Localization and expression of molt-inhibiting hormone and nitric oxide synthase in the central nervous system of the green shore crab, Carcinus maenas, and the blackback land crab, Gecarcinus lateralis.

In decapod crustaceans, molting is controlled by the pulsatile release of molt-inhibiting hormone (MIH) from neurosecretory cells in the X-organ/sinus gland (XO/SG) complex in the eyestalk ganglia (ESG). A drop in MIH release triggers molting by activating the molting gland or Y-organ (YO). Post-transcriptional mechanisms ultimately control MIH levels in the hemolymph. Neurotransmitter-mediated electrical activity controls Ca2+-dependent vesicular release of MIH from the SG axon terminals, which may be modulated by nitric oxide (NO). In green shore crab, Carcinus maenas, nitric oxide synthase (NOS) protein and NO are present in the SG. Moreover, C. maenas are refractory to eyestalk ablation (ESA), suggesting other regions of the nervous system secrete sufficient amounts of MIH to prevent molting. By contrast, ESA induces molting in the blackback land crab, Gecarcinus lateralis. Double-label immunofluorescence microscopy and quantitative polymerase chain reaction were used to localize and quantify MIH and NOS proteins and transcripts, respectively, in the ESG, brain, and thoracic ganglion (TG) of C. maenas and G. lateralis. In ESG, MIH- and NOS-immunopositive cells were closely associated in the SG of both species; confocal microscopy showed that NOS was localized in cells adjacent to MIH-positive axon terminals. In brain, MIH-positive cells were located in a small number of cells in the olfactory lobe; no NOS immunofluorescence was detected. In TG, MIH and NOS were localized in cell clusters between the segmental nerves. In G. lateralis, Gl-MIH and Gl-crustacean hyperglycemic hormone (CHH) mRNA levels were ~105-fold higher in ESG than in brain or TG of intermolt animals, indicating that the ESG is the primary source of these neuropeptides. Gl-NOS and Gl-elongation factor (EF2) mRNA levels were also higher in the ESG. Molt stage had little or no effect on CHH, NOS, NOS-interacting protein (NOS-IP), membrane Guanylyl Cyclase-II (GC-II), and NO-independent GC-III expression in the ESG of both species. By contrast, MIH and NO receptor GC-I beta subunit (GC-Iß) transcripts were increased during premolt and postmolt stages in G. lateralis, but not in C. maenas. MIH immunopositive cells in the brain and TG may be a secondary source of MIH; the release of MIH from these sources may contribute to the difference between the two species in response to ESA. The MIH-immunopositive cells in the TG may be the source of an MIH-like factor that mediates molt inhibition by limb bud autotomy. The association of MIH- and NOS-labeled cells in the ESG and TG suggests that NO may modulate MIH release. A model is proposed in which NO-dependent activation of GC-I inhibits Ca2+-dependent fusion of MIH vesicles with the nerve terminal membrane; the resulting decrease in MIH activates the YO and the animal enters premolt.

Distribution of NADPH-diaphorase activity in the central nervous system of the young and adult land snail Megalobulimus abbreviatus.

Nitric oxide (NO) is a gas produced through the action of nitric oxide synthase that acts as a neurotransmitter in the central nervous system (CNS) of adult gastropod mollusks. There are no known reports of the presence of NOS-containing neurons and glial cells in young and adult Megalobulimus abbreviatus. Therefore, NADPH-d histochemistry was employed to map the nitrergic distribution in the CNS of young and adult snails in an attempt to identify any transient enzymatic activity in the developing CNS. Reaction was observed in neurons and fibers in all CNS ganglia of both age groups, but in the pedal and cerebral ganglia, positive neurons were more intense than in other ganglia, forming clusters symmetrically located in both paired ganglia. However, neuronal NADPH-d activity in the mesocerebrum and pleural ganglia decreased from young to adult animals. In both age groups, positive glial cells were located beneath the ganglionic capsule, forming a network and surrounding the neuronal somata. The trophospongium of large and giant neurons was only visualized in young animals. Our results indicate the presence of a nitrergic signaling system in young and adult M. abbreviatus, and the probable involvement of glial cells in NO production.

Distribution and characterization of nitric oxide synthase in the nervous system of Triatoma infestans (Insecta: Heteroptera).

The biochemical characterization of nitric oxide synthase (NOS) and its distribution in the central nervous system (CNS) were studied in the heteropteran bug Triatoma infestans. NOS-like immunoreactivity was found in the brain, subesophageal ganglion, and thoracic ganglia by using immunocytochemistry. In the protocerebrum, NOS-immunoreactive (IR) somata were detected in the anterior, lateral, and posterior soma rinds. In the optic lobe, numerous immunostained somata were observed at the level of the first optic chiasma, around the lobula, and in the proximal optic lobe. In the deutocerebrum, NOS-IR perikarya were mainly observed in the lateral soma rind, surrounding the sensory glomeruli, and a few cell bodies were seen in association with the antennal mechanosensory and motor neuropil. No immunostaining could be detected in the antennal nerve. The subesophageal and prothoracic ganglia contained scattered immunostained cell bodies. NOS-IR somata were present in all the neuromeres of the posterior ganglion. Western blotting showed that a universal NOS antiserum recognized a band at 134 kDa, in agreement with the expected molecular weight of the protein. Analysis of the kinetics of nitric oxide production revealed a fully active enzyme in tissue samples of the CNS of T. infestans.

