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Keywords:

  • Nitric oxide synthase;
  • Glutamate;
  • NMDA receptors;
  • CNS;
  • Sepia officinalis;
  • Inking system.

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. NOS assay
  5. Cyclic GMP determination
  6. RESULTS
  7. Biochemical characterization of neuronal NOS from Sepia officinalis
  8. NMDA receptors and L-glutamate in Sepia brain
  9. NMDA-induced cyclic GMP formation in intact Sepia brain
  10. NOS-like immunoreactivity in the CNS of Sepia officinalis
  11. NOS-like immunoreactivity : ink sac
  12. NMDAR2/3-like immunoreactivity : CNS
  13. NMDAR2/3-like immunoreactivity : ink sac
  14. DISCUSSION
  15. Acknowledgements
  16. REFERENCES

Abstract Chemical, biochemical, and immunohistochemical evidence is reported demonstrating the presence in the brain of the cuttlefish Sepia officinalis of a Ca2+-dependent nitric oxide synthase, NMDAR2/3 receptor subunits, and glutamate, occurring in neurons and fibers functionally related to the inking system. Nitric oxide synthase activity was concentrated for the most part in the cytosolic fraction and was masked by other citrulline-forming enzyme(s). The labile nitric oxide synthase could be partially purified by ammonium sulfate precipitation of tissue extracts, followed by affinity chromatography on 2′, 5′-ADP-agarose and calmodulin-agarose. The resulting activity, immunolabeled at 150 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis by antibodies to rat neuronal nitric oxide synthase, depended on NADPH and tetrahydro-L-biopterin, and was inhibited by NG-nitro-L-arginine. NMDAR2/3 subunit-immunoreactive proteins migrating at 170 kDa could also be detected in brain extracts, along with glutamate (whole brain : 0.32 ± 0.03 μmol of glutamate/mg of protein ; optic lobes : 0.22 ± 0.04 ; vertical complex : 0.06 ± 0.06 basal lobes : 0.58 ± 0.04 ; brachial lobe : 0.77 ± 0.06 ; pedal lobe : 1.04 ± 0.08 ; palliovisceral lobe : 0.86 ± 0.05). Incubation of intact brains with 1.5 mM glutamate or NMDA or the nitric oxide donor 2-(N,N-diethylamino)diazenolate-2-oxide caused a fivefold rise in the levels of cyclic GMP, indicating operation of the glutamate-nitric oxide-cyclic GMP signaling pathway. Immunohistochemical mapping of Sepia CNS showed specific localization of nitric oxide synthase-like and NMDAR2/3-like immunoreactivities in the lateroventral palliovisceral lobe, the visceral lobe, and the pallial and visceral nerves, as well as in the sphincters and wall of the ink sac.

Following identification of nitric oxide (nitrogen monoxide ; NO) as a central mediator in glutamate/NMDA-dependent excitatory neurotransmission, operation of this signaling pathway has been demonstrated in a variety of neural patterns in humans and nonhuman mammals (Garthwaite et al., 1988 ; Vincent, 1994 ; Garthwaite and Boulton, 1995). In postsynaptic terminals, binding of glutamate to NMDA receptors leads to Ca2+ influx, which activates a calmodulin-dependent neuronal isoform of nitric oxide synthase (NOS ; EC 1.14.23.39) (Garthwaite et al., 1989). The latter becomes engaged in a complex sequence of electron transfer processes that are mediated by NADPH, FMN, FAD, and tetrahydro-l-biopterin (BH4) (Bredt and Snyder, 1994 ; Knowles and Moncada, 1994 ; Mayer and Hemmens, 1997) and ultimately account for the production of NO via the oxygen-dependent conversion of l-arginine to l-citrulline.

Concomitant with studies on mammalian CNS, evidence has accumulated in recent years revealing the occurrence of NOS in the nervous systems of various invertebrates, thus indicating that the NO-signaling pathway is phyletically widespread and preserved throughout evolution. NADPH-dependent diaphorase histochemistry and/or immunohistochemistry has been used to localize NOS in neurons innervating the pharynx of the planarian (Eriksson, 1996) and in the brain of some Crustacea (Johansson and Mellon, 1998), Uniramia (Elphick et al., 1995 ; Stengl and Zintl, 1996), and various mollusks, including Limax maximus (Gelperin, 1994), Helix aspersa (Cooke et al., 1994), Pleurobranchaea californica (Moroz and Gillette, 1996), Helix pomatia (Huang et al., 1997), Lymnaea stagnalis (Moroz and Roylance, 1993 ; Moroz et al., 1993, 1994), Sepia officinalis (Chirchery and Chichery, 1994), Loligo beekeri (Kimura et al., 1997), and Octopus vulgaris (Robertson et al., 1997). Many efforts have been directed at highlighting the role of NO in the invertebrate CNS, in the activation of chemosensory processing, including olfactory and feeding behavior (Gelperin, 1994 ; Elphick et al., 1995 ; Moroz and Gillette, 1996 ; Colasanti et al., 1997 ; Bicker 1998 ; Johansson and Mellon, 1998 ; Korneev et al., 1998), and in visual and tactile learning (Robertson et al., 1994, 1995, 1996). In only a few cases, however, has a detailed biochemical characterization of the enzyme been provided (Elphick et al., 1993, 1995 ; Moroz et al., 1996 ; Colasanti et al., 1997 ; Huang et al., 1997), and attention has not been focused on the possible cooccurrence with glutamate receptors, which has somewhat limited appreciation of the role of the glutamate/NO signaling pathway in invertebrate neurochemistry.

