Snail Helix Pomatia Research Articles

Topographic organization of efferent neurons with different neurochemical characters in the cerebral ganglia of the snail Helix pomatia

Topographic organization of efferent neurons with different neurochemical characters in the cerebral ganglia of the snail Helix pomatia

Time-dependent actions of aluminates on membrane and action potentials of snail neurons

Aluminum (Al) is one of the elements, which is frequently subjected to experiments, however, the neurological observations with it are rather conflicting. The cause of this controversiality is not known but relates to some human disorders such as Alzheimer's disease and others as well. We studied the time-dependent actions of AlCl3 base solutions on resting membrane potential (Em), input resistance (Rin) and spike shape in giant neurons of the snail Helix pomatia L. at pH 7.7 and room temperature (22-24 degrees C) by use of intracellular technique. We reported significant differences in the effectiveness of the various Al solutions depending on the time of storage before use in the experiments (0, 2 and 6 days at room temperature). Freshly prepared and applied Al solutions caused a significant and dose-dependent depolarization with a concomitant decrease of Rin and the amplitude of the action potentials, but the 6 days solutions induced a hyperpolarization. Ouabain (0.1 mM) antagonized the hyperpolarization. The pH (7.1 or 7.7) and the time of the storage in combination also modified the direct membrane effects. Our experiments show that Al can induce differential membrane effects depending on the presence of various aluminum compounds. Namely, the predominate aluminum-monomer at pH 7.7 the Al(OH)4- might cause depolarization but the polynuclear aluminum complexes after polymerization of the monomers could hyperpolarize the neuronal membrane. We suppose, that the time-dependent equilibrium of various aluminum complexes plays a role in generating controversies in this field and emphasize again the importance of standardization of the experimental protocol.

The possible roles of the monoaminergic system in the feeding of the snail Helix pomatia

The possible role of serotonin and dopamine in the feeding of Helix pomatia was studied applying immunocytochemical, biochemical, and behavioral techniques as well as bioassay experiments. Immunocytochemistry showed that dopamine-containing (thyrosin-hydroxylase-immunoreactive) neuronal elements of the crop and the gizzard belong to the intrinsic part, whereas serotonin-containing (serotonin-immunoreactive) neuronal elements belong to the extrinsic part of the gastrointestinal nervous system. Bioassay studies on the spontaneous contractions of the crop and the gizzard showed that dopamine affected only the longitudinal muscle contractions by increasing both the tonus and contractility, whereas serotonin was effective on both the longitudinal and circular muscle contractions. Serotonin increased the tonus and contractility of longitudinal muscles in the crop but decreased them in the gizzard. Serotonin decreased the tonus and contractility of the circular muscles in the crop but increased them in the gizzard. Serotonin effects on the circular muscle of the gizzard were concentration dependent between a range of 10(-5) M-3 x 10(-5) M. HPLC measurements of monoamines in starved and satiated animals showed that the concentration of both dopamine and serotonin significantly decreased in both the CNS and different parts of the gastrointestinal tract of satiated animals, suggesting a significant monoamine liberation during feeding. The injection of monoamines (10(-3) and 10(-2) M) into the body cavity of starved animals showed that only dopamine was able to induce feeding whereas serotonin increased the general activity of the animals suggesting that the initiation of feeding is rather dopamine than serotonin dependent.

Neurophysiological and Behavioral Responses to Olfactory Stimuli in the Snail Helix pomatia L.

Some aspects of olfactory sensitivity in the pulmonate Helix pomatia L. were studied by means of neurophysiological and behavioral methods. Single fiber recordings were carried out in the olfactory nerve of the posterior tentacles. Olfactory stimulations with different odors were performed by means of a continuous air stream. The order of neuronal sensitivity to different odors was as follows: ethanol ³ ethyl acetate > pentanol > hexanol > octanol > diethyl malonate > vanillin. Furthermore, the results revealed a relative specificity for some substances. A comparison between neurophysiological and behavioral data shows that those substances, which cause the highest increases in impulse frequency, also evoke a behavioral avoidance reaction.

Cloning and characterization of two cDNAs encoding sulfatases in the Roman snail, Helix pomatia.

