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.

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.

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.

Involvement of actin and Na+ -K+ ATPase in urine formation of the freshwater pulmonate Helisoma.

The site and process of urine formation in the renopericardial system of Helisoma have been investigated. Osmotic pressure and protein content of hemolymph from the heart, pericardial fluid from the pericardial cavity, prourine from the kidney sac, and urine from the ureter have been determined. Osmotic pressure is equal in hemolymph, pericardial fluid, and prourine, but less in urine. Protein content is similar in hemolymph and pericardial fluid, but much less in prourine and urine. Hemoglobin molecules are present in hemolymph and pericardial fluid but not in prourine. It is suggested that in Helisoma the kidney sac is the site of prourine formation, and prourine is an ultrafiltrate of hemolymph. The kidney epithelial cells contain 6- to 7-nm microfilaments which react with heavy meromyosin producing unidirectional arrowheads. Numerous actin filaments are present in the vicinity of the lateral cell membranes and basal processes. It is possible that the actin filaments regulate the extracellular spaces for prourine passage. It is postulated that the actin-rich kidney epithelium may generate hydrostatic pressure for ultrafiltration. Na+ -K+ ATPase is located on the luminal side of the kidney epithelium, which may regulate intracellular fluid level of the kidney epithelial cells, and thereby regulate their cell volume. Thus Na+ -K+ ATPase may be involved in the regulation of extracellular spaces in kidney epithelial cells. The enzyme may participate in the production of hyposmotic urine.