Recently, we reported the occurrence of a Ca2+/calmodulin-dependent NOS as well as of NMDARI glutamate receptors in the immature cells of the ink gland of Sepia officinalis (Palumbo et al., 1997). This finding hints at a possible regulatory role of the glutamate-NO neurotransmission pathway on melanin synthesis and related processes associated with the inking mechanism of cephalopods. As an extension of that study, we report herein that a Ca2+/calmodulin-dependent NOS, as well as NMDAR2/3 receptors and glutamate, is distinctly present in certain specific CNS regions and nervous pathways of Sepia officinalis related to and/or controlling the ink defense system.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. NOS assay
  5. Cyclic GMP determination
  6. RESULTS
  7. Biochemical characterization of neuronal NOS from Sepia officinalis
  8. NMDA receptors and L-glutamate in Sepia brain
  9. NMDA-induced cyclic GMP formation in intact Sepia brain
  10. NOS-like immunoreactivity in the CNS of Sepia officinalis
  11. NOS-like immunoreactivity : ink sac
  12. NMDAR2/3-like immunoreactivity : CNS
  13. NMDAR2/3-like immunoreactivity : ink sac
  14. DISCUSSION
  15. Acknowledgements
  16. REFERENCES

Animals

Specimens of Sepia officinalis were collected in the bay of Naples and kept in a controlled environment for 2 days. Dissections were performed using cold anesthesia.

Antibodies

Polyclonal antibodies to rat neuronal NOS were generated by Primm (Milan, Italy) in rabbits by injecting an ovalbumin-conjugated synthetic peptide that corresponds to the unique C-terminal sequence (amino acid residues 1,414-1,429) of the rat NOS-I protein (Bredt et al., 1991). The antiserum was affinity-purified using the peptide conjugated to Sepharose 4B-CNBr (Pharmacia LKB Biotechnology, Uppsala, Sweden) and concentrated to give a stock solution of 1.3 mg of protein/ml. Rabbit anti-NMDAR1 and anti-NMDAR2/3 were obtained from Chemicon International Inc. (Temecula, CA, U.S.A.). For comparison, commercial polyclonal antibodies against neuronal NOS from Alexis Corp. (Laufelfingen, Switzerland) raised against an N-terminal peptide of rat brain NOS conjugated to keyhole limpet hemocyanin were also used.

Enzyme preparation

Brain and optic lobes were dissected and immediately frozen in plastic tubes kept on dry ice. NOS preparation was carried out essentially as described (Mayer et al., 1990). When necessary, the supernatant from ammonium sulfate treatment was passed through a prepacked, disposable PD-10 column of Sephadex G-25 (Amersham Pharmacia, Uppsala, Sweden) and eluted with triethanolamine buffer. In the case of brain, NOS enzyme was partially purified by 2′, 5′-ADP-agarose (Sigma-Aldrich, Milan, Italy) and calmodulin-agarose (Sigma-Aldrich) chromatography (Mayer et al., 1990 ; Schmidt et al., 1991). When necessary, cytosolic and microsomal fraction homogenates were separated by centrifugation at 100,000 g for 1 h. The particulate fraction was washed with 1 M KCl to remove loosely bound cytosolic proteins. Preparation of rat cerebellum extract was performed as previously described (Bredt and Snyder, 1990). Total protein concentration in tissues was determined using a Bio-Rad protein assay reagent (Bio-Rad, Milan, Italy) with bovine serum albumin as a standard.

NOS assay

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. NOS assay
  5. Cyclic GMP determination
  6. RESULTS
  7. Biochemical characterization of neuronal NOS from Sepia officinalis
  8. NMDA receptors and L-glutamate in Sepia brain
  9. NMDA-induced cyclic GMP formation in intact Sepia brain
  10. NOS-like immunoreactivity in the CNS of Sepia officinalis
  11. NOS-like immunoreactivity : ink sac
  12. NMDAR2/3-like immunoreactivity : CNS
  13. NMDAR2/3-like immunoreactivity : ink sac
  14. DISCUSSION
  15. Acknowledgements
  16. REFERENCES

Samples (250 μl) were incubated for 1 h at 37°C in a total volume of 500 μl of a 50 mM Tris-HCl, pH 7.6, buffer containing 4.2 μM L-[U-14C]arginine monohydrochloride (1,665,000 dpm) (Amersham Italia, Milan, Italy), 0.5 mM NADPH, 0.25 mM CaCl2, 5 μM FAD, 10 μg/ml calmodulin, and 10 μM BH4. Each reaction was stopped by the addition of 4.5 ml of ice-cold 100 mM HEPES buffer containing 4 mM EDTA at pH 5.5 and passed through a 4-ml Dowex 50 (Na+ form) column that retains arginine. The radiolabeled citrulline generated was eluted with water and quantified by both liquid scintillation counting and HPLC analysis after derivatization with o-phthaldialdehyde (Godel et al., 1984).

Glutamate determination

Whole brains, dissected brain regions, or slices of optic lobes cut on ice were immediately transferred into oxygenated sea water or calcium-free artificial sea water. The medium was changed every 15 min, and at fixed intervals of time the tissues were removed and homogenized in 10 volumes of 5% trichloroacetic acid. The homogenate was centrifuged at 10,000 g for 10 min, and glutamate concentration was determined by HPLC after derivatization with o-phthaldialdehyde, as detailed above.

