Ingrith Deyrup-Olsen

Immunological and biological characteristics of the vasotocin-like activity in the head ganglia of gastropod molluscs

Extracts of cerebral and pleuro-pedal ganglia from two terrestrial slugs, Ariolimax columbianus and Limax maximus, and from the marine opisthobranch, Aplysia californica, contain immunoreactivity resembling that of a vasotocin or vasopressin. Radioimmunoassays using several antisera indicate that the immunoreactivity is not due to vasotocin, vasopressin, or any other known naturally occurring neurohypophyseal peptide. Immunoreactivity of extracts on a relatively nonspecific vasopressin antiserum is well correlated with activity on antidiuretic assays on rats. Both immunoreactivity and antidiuretic activity are adsorbed onto bovine neurophysin affinity columns. Thus these extracts contain one or more peptides that closely resemble the vertebrate antidiuretic hormones, vasotocin and vasopressin, both immunologically and pharmacologically. The amounts of immunoreactivity and antidiuretic activity in ganglion extracts do not appear to change during dehydration and rehydration. Although both ganglionic extracts and vasotocin stimulate exudation of fluid across the slug body wall, the present experiments provide no evidence that the vasotocin-like material(s) in these ganglia may participate as neurotransmitters or hormones in the regulation of fluid balance. This remains an attractive hypothesis.

Surface exudation in terrestrial slugs

1. Exudation by the skin of terrestrial slugs in vivo and in vitro was enhanced by tactile, chemical (acetylcholine, 5-hydroxytryptamine; hypotonic fluids), and electrical simulation. 2. Of total body fluid loss, most occurred through the skin rather than in the urine, especially under conditions of fluid overload. 3. Very large molecules, including exogenous dextrans (up to mol. wt 5 × 106) and endogenous hemocyanin were found to penetrate through the skin.

Ultrastructure and permeability characteristics of the membranes of mucous granules of the hagfish

Mucous granules secreted by the slime glands of the hagfish. Eptatretus stouti, were studied after ultrarapid cryofixation and freeze substitution in diverse media (osmium tetroxide in acetone; several aqueous glutaraldehyde-based media with or without osmium). Only freeze substitution with osmium tetroxide-acetone preserved the granules intact, allowing visualization of its single unit membrane. Tests of the rupture or stability of freshly secreted mucous granules in sea water and other aqueous media showed the membranes of the granules are permeable to all inorganic cations tested, ranging in relative mass from ammonium to barium. They are permeable to the univalent anions chloride, nitrate, and bicarbonate, but not to the di- or trivalent anions sulfate, phosphate, and citrate. Moreover, in solutions where nonpenetrant anions were present, rupture occurred if the osmotic pressure was below a critical level (about 800 mOsmol/l). The structural and permeability characteristics of the granules account for the explosive speed with which they rupture, releasing their mucous contents, on contact with sea water.

Polymer Gel Phase Transition: The Molecular Mechanism of Product Release in Mucin Secretion?

Mucus is a polymer gel resulting from the annealing and hydration of a variety of secretory products. Mucins which are the principal polymeric species in the mucus matrix, are condensed inside secretory granules and undergo massive swelling upon release. Upon exocytosis from goblet cells mucins swell following a typical first order kinetics that is characteristic of polymer gels (Verdugo, 1984; Tanaka and Fillmore, 1979). The swelling of mucus, which is the leading parameter for the control of mucus rheology, is driven by a Donnan potential (Tam and Verdugo, 1981). However the condensation of mucins inside the secretory granule is still not well understood. An important condition for mucin condensation is the shielding of their polyionic charges which depends upon the presence of large amounts of Ca2+ inside the secretory granule (Verdugo et. al., 1987). Although the eationic shield provided by calcium is expected, it is not sufficient to explain a specific molecular mechanism of mucins condensation. We have proposed that the condensation and decondensation of secretory mucins could be explained by a polymer gel phase transition mechanism. The experiments reported here were designed to test if the native polymer network contained in the giant mucin granule of the slugAriolimax columbianus can undergo polymer gel phase transition. Isolated granules were demembranated and suspended in solutions containing different water/glycerol ratios. The volume of the mucin polymer network was monitored by video- microscopy. Video recordings were stored in magnetic tape. Results indicate that the mucin network can expand as much as 600 fold. This volume change is reversible and characteristically discontinuous demonstrating the typical features of a polymer gel phase transition: The critical solvent/non-solvent ratio is 74/26% water/glycerol (pH 7, 20°C), and varies with pH, temperature and Ca2+ concentration.

