Intracellular degradation of C-peptides in molluscan neurons producing insulin-related hormones.

Single Light Green Cells (LGC) of Lymnaea stagnalis, expressing four genes encoding insulin-related peptides (MIPs) and C-peptides, and sections from the median lip nerve (MLN) were subjected to MALDI-MS. Mass spectra of LGCs and MLNs were almost identical. Masses corresponding to those of the MIPs and some C alpha-peptides could be distinguished. ProMIP III C alpha-peptide and C beta-peptides were not found. The spectra showed additional masses matching those of carboxyterminally truncated C alpha-peptides. Peptides with similar masses were isolated from MLN extracts by HPLC, using electrospray-MS screening. Amino acid sequence analysis revealed intact proMIP I, II and V C alpha-peptides and I, II C alpha-peptide 1-24, 1-22 and 1-15.

Structural and functional evolution of the vasopressin/oxytocin superfamily: vasopressin-related conopressin is the only member present in Lymnaea, and is involved in the control of sexual behavior.

It has been suggested that the gene duplication that led to the formation of the vasopressin/oxytocin two-gene family occurred early during vertebrate evolution. However, the existence of both vasopressin- and oxytocin-related peptides in invertebrates suggests that this duplication may have occurred much earlier, although there is no evidence for the co-occurrence of vasopressin- and oxytocin-related peptides in the same invertebrate species. We report here that in Lymnaea only the vasopressin-related peptide Lys-conopressin, but not an oxytocin-related peptide, is present. Moreover, it is very likely that an oxytocin-like cDNA or gene is absent. The conopressin gene is expressed in neurons that control male sexual behavior, and its gene products are present in the penis nerve and the vas deferens. Conopressin induces muscular contractions of the vas deferens and inhibits central neurons that control female reproductive behavior. Thus, although structurally related to vasopressin, conopressin has functional and behavioral characteristics typical for oxytocin. Physiological and receptor binding data suggest that conopressin and [Ile8]-conopressin, a synthetic oxytocin-like analog of conopressin, are functionally equivalent in Lymnaea, and that the chemical nature of the amino acid residue at position 8 does not result in a functional difference. Therefore, we suggest that invertebrates contain only a single member of the vasopressin/oxytocin gene family and that the amino acid change that distinguishes vasopressin from oxytocin is functionally neutral in invertebrates.

Purification and characterization of a novel tetradecapeptide that modulates oesophagus motility in Lymnaea stagnalis.

The isolated oesophagus of Lymnaea stagnalis was used as a bioassay preparation to identify peptides that modulate the activity of the oesophagus. Several modulatory messengers were detected from the oesophageal extract. One of these, having an excitatory action, was further purified from both the oesophagus and the central nervous system through high performance gel permeation chromatography followed by serial reversed phase high performance liquid chromatography. The purified peptide was chemically characterized through amino acid sequencing and mass spectrometry as GFRANSASRVAHGY-NH2.

The sodium influx stimulating peptide of the pulmonate freshwater snail Lymnaea stagnalis.

In Lymnaea stagnalis integumental Na+ uptake is stimulated by the sodium influx stimulating (SIS)-peptide. Its primary structure was determined as: SRTQSRFAS- YELMGTEGTECVTTKTISQICYQCATRHEDSFVQVYQECCKKEMGLREYCEEIYTELPIRSGLWQPN++ +. Antisera raised against parts of SIS-peptide stained neurons in the visceral, parietal, and pleural ganglia, and in the proximal parts of the intestinal, anal, and right internal pallial nerves. Locations and axon projection patterns of these neurons suggest that they represent the previously described neurosecretory yellow cells.

SKPYMRFamide, a novel FMRFamide-related peptide in the snail Lymnaea stagnalis.

In Lymnaea stagnalis, three members of the FMRFamide peptide family have been chemically identified in the central nervous system, and other members of the family are predicted by cDNA studies. The present study demonstrates the occurrence of even more FMRFamide-related peptides in this species by identifying a novel member of this family. The peptide was purified from brain extracts by three different HPLC steps. Its amino acid sequence has been determined as Ser-Lys-Pro-Tyr-Met-Arg-Phe-amide (SKPYMRFamide).