Cyclic GMP determination

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. NOS assay
  5. Cyclic GMP determination
  6. RESULTS
  7. Biochemical characterization of neuronal NOS from Sepia officinalis
  8. NMDA receptors and L-glutamate in Sepia brain
  9. NMDA-induced cyclic GMP formation in intact Sepia brain
  10. NOS-like immunoreactivity in the CNS of Sepia officinalis
  11. NOS-like immunoreactivity : ink sac
  12. NMDAR2/3-like immunoreactivity : CNS
  13. NMDAR2/3-like immunoreactivity : ink sac
  14. DISCUSSION
  15. Acknowledgements
  16. REFERENCES

Intact brains were incubated at 20°C in sea water (2 ml) with gentle agitation in the presence of glutamate, NMDA, 2-(N,N-diethylamino)diazenolate-2-oxide (DEA/NO), or D(-)-2-amino-5-phosphonopentanoic acid (D-AP5) + NMDA, as indicated in Results. After incubation for appropriate intervals of time, the tissues were immediately frozen in liquid nitrogen and cyclic GMP levels were determined by an enzyme immunoassay system under nonacetylation conditions according to the manufacturer’s instructions (Amersham).

Immunoblotting and immunohistochemistry

Immunoblotting of NOS and NMDA receptors was carried out as previously described (Palumbo et al., 1997). For immunohistochemistry, tissues were fixed by immersion in Bouin’s liquid for 6 h at room temperature and embedded in paraffin (Paraplast). Serial sagittal sections (7 μm) were processed by indirect immunoperoxidase methods and were treated with 0.3% H2O2 to block the endogenous peroxidase. After a 20-min incubation with normal goat serum to reduce background staining, the sections were placed overnight in a moist chamber at 4°C with the primary antibodies : rabbit polyclonal anti-NOS (1:250), rabbit polyclonal anti-NMDAR1 (1:100), and rabbit polyclonal anti-NMDAR2/3 (1:100). Afterwards, the sections were incubated for 1 h at room temperature with a biotinylated goat anti-rabbit antibody (Pierce, Milan, Italy) diluted 1:200 in 0.1 M phosphate buffer, pH 7.4, containing 0.9% NaCl. The sections were incubated with an avidin-peroxidase conjugate (Pierce) for 1 h at 1:200 dilution. The sections were examined on a Zeiss Axioscope microscope. In control experiments, the NOS immunoreaction failed when the antibody was preabsorbed with synthetic peptide 1,414-1,429 conjugated to ovalbumin (10-6M). The NMDAR2/3 immunoreaction was also abolished when the primary antibody was omitted or when it was substituted by normal rabbit serum.

Statistical analysis

Data were analyzed by two-tailed Student’s t test and are given as means ± SD. The probability level accepted for significance was p < 0.05.

Biochemical characterization of neuronal NOS from Sepia officinalis

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. NOS assay
  5. Cyclic GMP determination
  6. RESULTS
  7. Biochemical characterization of neuronal NOS from Sepia officinalis
  8. NMDA receptors and L-glutamate in Sepia brain
  9. NMDA-induced cyclic GMP formation in intact Sepia brain
  10. NOS-like immunoreactivity in the CNS of Sepia officinalis
  11. NOS-like immunoreactivity : ink sac
  12. NMDAR2/3-like immunoreactivity : CNS
  13. NMDAR2/3-like immunoreactivity : ink sac
  14. DISCUSSION
  15. Acknowledgements
  16. REFERENCES

Throughout this study, the term brain will be used to indicate specifically the complex of supraesophageal mass and subesophageal mass, as distinct from the optic lobes.

A cytosolic NOS-like activity converting radiolabeled L-arginine to L-citrulline was detected in the homogenates from the brain and optic lobes of Sepia officinalis. When tested in the crude supernatant immediately after homogenate preparation, this activity (0.41 ± 0.05 pmol of citrulline/mg of protein/60 min, n = 4) proved unusually labile compared with the NOS from the ink gland (Palumbo et al., 1997) and rat cerebellum, even in the presence of protease inhibitors. Rapid removal of the protein fraction from the supernatant by ammonium sulfate precipitation (Mayer et al., 1990), however, allowed a substantial increase of recovered activity (~40-fold). The effects of various additives on enzyme activities determined in ammonium sulfate precipitates of extracts from brain and optic lobes are shown in Fig. 1. Citrulline formation was markedly reduced by Ca2+ chelation with EGTA, as well as by calmodulin antagonists N-(6-aminohexyl)-1-naphthalenesulfonamide (W5), N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide (W13), and, to a much lesser extent, by trifluoperazine (TFP). NG-Nitro-L-arginine methyl ester (L-NAME), NG-nitro-L-arginine (L-NA), and NG-nitro-D-arginine (D-NA) failed to affect the arginine-converting activity to any significant extent, whereas NG-methyl-L-arginine (L-NMA) efficiently suppressed L-citrulline formation. Notably, unexpectedly high activities were observed when brain and optic lobe preparations were tested in the absence of added NADPH and BH4, critical cofactors of mammalian NOS (Mayer and Werner, 1995 ; Mayer and Hemmens, 1997). Omission of NADPH in similar NOS assays in rat cerebellum extracts, taken as positive controls, reduced L-citrulline production to 3.7 ± 1.5% of the control value (three separate experiments).

image

Figure 1. L-[14C]Arginine-L-[14C]citrulline converting activities in ammonium sulfate precipitates from brain (shaded columns) and optic lobes (open columns) of Sepia officinalis. Activities are expressed as percentages of control values obtained from reaction mixtures containing all cofactors required for neuronal NOS activity (see Materials and Methods). -NADPH, without β-NADPH ; -Ca2+, Ca2+-free, EGTA (4 mM)-containing mixture ; -BH4, without BH4. Additive concentrations : W5, 0.5 mM ; W13, 0.5 mM ; TFP, 0.2 mM ; L-NAME, 0.5 mM ; L-NA, 0.5 mM ; D-NA, 0.5 mM ; L-NMA, 0.5 mM. Data are mean ± SD (bars) values of four separate experiments. **p < 0.01.