The sulfatase from the snail Heli pomatia is widely used for analytical applications. We have investigated the content of sulfatases in H. pomatia, using a biochemical and a molecular approach. A 112-kDa protein from the intestinal juice of H. pomatia comigrated with sulfatase activity when chromatographed on Sephacryl S300 and concanavalin A-Sepharose. The N-terminal amino acid sequence of the protein was similar to one of three sulfatase motifs defined by sequence alignment of known sulfatases. Degenerate primers designed from the motifs and the N-terminal amino acid sequence obtained were used to generate PCR fragments and to isolate both a full-length and a 3'-truncated cDNA encoding H. pomatia sulfatases, designated SULF1 and SULF2. SULF1 consists of 503 amino acids and shows 53-55% identity to the mammalian arylsulfatase B. The amino acid sequence deduced from the 878-bp SULF2 cDNA fragment is 55% identical with SULF1. Both SULF1 and SULF2 contain the cysteine residue conserved in the active site of many sulfatases, which is known to be posttranslationally modified into formylglycine in eukaryotic sulfatases. However, the SULF1 and SULF2 cDNAs do not code for the protein purified. This indicates the presence of at least three sulfatase genes in H. pomatia.

Electrospray ionization mass spectrometry of zinc, cadmium, and copper metallothioneins: evidence for metal-binding cooperativity.

Electrospray ionization (ESI) mass spectra of both well-characterized and novel metallothioneins (MTs) from various species were recorded to explore their metal-ion-binding modes and stoichiometries. The ESI mass spectra of the zinc- and cadmium-binding MTs showed a single main peak corresponding to metal-to-protein ratios of 4, 6, or 7. These findings combined with data obtained by other methods suggest that these MTs bind zinc or cadmium in a single predominant form and are consistent with the presence of three- and four-metal clusters. An unstable copper-specific MT isoform from Roman snails (Helix pomatia) could be isolated intact and was shown to preferentially bind 12 copper ions. To obtain additional information on the formation and relative stability of metal-thiolate clusters in MTs, a mass spectrometric titration study was conducted. One to seven molar equivalents of zinc or of cadmium were added to metal-free human MT-2 at neutral pH, and the resulting complexes were measured by ESI mass spectrometry. These experiments revealed that the formation of the four-metal cluster and of the thermodynamically less stable three-metal cluster is sequential and largely cooperative for both zinc and cadmium. Minor intermediate forms between metal-free MT, Me4MT, and fully reconstituted Me7MT were also observed. The addition of increasing amounts of cadmium to metal-free blue crab MT-I resulted in prominent peaks whose masses were consistent with apoMT, Cd3MT, and Cd6MT, reflecting the known structure of this MT with two Me3Cys9 centers. In a similar reconstitution experiment performed with Caenorhabditis elegans MT-II, a series of signals corresponding to apoMT and Cd3MT to Cd6MT species were observed.

Topographic organization of efferent neurons with different neurochemical characters in the cerebral ganglia of the snail Helix pomatia.

This study provides a description of the organization of neurons efferent to different head areas in the cerebral ganglia of Helix pomatia, revealed by simultaneous Ni-lysine and Co-lysine back-filling of different pairs of cerebral nerves. The backfills show that labeled cerebral neurons that innervate the head areas are concentrated in seven representation foci distributed in different parts of the cerebral ganglia. Almost each head area is represented in each focus. At a gross level, the representation of the different head areas in the representation foci shows a topographic arrangement. Each focus is constituted by neurochemically different groups of neurons. All head areas are innervated by serotonin-containing fibers from a single focus (Focus 2) and by dopamine-containing fibers from Foci 1, 2, and 4. However, they are innervated by CARP and FMRFamide-containing fibers from all of the foci. The combination of retrograde labeling with 5, 6-dihydroxytriptamine induced pigment labeling of serotonin-containing neurons or with fluorescence tyrosinehydroxylase immunocytochemistry to detect dopamine-containing neurons showed that the different head areas are topographycally represented in the clusters of both the serotonin- and dopamine-containing cells. The combination of Ni-lysine backfillings from different cerebral nerves with fluorescence CARP and FMRFamide immunocytochemistry revealed that the head areas are represented also in both the CARP and FMRFamide immunoreactive groups of neurons in the different foci.

Topographic organization of serotonergic and dopaminergic neurons in the cerebral ganglia and their peripheral projection patterns in the head areas of the snail Helix pomatia.