Some polypeptides in the nervous system of the marine worm, Nereis diversicolor, are related to the sodium influx stimulating peptide of the pulmonate freshwater snail, Lymnaea stagnalis.

Total mRNA, extracted from brain of the marine worm, Nereis diversicolor (Annelida, Polychaeta), was translated either in vitro using a rabbit reticulocyte lysate or in ovo (Xenopus laevis oocyte). The synthesized polypeptides were analyzed by electrophoresis and Western blotting techniques using polyclonal antisera raised against three peptides: sodium influx stimulating peptide (SISP) sequences 10-19 and 67-76 and a monoclonal antibody raised against purified native SISP (1-77) of Lymnaea stagnalis. Among the products translated in vitro, three polypeptides of 80, 72, and 64 kDa were recognized by the anti-SISP (10-19) polyclonal antiserum and by the monoclonal antiserum, but not by anti-SISP (67-76). Some of the in ovo translated products showed almost identical immunoreactivity to both the anti-SISP (10-19) and the monoclonal antibody. These polypeptides have molecular masses of 80, 72, and 43 kDa. No polypeptides were recognized by anti-SISP (67-76). Western blotting analysis of brain extracts revealed a number of proteins that reacted with the antiserum raised against SISP (10-19) and the monoclonal antiserum. Several perikarya of brain ganglionic nuclei and ventral nerve cord were immunoreactive to anti-SISP (10-19). The monoclonal antiserum gave similar results, although with a less intense immunoreaction. The infracerebral region was also stained, suggesting that the immunoreactive material is released as a true neurohormone into the hemolymph. The largest polypeptides, in particular those translated from brain mRNA, could be neuropeptide precursors containing a SISP-related sequence.

Evolution of the vasopressin/oxytocin superfamily: characterization of a cDNA encoding a vasopressin-related precursor, preproconopressin, from the mollusc Lymnaea stagnalis.

Although the nonapeptide hormones vasopressin, oxytocin, and related peptides from vertebrates and some nonapeptides from invertebrates share similarities in amino acid sequence, their evolutionary relationships are not clear. To investigate this issue, we cloned a cDNA encoding a vasopressin-related peptide, Lys-conopressin, produced in the central nervous system of the gastropod mollusc Lymnaea stagnalis. The predicted preproconopressin has the overall architecture of vertebrate preprovasopressin, with a signal peptide, Lys-conopressin, that is flanked at the C terminus by an amidation signal and a pair of basic residues, followed by a neurophysin domain. The Lymnaea neurophysin and the vertebrate neurophysins share high sequence identity, which includes the conservation of all 14 cysteine residues. In addition, the Lymnaea neurophysin possesses unique structural characteristics. It contains a putative N-linked glycosylation site at a position in the vertebrate neurophysins where a strictly conserved tyrosine residue, which plays an essential role in binding of the nonapeptide hormones, is found. The C-terminal copeptin homologous extension of the Lymnaea neurophysin has low sequence identity with the vertebrate counterparts and is probably not cleaved from the prohormone, as are the mammalian copeptins. The conopressin gene is expressed in only a few neurons in both pedal ganglia of the central nervous system. The conopressin transcript is present in two sizes, due to alternative use of polyadenylylation signals. The data presented here demonstrate that the typical organization of the prohormones of the vasopressin/oxytocin superfamily must have been present in the common ancestors of vertebrates and invertebrates.

The bioelectrical activity of the body wall of the pulmonate freshwater snail Lymnaea stagnalis: effects of neurotransmitters and the sodium influx stimulating neuropeptides.