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To remove NADPH and other possible endogenous cofactors, the ammonium sulfate precipitate from brain extracts was chromatographed on a Sephadex G-25 column. However, the resulting citrulline-forming activity displayed only a low dependence on exogenous NADPH and BH4 (Fig. 2), suggesting the presence of NOS-independent, arginine-converting activities. Consistent with this view, L-ornithine and L-valine, specific inhibitors of arginase (Di Iulio et al., 1997 ; Giraldez and Zweier, 1998), and O-methylisourea, an arginine deiminase blocker (Smith et al., 1978), caused suppression of citrulline formation to a variable extent, whereas microsomal cytochrome P450 inhibitors, sodium cyanide and sodium azide, exerted little or no effect.

image

Figure 2. L-[14C]Arginine-L-[14C]citrulline converting activities in the ammonium sulfate precipitate from brain of Sepia officinalis after chromatography of the extract on a Sephadex G-25 column. Activities are expressed as percentages of control values obtained from reaction mixtures containing all cofactors required for neuronal NOS activity (see MATERIALS AND METHODS). -NADPH, without β-NADPH ; -BH4, without BH4. Additive concentrations : L-ornithine, 1.5 mM ; L-valine, 50 mM ; O-methylisourea, 5 mM ; sodium cyanide, 0.1 mM ; sodium azide, 0.1 mM. Data are mean ± SD (bars) values of four separate experiments. *p < 0.05 ; **p < 0.01.

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To purify the brain activity, the ammonium sulfate precipitate was chromatographed on 2′, 5′-ADP-agarose and the retained fraction was passed on calmodulinagarose (Schmidt et al., 1991). After the entire chromatographic sequence, only a low percentage of the maximal activity could be recovered in the absence of added NADPH and BH4, respectively (Fig. 3), and ~80% inhibition of citrulline formation was observed in the presence of L-NA. In some preparations, the degree of purification of the NOS was not satisfactory, as indicated by relatively high residual activities (45-61%) in the absence of added NADPH. In other cases, considerable losses of activity were observed, on account of a marked instability of the enzyme.

image

Figure 3. L-[14C]Arginine-L-[14C]citrulline converting activities in the partially purified NOS from brain of Sepia officinalis. The ammonium sulfate precipitate was purified by affinity chromatography on 2′,5′-ADP-agarose and calmodulin-agarose. Values of relative NOS activities are expressed as percentages of control values obtained from reaction mixtures containing all cofactors required for neuronal NOS activity (see MATERIALS AND METHODS). -NADPH, without β-NADPH ; -BH4, without BH4 ; -Ca2+, Ca2+-free, EGTA (4 mM)-containing mixture. Additive concentrations : L-NA, 0.5 mM ; W13, 0.5 mM. Data are mean ± SD (bars) values of three separate experiments. **p < 0.01.

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On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), an antibody prepared against a peptide from the C-terminal region of mammalian NOS (see MATERIALS AND METHODS) revealed a distinct NOS-immunoreactive protein in the purified fraction at 150 kDa (Fig. 4), close in size to rat brain NOS. The same band was also detected in the crude extract of brain and optic lobes. Immunoblotting experiments with a commercial polyclonal antibody raised against an N-terminal peptide from rat neuronal NOS failed to detect crossreactive material. PAGE analysis under nondenaturing conditions showed an immunoreactive band at 200 kDa (data not shown), similar to that from rat cerebellum. Analysis of the particulate fraction of Sepia brain showed that neuronal NOS was not present to any significance.

image

Figure 4. Western blot analysis of partially purified NOS from brain of Sepia officinalis after SDS-PAGE and immunolabeling with antibodies (1:500) prepared against a peptide from the C-terminal region of mammalian NOS (see Materials and Methods). Numbers on the left indicate the positions of prestained molecular mass markers shown as kilodaltons.

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NMDA receptors and L-glutamate in Sepia brain

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. NOS assay
  5. Cyclic GMP determination
  6. RESULTS
  7. Biochemical characterization of neuronal NOS from Sepia officinalis
  8. NMDA receptors and L-glutamate in Sepia brain
  9. NMDA-induced cyclic GMP formation in intact Sepia brain
  10. NOS-like immunoreactivity in the CNS of Sepia officinalis
  11. NOS-like immunoreactivity : ink sac
  12. NMDAR2/3-like immunoreactivity : CNS
  13. NMDAR2/3-like immunoreactivity : ink sac
  14. DISCUSSION
  15. Acknowledgements
  16. REFERENCES

SDS-PAGE analysis of extracts from Sepia brain revealed a single sharp band at 170 kDa immunoreactive to NMDAR2/3 receptor subunit antibodies (Fig. 5). A similar immunoreactive band was detectable in extracts from optic lobes. An exceedingly weak response ruled out the presence in the brain of the R1 subunit.

image

Figure 5. Western blot analysis of extracts from brain of Sepia officinalis after SDS-PAGE and immunolabeling with antibodies to NMDAR2/3 (1:500). Numbers on the left indicate the positions of prestained molecular mass markers shown as kilodaltons.