Ultrastructure and lysis of mucin-containing granules in epidermal secretions of the terrestrial slug Ariolimax columbianus (Mollusca: Gastropoda:Pulmonata)

SummaryThe giant mucous cells in the skin of the terrestrial banana slug Ariolimax columbianus secret intact granules containing mucins. Electron microscopy, after ultrarapid freezing and freeze-substitution in osmium, shows that the secreted granules are bounded by two distinct membranes, presumably derived from the Golgi apparatus and the plasmalemma. Relatively stable, intact granules can be obtained in great quantity in our in vitro system. Rapid lysis of the granules was induced by adenosine triphosphate. At much higher concentrations, adenosine diphosphate and 5′-adenylimido-diphosphate also caused lysis. Other nucleotides and related compounds, as well as 1,4,5-inositol triphosphate and molluscan neurotransmitters and neuropeptides, had no effect on the granules. The stability of secreted granules varied with the ionic composition of the isosmotic medium in which they were suspended. When the predominant cation in the medium was potassium, and calcium was also present, granules lysed if exposed to shear stress (stirring of the suspension). This did not occur if sodium was the major cation present. None of the other ions in the suspension media had detectable effects on the stability of the granules.

The channel cell of the terrestrial slug Ariolimax columbianus (Stylommatophora, Arionidae).

SummaryStudies were carried out to identify the route by which macromolecules and large volumes of fluid traverse the skin of terrestrial gastropods. Electron micrographs of the skin of the banana slug Ariolimax columbianus demonstrated that carbon particles can enter large, specialized cells and pass thence to the exterior. These cells, which are termed channel cells, range up to 500 μm in length; they reach from the external surface of the skin to deep within the subepithelial interstitium. At the light-microscope level they show a large central channel or reservoir apparently filled with homogeneous fluid; after injection of ink into the body cavity this central channel becomes ink-filled. Electron micrographs show cisternae of the smooth endoplasmic reticulum, opening from the cell surface and occasionally traversing the entire cytoplasmic layer. The neurohormone arginine vasotocin stimulates fluid and particle movement through the channel cell; this response is inhibited by norepinephrine. Fluid output is dependent on the presence of a transwall hydrostatic pressure gradient of about 7 torr or above, as well as on activation of the channel cells.

An insulin-like substance in the blood of the slug Prophysaon foliolatum (Arionidae) in the course of tail regeneration

Abstract 1. 1. Studies have been carried out to search for the presence of insulin-like substances (ILS) in a terrestrial slug, Prophysaon foliotatum (Arionidae), which has the unusual capacity to autotomize and regenerate the terminal section (tail) of the body. 2. 2. An ILS in Prophysaon blood was revealed by a radioimmunoassay developed for salmon insulin. The ILS levels were elevated 8 weeks after autotomy (means 231–300% of control values). 3. 3. Since the tail is a major site of glycogen storage, the results may indicate a role of Prophysaon ILS in glycogen metabolism and/or growth and regeneration.

Blood venting through the pneumostome in terrestrial slugs

Abstract 1. Terrestrial slugs eject blood through the pneumostome in response to injection into the hemocoel of acetylcholine, 5-hydroxytryptamine, dopamine, and histamine. 2. This blood-venting response can be initiated also by osmotic, electrical, and mechanical stimuli. 3. Normally the response terminates rapidly; it can be elicited repeatedly in a given individual. Full recovery occurs within a few days. 4. The mechanism resembles blood ejection through the hemal pore of Lymnea stagnalis . Possible physiological roles of the response in slugs are discussed.

Product release by mucous granules of land slugs: II. species diversity in triggering of mucous granule rupture

Epithelial cells of the body wall of several species of terrestrial snails and slugs secrete mucus in membrane-bound granules. On emergence from mucous cells, the granules are triggered to rupture and release their contents, forming the protective mucous layer. In three species of slugs of the Superfamily Arionoidea (Ariolimax columbianus, Arion ater, Prophysaon foliolatum), ATP at micromolar concentrations triggered granule rupture through processes dependent on activation of calcium channels. Granules of a fourth species in this superfamily (Philomycus carolinianus) were insensitive to ATP. Granules secreted by slugs of the Superfamily Limnoidea (Limax maximus, Deroceras reticulatum) were also entirely unaffected by ATP, and showed no evidence of a role of calcium channels in mucous granule sensitivity. Instead, they ruptured when immersed in media of elevated pH, a process blocked by nigericin. The slugs of the two superfamilies represented taxa quite widely separated in evolutionary origin. Nevertheless, the diversity of granule function was not expected, considering the crucial and presumably ancient role of mucus in protection of cell surfaces, not only in slugs and other molluscs but throughout the range of animal taxa.