The distribution of monoaminergic neurons within the cerebral ganglia was investigated in the pulmonate snail Helix pomatia. Simultaneous serotonin and tyrosine hydroxylase double immunostaining revealed that the immunoreactive cell groups are concentrated in a putative monoaminergic center on the ventral surface of the cerebral ganglia. Simultaneous cobalt (Co)- and nickel (Ni)-lysine backfills of cerebral nerves were combined with 5, 6-dihydroxytryptamine pigment-labelling of serotonergic neurons, or with fluorescence immunocytochemistry of dopaminergic neurons. This showed that the serotonergic and dopaminergic cell groups can be divided into smaller subgroups on the basis of their axonal projections into different cerebral nerves. These subgroups show a topographic organization within the serotonergic and dopaminergic neuronal clusters. In the serotonergic system, the different regions of the head are represented in a rostrocaudal direction, whereas a caudorostral organization is characteristic for the dopaminergic system. No serotonin- or dopamine-immunoreative cell bodies but numerous fibers were observed in the head areas, indicating that these are innervated by cerebral monoaminergic neurons and show different innervation patterns. Serotonin-immunoreactive fibers mostly innervate muscle fibers, whereas dopamine-immunoreactive processes do not innervate effector cells, but terminate within the nerve branches of the head areas. On the basis of their innervation pattern, we suggest that dopaminergic neurons may take part in en route modulation of sensory afferent and efferent processes in an as yet unknown manner. The serotonergic neurons, on the other hand, may play a direct role in the modulation of muscle function.

Effects of veratridine and its derivatives on the Na-conducting channels in Helix neurons

Effects of veratrum alkaloids were studied on the Na-channels of the land snail Helix pomatia. It was found that veratridine and its analogues depolarize the membrane due to the increased Na-permeability. The inactivation was shifted right along the voltage axis and the recovery from the inactivation was faster after veratridine treatment. After alkaloid treatment the selectivity of the Na-channel decreased, however, the selectivity sequence was not altered. The activation curve was not shifted. Veratridine derivatives, which appeared to be more effective on insects, had almost no effect on the Na-current.

Assembly and architecture of invertebrate cytoplasmic intermediate filaments reconcile features of vertebrate cytoplasmic and nuclear lamin-type intermediate filaments

The two major intermediate filament (IF) proteins from the esophagus epithelium of the snail Helix pomatia and the two major IF proteins from muscle tissue of the nematode Ascaris suum were investigated under a variety of assembly conditions. The lowest-order complexes from each of the four protostomic invertebrate (p-INV) IF proteins are parallel, unstaggered dimers involving two-stranded α-helical coiled coil formation of their ∼350 amino acid residue central rod domain (i.e. long-rod). In the electron microscope these are readily recognized by their distinct ∼56 nm long rod with two globular domains (i.e. representing the non-helical carboxy-terminal tail domain of the p-INV IF proteins) attached at one end, closely resembling vertebrate lamin dimers. The next-higher-order oligomers are tetramers, which are easily recognized by their two pairs of globular tail domains attached at either end of a ∼72 nm long central rod portion. According to their size and shape, these tetramers are built from two dimers associated laterally in an antiparallel, approximately half-staggered fashion via the amino-terminal halves of their rod domains. This is similar to the NN-type tetramers found as the most abundant oligomer species in all types of vertebrate cytoplasmic IF proteins, which contain a ∼310 amino acid residue central rod domain (i.e. short-rod). As a first step toward filament formation, the p-INV IF tetramers anneal longitudinally into protofilaments by antiparallel CC-type association of the carboxy-terminal halves of their dimer rods. The next step involves radial growth, occurring initially through lateral association of two four-chain protofilaments into octameric subfibrils, which then further associate into mature, full-width filaments. Head-to-tail polymers of dimers and paracrystalline fibers commonly observed with vertebrate lamins were only rarely seen with p-INV IF proteins. The globular domains residing at the carboxy-terminal end of p-INV IF dimers were studding the surface of the filaments at regular, ∼24.5 nm intervals, thereby giving them a “beaded” appearance with an axial periodicity of about 24.5 nm, which is ∼3 nm longer than the corresponding ∼21.5 nm repeat pattern exhibited by short-rod vertebrate IFs.

Electrophysiological responses to thermal stimuli in peripheral nerves of the snail Helix pomatia L.

1. In Helix pomatia single units of the inner lip nerves and the pedal nerves were recorded during thermal stimulation of the lip region and the epithelium of the sole, respectively. 2. 13.3% of all single units recorded from the inner lip nerves and 11.7% of the pedal nerve fibres proved to be sensitive to thermal stimuli. 3. These results suggest that peripheral thermoresponsive structures contribute to the control of behavior leading to avoidance of unfavorable ambient temperatures.