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L-Glutamate concentration in homogenates of whole brains, optic lobes, and selective areas of the brain of Sepia officinalis was then determined by HPLC. Mean values (in μmol of glutamate/mg of protein) were 0.32 ± 0.03 in whole brain, 0.22 ± 0.04 in optic lobes, 0.65 ± 0.06 in vertical complex, 0.58 ± 0.04 in basal lobes, 0.77 ± 0.06 in brachial lobe, 1.04 ± 0.08 in pedal lobe, and 0.86 ± 0.05 in palliovisceral lobe. Incubation in sea water at 20°C for 90 min resulted in a significant decrease in glutamate concentration : 0.16 ± 0.03 in whole brain, 0.13 ± 0.03 in optic lobes, 0.35 ± 0.04 in vertical complex, 0.33 ± 0.03 in basal lobes, 0.27 ± 0.04 in brachial lobe, 0.35 ± 0.02 in pedal lobe, and 0.45 ± 0.03 in palliovisceral lobe. Comparable glutamate decay profiles were observed following similar incubation of brains and optic lobes in Ca2+-free artificial sea water.

Antisera raised against glutamate (Ottersen and Strom-Mathisen, 1985) were used for immunohistochemical analysis of glutamate in brain specimens before and after incubation for 90 min in sea water and in Ca2+-free artificial sea water. Before incubation, a conspicuous glutamate-like immunoreactivity was found in the somata of positive neurons and in fibers, whereas after incubation the immunoreactivity remained in terminal endings and there was a considerable loss from cell bodies in both media (data not shown).

NMDA-induced cyclic GMP formation in intact Sepia brain

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. NOS assay
  5. Cyclic GMP determination
  6. RESULTS
  7. Biochemical characterization of neuronal NOS from Sepia officinalis
  8. NMDA receptors and L-glutamate in Sepia brain
  9. NMDA-induced cyclic GMP formation in intact Sepia brain
  10. NOS-like immunoreactivity in the CNS of Sepia officinalis
  11. NOS-like immunoreactivity : ink sac
  12. NMDAR2/3-like immunoreactivity : CNS
  13. NMDAR2/3-like immunoreactivity : ink sac
  14. DISCUSSION
  15. Acknowledgements
  16. REFERENCES

Exposure of intact brains to 1.5 mM glutamate, 1.5 mM NMDA, or 1.5 mM DEA/NO caused a rapid increase in the levels of cyclic GMP (Fig. 6), all peaking within 1 min and then rapidly dropping to basal values within 3 min. NMDA concentration was such as to induce a well detectable stimulatory effect, as determined in preliminary experiments, in which the compound was tested in the range of concentrations of 0.5-3.0 mM. Glutamate and DEA/NO were used at the same concentration for comparative purposes. Significant suppression of the activating effect of NMDA was observed in the presence of the NOS inhibitor L-NA, as well as the glutamate/NMDA antagonist D-AP5. A less pronounced inhibitory effect on NMDA-induced stimulation of cyclic GMP formation was observed in the presence of the less active analogue of L-NA, D-NA (Wang et al., 1999).

image

Figure 6. Time course of formation of cyclic GMP in Sepia brains. Brains were incubated at 20°C in sea water containing either of the following substances : NMDA (1.5 mM) (●) ; glutamate (1.5 mM) (▴) ; DEA/NO (1.5 mM) (▪) ; D-AP5 (10 mM) + NMDA (1.5 mM) (X). Brains were also preincubated for 30 min with 10 mM L-NA (♦) or 10 mM D-NA (□) and subsequently incubated with NMDA (1.5 mM). Cyclic GMP levels were determined in groups of organs (n = 3) collected at fixed intervals of time. All experiments were run in triplicate, and data represent the mean ± SD (bars) values.

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NOS-like immunoreactivity in the CNS of Sepia officinalis

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. NOS assay
  5. Cyclic GMP determination
  6. RESULTS
  7. Biochemical characterization of neuronal NOS from Sepia officinalis
  8. NMDA receptors and L-glutamate in Sepia brain
  9. NMDA-induced cyclic GMP formation in intact Sepia brain
  10. NOS-like immunoreactivity in the CNS of Sepia officinalis
  11. NOS-like immunoreactivity : ink sac
  12. NMDAR2/3-like immunoreactivity : CNS
  13. NMDAR2/3-like immunoreactivity : ink sac
  14. DISCUSSION
  15. Acknowledgements
  16. REFERENCES

A separate set of experiments was aimed at localizing NOS activity in the CNS of Sepia. The CNS of Sepia officinalis is complex and highly centralized and consists of brain and optic lobes. The brain is arranged around the esophagus with supraesophageal and subesophageal masses, which are connected laterally by a region that may be considered as periesophageal. Each of these parts is subdivided further into 38 lobes (Budelmann, 1995). The optic lobes are the largest lobes of the CNS and are connected with brain on either side of the periesophageal parts by short optic tracts. On each optic tract, there are three structures, i.e., the olfactory lobe, the peduncle lobe, and the optic glands. In the present study, the interest was focused on those nervous lobes situated in the subesophageal mass that are specifically involved in the control of body movements and visceral functions.