A method is described to dissect segments of the head skin of Lymnaea stagnalis. These skin segments were used to study the effects of neurotransmitters and of the Lymnaea sodium influx stimulating (SIS) peptides on ion transport, using the Ussing-cell technique. The electrical activity of the segments, with Lymnaea ringer solution at both sides, was low: the mean electrical potential difference (PD) across the skin was 0.7 +/- 0.6 mV (inside positive) at a resistance of 160 +/- 57 ohm.cm2 (inward short-circuit current, SCC, 4.2 +/- 3.0 microA/cm2; n = 25). Acetylcholine, adrenalin, noradrenalin, histamine, dopamine, and GABA, at 10(-5) M, did not affect skin resistance and PD. 5-Hydroxytryptamine-HCl (5-HT), however, in a dose-dependent way, increased the PD, SCC, and, to a much lesser extent, the resistance of skin segments. Extracts of the medium lip nerves, which contain the SIS peptides, had similar effects, but of much longer duration. Effects comparable to those of 5-HT and the SIS peptides could also be brought about by cAMP analogs. The inward current stimulation by 5-HT, SIS peptides, and cAMP was abolished by ouabain. The inward current induced by 5-HT and the SIS peptides was partly (10-25%) inhibited by amiloride. The presence of tetrodotoxin (10(-6) M) did not prevent the inward current stimulation by 5-HT and the SIS peptides.

Neurohormonal control of Na+ and Cl- metabolism in the pulmonate freshwater snail Lymnaea stagnalis.

In Lymnaea stagnalis, extirpation of the cerebral ganglia resulted in a significant decrease of the Na+ and Cl- concentrations in the haemolymph. In intact snails, injection of an extract of the cerebral ganglia stimulated the Na+ influx from the external medium in a dose-dependent way. No effect on the Cl- influx was found. Extract injections did not affect the Na+ efflux. The Na+ influx stimulating activity, in relatively high concentrations, was also present in extracts of the median lip nerve (an important neurohaemal area, originating from the cerebral ganglia). The influx-stimulating activity was heat-stable and destroyed by Pronase treatment. It is suggested that in L. stagnalis, Na+ uptake from the medium is controlled by a neurohormone produced by the cerebral ganglia.

Studies of the characteristics, distribution and physiological role of a large cardioactive peptide in Lymnaea stagnalis.

Large cardioactive peptide (LCP, heat-stable, mol. wt greater than or equal to 1500) occurs in the ventricle and CNS, and in high concentrations in the auricle. The LCP haemolymph concentrations in fed animals are ca. 35 times higher than those in starved snails. The excitatory effects of LCP on auricle, ventricle and oesophagus are similar to those of some (putative) neurotransmitters. LCP has no effects on the penis retractor muscle. Its effects on the auricle are much more prolonged than those of the transmitters. It is suggested that LCP is a neurohormone, which is transported to the auricle via the nervus intestinalis, and released in response to feeding stimuli.

A morphological, enzyme-cytochemical, and physiological study of the blood-gonad barrier in the hermaphroditic snail Lymnaea stagnalis.

In the hermaphroditic pulmonate snail Lymnaea stagnalis a blood-gonad (blood-testis) barrier appears to exist. Septate junctions between Sertoli cells and epithelial cells of the neck areas of the gonadal acini constitute this barrier; they separate the male from the female compartment. Experiments with tracer substances (colloidal gold particles, lanthanum nitrate, tannic acid) showed that the basal lamina around the acini hardly forms a barrier; only the larger colloidal gold particles do not pass this lamina. Physiological, the blood-gonad barrier is apparent in studies on the composition of gonadal fluid, which differs considerably from that of haemolymph. The osmolarity and the concentration of protein and amino acids in gonadal fluid exceed those of haemolymph. As to the major ions, in the gonadal fluid Na+ is partly replaced by K+, and HCO-3 is almost totally replaced by Cl-. Such a distribution of HCO-3 and Cl- is indicative of metabolic acidosis. The cytochemical localization of carbonic anhydrase activity in cells lining the acinar lumen (Sertoli cells, epithelial cells) suggests that these cells are involved in the process of ion exchange. The metabolic acidosis in the gonad might result from the anaerobic production of lactate and succinate by Sertoli cells; these cells lack the enzymes cytochrome oxidase, lactate dehydrogenase, and succinate dehydrogenase. Spermatogenic cells, on the other hand, do possess these enzymes. This probably indicates that these cells metabolize lactate and succinate secreted by Sertoli cells.