Steroid sulphatase: expression, isolation and inhibition for active-site identification studies

Steroid sulphatase, which can hydrolyse 3-hydroxysteroid sulphates, has important roles in several physiological and pathological processes. A number of steroid sulphatase inhibitors have now been developed, of which the most potent to date is oestrone-3- O-sulphamate (EMATE). This inhibitor inactivates steroid sulphatase in an irreversible, time- and concentration-dependent manner. In order to be able to use a radiolabelled derivative of EMATE to study the active site, it will be essential to prepare the steroid sulphatase in a pure form. For this, attempts have been made to express the protein, using the steroid sulphatase cDNA, in the pGEX2T expression system and also to express a mutant form of the protein, in which the putative membrane-spanning domain was deleted, in CHO cells. In addition, a soluble steroid sulphatase has been identified from the snail Helix pomatia. This steroid sulphatase is inhibited by EMATE in an irreversible manner, similar to the human steroid sulphatase and appears to possess a histidine residue at its active site. The expression and/or isolation of a steroid sulphatase, in conjunction with the use of a radiolabelled derivative of EMATE should allow important new information about the active site of this enzyme and the mechanism of its inactivation to be obtained.

Excitability increase in withdrawal interneurons after conditioning in snail.

Membrane mechanisms of conditioning of the defensive reflex in the snails Helix pomatia and H. lucorum were investigated. Tapping on the shell was used as a conditioned stimulus, which under normal conditions produces no defensive reaction. A light blow of air into the pneumostome, called the defensive closure reaction, was used as an unconditioned stimulus. When a combination of conditioned and unconditioned stimuli were presented with 2-4 min interval, the reflex developed over a period of 3 days. The separate conditioned and unconditioned stimuli presented randomly were used as an active control. The electrical characteristics of identified interneurons involved in this defensive behavior were then measured in an isolated preparation. There was shown to be a decrease in the threshold of action potential generation from 20.5 to 16.3 mV and depolarizing shift of membrane potential from -62.1 to -57.0 mV. The electrical characteristics of withdrawal interneurons of active control snails did not differ from those in intact animals. All results show an increase in excitability of withdrawal interneurons after associative learning.

The organization of serotonin-, dopamine-, and FMRFamide-containing neuronal elements and their possible role in the regulation of spontaneous contraction of the gastrointestinal tract in the snail Helix pomatia.

The distribution of serotonin-, tyrosine hydroxylase-, and FMRFamide-immunoreactive neuronal elements, as well as the concentrations of serotonin and dopamine in the different parts of the gastrointestinal tract, were studied in the snail Helix pomatia. The sensitivity of the spontaneous contractions of the alimentary tract to serotonin, dopamine, and FMRFamide was also tested. Serotonin-, tyrosine hydroxylase-, and FMRFamide-immunoreactive elements could be demonstrated in each part of the gastrointestinal tract, but they showed different innervation patterns. Serotonin- and tyrosine hydroxylase-immunoreactive elements were dominant in the submucosal layer, whereas FMRFamide-immunoreactive elements were dominant in both the mucosal and submucosal layers. Tyrosine hydroxylase-immunoreactive elements were confined to the longitudinal muscle trabeculae of submucosa, whereas serotonin-immunoreactive elements were distributed throughout the submucosal layer. No serotonin-immunoreactive cell bodies, but only fibers, could be detected in the gastrointestinal tract, and therefore they represent extrinsic elements. Tyrosine hydroxylase- and FMRFamide-immunoreactive cell bodies represent intrinsic elements of the tract. The occurrence and density of the serotonin- and tyrosine hydroxylase-immunoreactive elements showed significant differences in the different parts of the alimentary tract, in accordance with HPLC assays, which revealed a significant frontocaudal decrease in both the serotonin (from 2.11 to 1.21 pM/mg) and dopamine (from 3.28 to 0.52 pM/mg) contents of the different parts of the alimentary tract. Dopamine at 10(-5) M concentration proved to be effective only on the longitudinal muscles by increasing the tone and frequency of contractions, but was ineffective on the circular muscles. Serotonin affected both the longitudinal and circular muscles. Serotonin at 10(-5) M concentration decreased the tone and increased the frequency of low-amplitude contractions of the longitudinal muscles of the esophagus and the gizzard but increased both the tone and frequency of the crop. Serotonin at 10(-9) M concentration slightly decreased the tone and blocked the contractions of the circular muscles in the crop but at 10(-5) M concentration induced contractions of the circular muscles in the gizzard. FMRFamide at 10(-6) M concentration decreased the tone and was shown to block the contractions of both the longitudinal and circular muscles.