NOS immunoreactive cells and fibers were widely distributed in all regions of the palliovisceral lobe, i.e., the posterior subesophageal mass. Immunopositive neurons and fibers were observed, particularly, in the lateroventral palliovisceral lobe. These immunoreactive cells have a diameter of ~10-20 μm and are spherical with large nuclei and little cytoplasm. They are localized in the inner region of the lobe and send their immunopositive axons into the neuropil of the central palliovisceral lobe (Fig. 7A), where they join immunoreactive fibers arising from small neurons of the inner region of the palliovisceral lobe. These fibers enter the pallial and visceral nerves and leave the subesophageal mass. In the visceral lobe, numerous immunopositive neurons form a thick wall of small cells. These are spherical or polygonal in shape, with a diameter of ~10-15 μm (Fig. 7B), and send their positive axons into the neuropil at the back of the posterior subesophageal mass. This region provides the roots of the visceral nerves that are seen as immunopositive. No positivity was found in the larger neurons.

image

Figure 7. NOS-like immunoreactivity in the palliovisceral lobe and the ink sac. A : Transverse section of palliovisceral lobe showing the region of the lateroventral palliovisceral lobe. Small immunoreactive spherical neurons (large arrows) send their immunopositive axons into the neuropil of the central palliovisceral lobe (small arrows). B : Transverse section of the visceral lobe. Polygonal immunopositive neurons are seen (large arrows). The immunoreactive axons (small arrows) provide the roots of the visceral nerves. Note the absence of immunopositivity in the larger neurons (asterisk). C : Transverse section of the sphincter region of the ink sac wall. Immunoreactive nerve endings make contacts with outer circular muscle layers (arrows). D : Transverse section of the ink sac wall showing immunoreactive fibers that make contacts with the trunks of the longitudinal muscle layers (arrows). Scale bars = 25 μm (A), 15 μm (B), and 5 μm (C and D).

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NOS-like immunoreactivity : ink sac

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. NOS assay
  5. Cyclic GMP determination
  6. RESULTS
  7. Biochemical characterization of neuronal NOS from Sepia officinalis
  8. NMDA receptors and L-glutamate in Sepia brain
  9. NMDA-induced cyclic GMP formation in intact Sepia brain
  10. NOS-like immunoreactivity in the CNS of Sepia officinalis
  11. NOS-like immunoreactivity : ink sac
  12. NMDAR2/3-like immunoreactivity : CNS
  13. NMDAR2/3-like immunoreactivity : ink sac
  14. DISCUSSION
  15. Acknowledgements
  16. REFERENCES

In the wall of the ink sac, immunoreactive fibers, arising from the visceral nerve, give off a branch, the ink sac nerve, that reaches the ink sac duct and splits up, sending branches to the anterior and posterior regions of the wall of the ink sac. Most of these fibers contact the outer circular muscle layer in the regions of the anterior and posterior sphincters (Fig. 7C), and only some positive fibers are seen in the inner longitudinal muscle layer. All along the wall of the ink sac, below the sphincters, immunoreactive fibers make contact with the trunks of the longitudinal muscle layer (Fig. 7D). No immunopositivity was observed in the circular muscle layer of the wall of the ink sac.

NMDAR2/3-like immunoreactivity : CNS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. NOS assay
  5. Cyclic GMP determination
  6. RESULTS
  7. Biochemical characterization of neuronal NOS from Sepia officinalis
  8. NMDA receptors and L-glutamate in Sepia brain
  9. NMDA-induced cyclic GMP formation in intact Sepia brain
  10. NOS-like immunoreactivity in the CNS of Sepia officinalis
  11. NOS-like immunoreactivity : ink sac
  12. NMDAR2/3-like immunoreactivity : CNS
  13. NMDAR2/3-like immunoreactivity : ink sac
  14. DISCUSSION
  15. Acknowledgements
  16. REFERENCES

Intense NMDAR2/3-like immunoreactivity was found in the palliovisceral lobe, overlapping that of NOS to some extent. In the lateroventral palliovisceral lobe, the immunopositive cells are localized in the inner region, as observed in the case of NOS-like immunoreactivity. These neurons are small in size with a diameter of ~10-20 μm (Fig. 8A). Few immunopositive fibers were detected in the neuropil. These fibers extended to the neuropil of the central palliovisceral lobe, and some of them entered the visceral nerves. In the visceral lobe, the immunopositive cells were small, with a diameter of ~10-15 μm, morphologically similar to those immunoreactive to NOS and widely scattered in the cortical cell layer of the lobe (Fig. 8B and C). The immunopositive axons enter the visceral nerves (Fig. 8B). No positivity was found in the larger neurons. The antibody against subunit 1 of the NMDA receptor did not show any positivity when tested on sections of the palliovisceral lobe.

image

Figure 8. NMDAR2/3-like immunoreactivity in the palliovisceral lobe and the ink sac. A : Transverse section of palliovisceral lobe showing the region of the lateroventral palliovisceral lobe. Spherical immunopositive neurons are seen (arrows). B : Transverse section of the visceral lobe. Immunopositive neurons (large arrows) send their immunopositive axons in the neuropil (small arrows) from which arise the visceral nerves. C : Transverse section of the visceral lobe showing the numerous immunopositive neurons (large arrows) and nerve endings on immunonegative neurons (small arrows). Spherical and polygonal neurons are seen. Note the absence of immunopositivity in the larger neurons (asterisk). D : Transverse section of the sphincter region of the ink sac wall. Immunoreactive nerve endings make contacts with inner longitudinal muscle layers (arrows). E : Transverse section of the ink sac wall showing immunoreactive fibers that make contacts with the trunks of the longitudinal muscle layers (arrows). Scale bars = 10 μm (A), 20 μm (B), 25 μm (C), and 5 μm (D and E).

