of thin-sections and freeze-fracture replicas of these membranes suggest that they are gap junctions, which join the DB cells with each other. It is likely that gap junction-mediated cell to cell communication is involved in the activation of the DB cells for their role(s) in reproduction.

Extracellular application of cobalt: a fast and simple method for delineating invertebrate neurosecretory pathways.

The extracellular cobalt backfilling technique was shown to be an excellent method to obtain cobalt backfills of invertebrate neurosecretory cells (NSCs). Aqueous cobalt was placed in an extracellular suction electrode into which a portion of a neurohaemal (NH) region containing the axons and/or terminals of NSCs was drawn. Spontaneously discharging extracellular action potentials were recorded as the cobalt was applied to the NH region, and the greater the electrical activity, the more extensive the cobalt backfilling. The greatest success occurred with 0.5 M cobalt chloride dissolved in physiological saline. No backfills were obtained in the absence of electrical activity. This technique was shown to backfill NSCs terminating in NH regions of the insect, Rhodnius prolixus, the isopod crustacean, Oniscus asellus, and the freshwater pulmonate snail, Helisoma. Combined with a light insensitive silver intensification method, this paper describes a relatively fast and simple method for delineating invertebrate neurosecretory pathways.

A study on the cytoplasmic granules of the pericardial gland cells of some bivalve molluscs.

The pericardial glands of three bivalve molluscs are composed of convoluted epithelium that appears as pouches on the auricles of Mytilus and as tubules in the connective tissue at the anterior-lateral sides of the pericardial cavity of Mercenaria and Anodonta. The pericardial gland cells are attached to each other by many randomly placed desmosome-like cell junctions and gap junctions. Belt-desmosomes that are characteristic of epithelial cells were not observed. The basal membrane of these cells is invaginated producing complex interdigitating cytoplasmic processes and filtration slits. The pericardial gland cells stain for the presence of iron with Prussian blue stain. Electron-dense and electron-lucent granules of various diameters are present in the cytoplasm. Many electron-dense granules contain ferritin-like particles in which the presence of iron has been demonstrated by microanalysis. It is suggested that these particles are the iron storage protein ferritin since they contain iron, and are water soluble, heat stable, and morphologically similar to mammalian ferritin. Ferritin particles are probably both synthesized and broken down by the pericardial gland cells; thus the pericardial gland cells may be involved in iron homeostasis in these molluscs.

The effect of the brain and dorsal bodies of Helisoma duryi (mollusca: pulmonata) on albumen gland synthetic activity in vitro.

Albumen gland explants from Helisoma duryi can be maintained in a liquid culture medium for at least 96 hr. This allows measurement of incorporation of [14C]glucose into newly synthesized polysaccharides both retained in the tissue and released into the medium. The albumen gland basal synthetic activity is much higher in reproductively active than in reproductively inactive snails (maintained in constant darkness, virgin, or without dorsal bodies). The presence of the whole brain or cerebral ganglia (with dorsal bodies) of reproductively active snails in the culture medium stimulates polysaccharide synthesis in albumen gland explants from snails in constant darkness. Addition of dorsal body homogenate to the culture medium stimulates polysaccharide synthesis in albumen gland explants from snails without dorsal bodies. These results show that an endocrine factor from the dorsal bodies and possibly a neuroendocrine factor(s) from the cerebral ganglia directly stimulate polysaccharide synthesis in the albumen gland.

Cell contacts between follicle cells and the oocyte of Helisoma (Mollusca, Pulmonata).

The cell contacts between follicle cells, and follicle cells and oocytes of egg-laying populations of Helisoma duryi and non-egg-laying populations of H. trivcolvis have been studied. Scanning electron microscopy reveals that four to six follicle cells envelop a single developing oocyte. Thin sections and lanthanum impregnations demonstrate apical zonulae adherentes followed by winding pleated-type septate junctions between follicle cells. Gap junctions and septate junctions have been found between follicle cells and vitellogenic oocytes. Freeze-fracture replicas show relatively wide sinuous rows of septate junctional particles, and nemerous large gap junctional particle aggregates on the P-face between vitellogenic oocytes and follicle cells. Septate and gap junctions between immature or nonvitellogenic oocytes and follicle cells are fewer compared to those in vitellogenic oocytes. Similarly, the junctional complexes are less developed in non-egg-laying H. trivolvis compared to those in egg-laying H. duryi. It is possible that intimate interaction between follicle cells and a developing oocyte is necessary for the maturation of the oocyte. The junctional complexes could be involved in the interaction of the follicle cells and the oocyte, and they must disassemble at the onset of ovulation. Rhombic particle arrays and nonjunctional ridges of particles have been found in the basal part of the oolemma.

Relative humidity affects the intercellular spaces and cell contacts of the kidney epithelium of the terrestrial snail Helix aspersa müller.

The effects of relative humidity on hemolymph osmolarity and on kidney ultrastructure are explored in Helix aspersa. The snails are active at 95% relative humidity and less active at 50% relative humidity. The hemolymph osmotic pressure increases with the decrease of relative humidity. Pericardial fluid and hemolymph collected from the heart contain similar amounts of total proteins, and both fluids display hemocyanin molecules in negatively stained preparations. When the snails are kept in an atmosphere of 95% relative humidity, numerous wide intercellular spaces are observed in the single-layered-kidney epithelium. The spaces are almost absent when the snails are kept at 50% relative humidity. It is suggested that prourine is formed through a paracellular junctional pathway across the single-layered kidney epithelium, and that the pericardial cavity is not the site of prourine formation. The septate junctions joining the kidney epithelial cells form a continuous belt of intimate contact in the paracellular pathway of prourine. Long septate junctions with many septa are present in the kidneys of snails from the atmosphere of 50% relative humidity, whereas short septate junctions with fewer septa are found in the kidneys of snails from the atmosphere of 95% relative humidity. It is possible that the longer septate junctions with many septa reduce prourine formation across the kidney sac epithelium.