The Bronchorelaxant Effect of Helicidine, a Helix Pomatia Extract, Involves Prostaglandin E2 Release

Helicidine is a biological extract prepared from the snail Helix pomatia L. and used in man as an anti-tussive agent. However, its mechanisms of action are not fully defined. In this study, we have investigated a possible relaxant effect of helicidine on guinea-pig airway smooth muscle and evaluated the role of prostanoids and airway epithelium in this relaxation. H. pomatia extract (0.001–1 mg/ml) induced a dose-dependent relaxation of guinea-pig trachea pre-contracted with histamine both in the presence and absence of tracheal epithelium. No significant difference in dose-dependency or magnitude of the relaxation was observed between tracheal segments with or without epithelium (maximal relaxant response of 35 ± 7 and 25 ± 7.5%, respectively). Relaxation of the trachea induced by H. pomatia extract (0.001–1 mg/ml) was inhibited by pretreatment with the cyclooxygenase inhibitor, indomethacin, both in the presence or absence of tracheal epithelium. H. pomatia extract (1 mg/ml) induced a marked and significant increase in prostaglandin E 2 release in tracheal segments with and without epithelium. These results indicate that helicidine possesses a bronchorelaxant activity which is independent of epithelium integrity and which is partly mediated by the release of the relaxant prostanoid, prostaglandin E 2. The origin of prostaglandin E 2 production in the airways remains to be defined.

Feeding behaviour of juvenile snails (Helix pomatia) to four plant species grown at elevated atmospheric CO2

The feeding behaviour of juveniles of the land snail Helix pomatia was examined in model plant communities consisting of Trifolium repens , Hieracium pilosella , Bromus erectus and Prunella vulgaris that are common species in extensively managed calcareous grasslands in the Swiss Jura mountains. The plant communities were grown either at ambient (350 ppm) or elevated (600 ppm) CO 2 concentrations. Leaves of T. repens and P. vulgaris grown in elevated atmospheric CO 2 had a lower specific leaf area, and leaves of T. repens had lower percentage N on a dry weight basis than leaves grown under ambient CO 2 concentration. Snails fed on all four plant species, but showed a overwhelming preference for T. repens (percentages of total biomass consumed were 91.9% at 350 ppm and 97.6% at 600 ppm). The species-specific feeding intensity of juvenile H. pomatia did not differ between the two treatments. The total dry weight of T. repens consumed by the snails was marginally greater ( P = 0.06) at elevated CO 2 , but there were no significant differences in leaf N or leaf area eaten. These findings are similar to numerous other studies showing that invertebrates increase their consumption of plant material to balance reductions in plant N concentrations at elevated CO 2 . The leaf biomass, leaf area and amount of nitrogen consumed of the other three plant species did not differ significantly between the two CO 2 treatments. Helix pomatia that fed on plants grown at elevated CO 2 atmosphere showed a larger increase in relative wet weight than those that fed on plants from ambient CO 2 conditions. However, the weight gain of H. pomatia was poorly correlated with amount of plant tissue consumed, so we suggest that the effect of CO 2 on weight gain in H. pomatia was due to a change in the quality of T. repens leaves.

Characteristics of haemolymph of Helix pomatia from different districts of central Europe

Haemolymph properties of garden snail (Helix pomatia) — a species currently used in ecotox‐icological studies for bioindication — were studied during vegetation of 1995. The snail specimens came from the Hercynian District of Bohemia, the Pontico‐Pannonian District of southern Moravia (both in Czech Republic) and from the Alps near Lunz am See (Austria). Total impact of heavy metals (Cd, Pb, Cu, Zn) on garden snail and, in some biochemical parameters of its haemolymph (contents of total protein, glucose and urea and activity of five so called indicator enzymes — AST, ALT, LDH, CHE, AP), were monitored. Snails from the area of Bohemian Hercynics showed significantly increased values of Pb and urea contents — contrary to, snails from the Alps, which exhibited a significantly increased Cd content as well as raised activity of two enzymes (ALT and CHE). Comparatively low value were found in snails from southern Moravia.