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NMDAR2/3-like immunoreactivity : ink sac

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. NOS assay
  5. Cyclic GMP determination
  6. RESULTS
  7. Biochemical characterization of neuronal NOS from Sepia officinalis
  8. NMDA receptors and L-glutamate in Sepia brain
  9. NMDA-induced cyclic GMP formation in intact Sepia brain
  10. NOS-like immunoreactivity in the CNS of Sepia officinalis
  11. NOS-like immunoreactivity : ink sac
  12. NMDAR2/3-like immunoreactivity : CNS
  13. NMDAR2/3-like immunoreactivity : ink sac
  14. DISCUSSION
  15. Acknowledgements
  16. REFERENCES

In the regions of the anterior and posterior sphincters, antibodies to NMDAR2/3 labeled few fibers from the visceral nerves making contact with the outer circular muscle layer. Somewhat more intense and widely distributed immunoreactivity was detected in fibers in the inner longitudinal muscle layer (Fig. 8D). All along the wall of the ink sac and below the sphincters, NMDAR2/3-immunopositive fiber make contact with the trunks of the longitudinal muscle layer, in analogy with NOS-positive fibers (Fig. 8E). No NMDAR2/3 immunopositivity was observed in the circular muscle layer of the ink sac wall. The antibody against subunit 1 of the NMDA receptor did not show any positivity when tested on sections of the ink sac.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. NOS assay
  5. Cyclic GMP determination
  6. RESULTS
  7. Biochemical characterization of neuronal NOS from Sepia officinalis
  8. NMDA receptors and L-glutamate in Sepia brain
  9. NMDA-induced cyclic GMP formation in intact Sepia brain
  10. NOS-like immunoreactivity in the CNS of Sepia officinalis
  11. NOS-like immunoreactivity : ink sac
  12. NMDAR2/3-like immunoreactivity : CNS
  13. NMDAR2/3-like immunoreactivity : ink sac
  14. DISCUSSION
  15. Acknowledgements
  16. REFERENCES

In addition to escape jetting and coloration changes, the adaptive response of cephalopods to predation and other threatening situations relies on a peculiar inking system that has been the subject of considerable interest and controversy since antiquity. In the cuttlefish, Sepia officinalis, as well as in Octopus and Loligo, the ink is a suspension of brown-to-black, insoluble melanin, derived by tyrosinase-catalyzed oxidation of tyrosine (Prota, 1992), which the animal produces and accumulates in an unusual effector organ, the ink sac. The latter is situated on the underside of the visceral mass with its open end toward the funnel and comprises the ink gland, which is specifically responsible for the synthesis of the pigment, and a reservoir with sphincters, where the ink accumulates (Tompsett, 1939).

The results described in the present article, together with preliminary data from other studies in progress in our laboratories, indicate for the first time the occurrence of NOS and glutamate NMDAR2/3 receptors in those regions of the Sepia nervous system controlling the inking system and provide evidence for the existence of the excitatory glutamate/NO/cyclic GMP signaling pathway.

Comparative analysis of glutamate levels in selective areas of the brain revealed higher values in those lobes of the subesophageal mass involved in the control of the inking system. In all cases, glutamate occurred in two apparently distinct pools : one that is easily lost during incubation in sea water, and another, presumptively vesicular, that is more strongly retained and occurs in high concentrations in the palliovisceral lobe. A detailed investigation of the mechanisms underlying the observed loss of metabolic glutamate was out of the scope of the present study. Within the limitations of the approach used, it appeared that the phenomenon was independent of the presence of Ca2+ in the incubation medium, reflecting conceivably higher rates of efflux and washout from perikarya than from synaptic vesicles (Ottersen and Storm-Mathisen, 1985).

A comparison of the properties of Sepia neuronal NOS with those of NOS activities from other invertebrates and rat cerebellar NOS is shown in Table 1. These data point to a considerable diversity of structural and biochemical properties represented by neuronal NOS activities in the CNS of invertebrates, which might entail more or less subtle functional variations of possible evolutionary significance. The presence of relatively high levels of NADPH-independent citrulline-producing enzymes accounts for most of the difficulties encountered during purification of Sepia neuronal NOS. The effects of specific inhibitors of arginine-converting, citrulline-forming enzymes suggest that arginase may contribute to the overall activity in Sepia brain, whereas cytochrome P450-like enzymes do not seem to be involved. Based on the extent of inhibitory effects, moreover, it is possible that additional unidentified enzyme activities (e.g., arginine deiminase) account for arginine conversion under the specific conditions of the NOS assay, a problem that is not encountered with mammalian brain NOS.

Table 1. Comparison of properties of neuronal NOS from Sepia, other invertebrates, and rat cerebellum
 SepiaHelixaPleurobrancheabSchistocercac,eDrosophiladHydrafDugesiagRat h,i
  1. aHuang et al. (1997) ;

  2. bMoroz et al. (1996) ;

  3. cElphick et al. (1995) ;

  4. dRegulski and Tully (1995) ;

  5. eElphick et al. (1993) ;

  6. fColasanti et al. (1997) ;

  7. gEriksson (1996) ;

  8. hBredt and Snyder (1990) ;

  9. iSchmidt et al. (1991).