Nucleotidase activities in membrane preparations of nervous ganglia and digestive gland of the snail Helix aspersa.

Nucleotide-metabolizing enzymes play an important role in the regulation of nucleotide levels. In the present report, we demonstrated an enzyme activity with different kinetic properties in membrane preparations of the nervous ganglia and digestive gland from Helix aspersa. ATPase and ADPase activities were dependent on Ca2+ and Mg2+ with pH optima approximately 7.2 and between 6.0 and 8.0 in digestive gland and nervous ganglia, respectively. The enzyme activities present in membrane preparations of these tissues preferentially hydrolyzed triphosphate nucleotides. In nervous ganglia, the enzyme was insensitive to the classical ATPases inhibitors. In contrast, in digestive gland, N-ethylmaleimide (NEM) produced 45% inhibition of Ca(2+)-ATP hydrolysis. Sodium azide, at 100 microM and 20 mM, inhibited Mg(2+)-ATP hydrolysis by 36% and 55% in digestive gland, respectively. The presence of nucleotide-metabolizing enzymes in these tissues may be important for the modulation of nucleotide and nucleoside levels, controlling their actions on specific purinoceptors in these species.

Different sensitivity of Ca(2+)-ATPase and cholinesterase to pure and commercial pesticides in nervous ganglia of Phyllocaulis soleiformis (Mollusca).

We measured the effects in vitro of pure and commercial pesticides on Ca(2+)-activated ATPase and cholinesterase (ChE) activities in the nervous system of the slug Phyllocaulis soleiformis. The pesticides used in this study included carbamate and organophosphates, which acts as reversible and irreversible anticholinesterases, respectively. Both enzymes were insensitive to pure carbofuran (1 mM), glyphosate (1 mM) and malathion (120 microM). However, the carbamate carbofuran, in the commercial formulation Furandan 350S, inhibited ATPase and ChE activities. The organophosphate glyphosate used in the commercial preparation of Gliz 480CS inhibited ATPase activity and increased cholinesterase activity. These effects are likely due to the action of adjuvant substances of the chemical formulation. The commercial formulation (Malatol 500CE) did not alter enzymes activities. Our results suggest that cholinesterase present in the slug nervous tissue has a different behavior to those identified in vertebrate nervous tissue, since it was insensitive to pure compounds, known as anticholinesterases in vertebrates. Considering the insensitivity of the Ca(2+)-activated ATPase, we suggested that the purinergic neurotransmission and other roles of ATP might not be affected by the pure pesticides tested.

Some kinetic and toxicological characteristics of thoracic ganglia cholinesterase of Chasmagnathus granulata (Decapoda, Grapsidae).

In this study, some kinetic and toxicological parameters of the thoracic ganglia cholinesterase from the estuarine crab Chasmagnathus granulata were determined. Effects of the type and concentration of substrate, pH (6.80-8.50), incubation temperature (5-35 degrees C) and eserine on the enzyme activity were studied. Enzymatic activity was higher at pH 7.40 and 8.00 and significantly reduced at lower temperatures (5-10 degrees C). Employing acetylthiocholine iodide (ATCh) as substrate, the K(m) and Vmax were estimated as 0.28 mM and 1.75 mumol.mg protein-1.min-1, respectively. A 14.11 and 24.51% decrease in enzyme activity were registered at 4.62 and 9.24 mM of ATCh, respectively. Using propionylthiocholine iodide as substrate, the K(m) and Vmax were estimated as 0.16 mM and 0.91 mumol.mg protein-1.min-1, respectively. The IC50 for eserine was estimated as 5.3 x 10(-4) mM. The Ki estimated for eserine (8.10 mM-1.min-1) indicates that the thoracic ganglia cholinesterase from C. granulata showed a low ability to generate an irreversible enzyme-inhibitor complex. The higher enzymatic activity registered with ATCh and the enzyme inhibition observed at high concentration of this substrate, suggest that thoracic ganglia cholinesterase from C. granulata is an acetylcholinesterase.

Distribution of NADPH-diaphorase in the perioesophageal ganglia of the snail, Helix aspersa.

In this paper we discuss the anatomical localization of NADPH-diaphorase using Nitroblue tetrazolium in perioesophageal ganglia of Helix aspersa. Our results show that the reaction is present in neurons and fibers of the procerebrum, some positive neurons are found in mesocerebrum, and there were positive fibers in the neuropile of postcerebrum and mesocerebrum; likewise, immunopositive fibers were found in the neuropile of pedal, pleural and parietal ganglia. The presence of NADPH-diaphorase in the interneurons of procerebrum suggests the participation of this enzyme in the production of nitric oxide for the processing of the olfactory information, as has been suggested in mammalian olfactory tissue.