Approach to the use of immobilized sulfatase for analytical purposes and for the production of desulfo-glucosinolates

A commercial crude sulfatase from snail ( Helix pomatia) was purified by ion exchange, affinity and FPLC gel-filtration chromatography. The enzyme isolated, which was ca. 37-fold more active than the commercial one, was covalently immobilized on nylon 6.6 by the cross linking method. Immobilized sulfatase was used to produce some desulfo-glucosinolates (DSGLs) on the gram-scale, starting from glucotropaeolin, glucoraphenin and epi-progoitrin, and for HPLC analyses of glucosinolates (GLs) contained in cruciferous material and rapeseed in particular. The immobilized enzyme, as well as allowing the standardization of the HPLC method for GLs analysis, seems to be an efficient system for producing DSGLs which are interesting starting materials to prepare bio-active structures through further chemical modifications.

Odor-taste associative learning of the slug brain: Modulation by gustatory input of the activity in the procerebrum

Odor input evokes characteristic, time-evolving (non-stationary) events in the spontaneously active central ganglia of the snail Helix pomatia. Assuming stationarity for the signals, one could, as the first approach, apply the Fourier-based methods, frequency amplitude characteristics (FAC) measures, for analyzing such events. We could thus for the first time describe such events in frequency and amplitude and show that the frequency, at which power increases most, is specific to the odor or its class [Comp. Biochem. Physiol. 123A (1999a) 95; Comp. Biochem. Physiol. 124A (1999b) 297]. Wavelet tools assume no record stationarity and are suitable for describing the dynamically evolving brain electrical signals precisely and quantitatively. We, therefore, tested these tools for the typical odor experiments with the procerebrum (PC), the pedal ganglion (PG) and the visceral ganglion (VG) of the Helix, which we earlier analyzed by the FAC measures and compared both results. The two basic findings of the present wavelet analysis are as follows: (i) the wavelet energy fluctuations clearly visualize dynamical interactions among the major bands (0.1–3.1 Hz), implying a possible ‘mutual exclusion’ between slow components <0.8 Hz and faster ones >0.8 Hz. (ii) Entropy behavior was characteristically different for each of the three brain regions. Only in PC the response to aversive odorants (decrease of entropy=more ordered state) is differentiated in entropy from that to attractive ones (increase of entropy=more disordered or more complexly ordered state) indicating the odor-discriminating function of this region. In VG entropy of the intrinsic activity is so high (highly disordered state) due to the strong wideband activity reaching >50 Hz that odor stimulation results mainly in lowering of entropy (=more ordered state) regardless of the nature of the odor. In PG, however, odor presentation generally increases entropy due to the robust, wide-band activation at >3 Hz (sensorimotor function) that is generated as a secondary, but dominant and robust, response. In respect to describing time evolution of different frequency band components the present wavelet tools can much more sensitively do so, as compared with the FAC measures. They can also characterize a change in the neuroelectrical state in terms of entropy.

Biochemical characterization and histochemical localization of nitric oxide synthase in the nervous system of the snail, Helix pomatia.

Nitric oxide synthase (NOS) in the snail Helix pomatia was characterized by biochemical and molecular biological techniques and localized by histochemical methods. Central ganglia contained particulate paraformaldehyde-sensitive and cytosolic paraformaldehyde-insensitive NADPH-diaphorase. The cytosolic NADPH-diaphorase activity coeluted with NOS activity. The activity of NOS was dependent on Ca2+ and NADPH and was inhibited by N(G)-nitro-L-arginine (L-NNA). Proteins purified by 2',5'-ADP affinity chromatography were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and migrated at 150, 60, 40, and 30 kDa. An antibody to mammalian NOS exclusively labeled the 60-kDa protein. Characterization of the cDNA of the corresponding 60-kDa NOS-immunoreactive protein revealed no sequence homology with any known NOS isoform. The recombinant protein exhibited Ca2+- and NADPH-dependent NOS activity, which was partially inhibited by EGTA and L-NNA. Histochemistry showed NADPH-diaphorase activity in discrete regions of the central and peripheral nervous system. About 60% of the NADPH-diaphorase-positive neurons colocalize with immunoreactive material detected by antibodies to mammalian NOS. Comparison of organs showed the highest NADPH-diaphorase activity in the nervous system, whereas moderate activity was present in muscle tissue, digestive tract, and gonads. Our study suggests the presence of NOS and a putative NOS-associated/regulating protein in mollusk nervous tissue.