Elution in 2′,5′-ADP-agarose 10 mM NADPH 10 mM NADPH 10 mM NADPH c 10 mM NADPH h
Molecular mass (kDa, native protein)200250152 d 200h, 279 i
Molecular mass (kDa, denatured protein)15060135 c 150h, 155 i
Ca2+ -dependentYesYesNoYes eYesNoYes h
Calmodulin-dependentYesYesYes eNoNoYes h
Inhibited by l-NAME, l-NA, or l-NMA YesYesYesYes cYesYesYes h

Apart from the biochemical interest connected with arginine metabolism in Sepia brain, these results provide caveats about possible pitfalls in NOS determination in invertebrates. Use of the radiolabeled arginine-conversion assay in the presence of other citrulline-forming enzymes may lead to overestimation, or even confusion, regarding authentic NOS activity (Di Iulio et al., 1997 ; Choi et al., 1998 ; Giraldez and Zweier, 1998 ; Wu and Morris, 1998), an issue that seems of particular relevance to the field of invertebrate NOS. For example, a substantial retention of activity in the absence of added NADPH has been reported for enzymes from Helix pomatia (67%) (Huang et al., 1997), Pleurobranchaea (40%) (Moroz et al., 1996), insects (80%) (Elphick et al., 1993), and Sepia ink gland (nearly 100%) (Palumbo et al., 1997). This behavior was ascribed to the presence of sufficient levels of endogenous NADPH in the tissue extracts, because in neither case was the enzyme purified to homogeneity. In view of our results, an alternative interpretation seems likely, in which prominent NADPH-independent l-citrulline-forming activities coexist with neuronal NOS and interfere with its determination.

It may also be relevant to notice, in this connection, that whereas in mammalian CNS NOS immunohistochemistry and NADPH-diaphorase histochemistry label identical neurons (Dawson et al., 1991 ; Saffrey et al., 1992), in invertebrate CNS evidence for a colocalization of NOS immunoreactivity and NADPH-diaphorase activity is largely incomplete and often missing. In Helix aspersa, for example, NADPH-diaphorase positive neurons do not exhibit NOS immunoreactivity at all (Cooke et al., 1994), and in Helix pomatia the cellular localization of NADPH-diaphorase activity and NOS-immunoreactive material is not identical (Huang et al., 1997). In Lymnaea stagnalis, moreover, Moroz et al. (1994) found fewer NOS-immunoreactive neurons with respect to those labeled by NADPH-diaphorase histochemistry, although a good correlation between NADPH-diaphorase staining and nitrate and nitrite levels in single neurons of Pleurobranchaea californica was taken to support the correspondence between NADPH-diaphorase and NOS activity (Cruz et al., 1997). The same uncertainty applies to Sepia and other cephalopods (Chichery and Chichery, 1994 ; Kimura et al., 1997 ; Robertson et al., 1997). Especially relevant is the reported lack of NADPH-diaphorase-positive reactions in the NOS-immunoreactive neurons of the subesophageal mass of Sepia officinalis, in contrast with an intense positive staining in the neuropils of the spines of the peduncle and the posterior and anterior basal lobes (Chichery and Chichery, 1994), an observation that has been confirmed by us (data not shown). Explanations of these inconsistencies belong at present to the realm of conjecture, but could possibly rest on the low levels of NOS and NADPH-diaphorase activities in Sepia brain.

A comment is warranted regarding the use of antibodies to mammalian NOS for immunohistochemical analysis of invertebrate NOS. As biochemical investigations expand current knowledge of neuronal NOS in invertebrate CNS, structural differences from the mammalian counterpart(s) become increasingly evident, which would make immunoreactivity not entirely unambiguous, as properly pointed out by Huang et al. (1997). A note of caution is also suggested by the different immunolabeling of the two antibodies used in the present study, which can be explained in light of the closer similarities of the C-terminal regions in the various NOS isoforms compared with the N-terminal regions (Knowles and Moncada, 1994). Yet the observed association of the immunoreactive band at 150 kDa in SDSPAGE of the partially purified fraction with NOS activity, together with the size analogy with the rat brain 150-kDa NOS-immunoreactive material, offers a sufficient argument to correlate the 150-kDa immunoreactive protein in Sepia with NOS.

Apart from the first isolation and biochemical characterization of a neuronal NOS from a cephalopod, a major outcome of the present study is the demonstration of the localization of NOS-like and NMDAR2/3-like immunoreactivities in some brain regions specifically responsible for the control of the inking system. The possible functional and behavioral implications of this finding are currently under investigation in our laboratories.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. NOS assay
  5. Cyclic GMP determination
  6. RESULTS
  7. Biochemical characterization of neuronal NOS from Sepia officinalis
  8. NMDA receptors and L-glutamate in Sepia brain
  9. NMDA-induced cyclic GMP formation in intact Sepia brain
  10. NOS-like immunoreactivity in the CNS of Sepia officinalis
  11. NOS-like immunoreactivity : ink sac
  12. NMDAR2/3-like immunoreactivity : CNS
  13. NMDAR2/3-like immunoreactivity : ink sac
  14. DISCUSSION
  15. Acknowledgements
  16. REFERENCES
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