Melanogenesis in the ink gland of Sepia officinalis

Among the various melanin-producing systems, the ink gland of the cuttlefish (Sepia officinalis) has traditionally been regarded as a most convenient model system for the studies of melanogenesis. The ink gland is a highly specialized organ with immature cells in the inner portion, from where the cells gradually mature, migrate towards the outer portion of the gland and become competent to produce melanin giving rise to particulate melanosomes. When cell maturation is complete, melanin is secreted into the lumen of the gland, accumulated into the ink sac and ejected on demand. Biochemical studies carried out over the past two decades have shown that the ink gland contains a variety of melanogenic enzymes, including tyrosinase, a peculiar dopachrome rearranging enzyme (which catalyses the rearrangement of dopachrome to 5,6-dihydroxyindole) and a peroxidase (presumably involved in the later stages of melanin biosynthesis). These enzymes are functionally interactive in close subcellular compartments of ink gland cells and appear to act in a concerted fashion during the process of melanogenesis in the mature portion of the gland. More recent studies have revealed that ink production and ejection are affected and modulated by the N-methyl-D-aspartate (NMDA)-nitric oxide (NO)-cyclic GMP (cGMP) signalling pathway. Glutamate NMDA receptor and NO synthase, the enzyme responsible for the synthesis of NO, have been detected by biochemical and immunohistochemical techniques in immature ink gland cells. Stimulation of NMDA receptors caused a marked elevation of cGMP levels, activation of tyrosinase and increased melanin synthesis in the mature portion of the gland, via the NO-guanylyl cyclase interaction. This signalling is also present in different regions of the nervous system in Sepia and in certain neural pathways controlling contraction of the ink sac sphincters and wall muscle in the ejection mechanism. Overall, these and other findings allowed elaboration of an improved model of melanin formation in Sepia, which underscores the complex interplay of melanogenic enzymes and regulatory factors, highlighting both the similarities and the differences with melanogenesis in mammals.     

Nitric oxide synthase in the nervous system and ink gland of the cuttlefish Sepia officinalis: molecular cloning and expression

Nitric oxide (NO) signaling is involved in numerous physiological processes in mollusks, e.g., learning and memory, feeding behavior, neural development, and defence response. We report the first molecular cloning of NOS mRNA from a cephalopod, the cuttlefish Sepia officinalis (SoNOS). SoNOS was cloned using a strategy that involves hybridization of degenerate PCR primers to highly conserved NOS regions, combined with RACE procedure. Two splicing variants of SoNOS, differing by 18 nucleotides, were found in the nervous system and the ink gland of Sepia. In situ hybridization shows that SoNOS is expressed in the immature and mature cells of the ink gland and in the regions of the nervous system that are related to the ink defence system.     

Fine Structure of Melanogenesis in the Ink Sac of Sepia offidnalis

The ink sac epithelium of the cuttlefish Sepia officinalis was investigated by electron microscopy. Melanogenesis in a simplified view seems to follow the general scheme of melanin formation in vertebrates. First, a membrane-bound protein matrix is formed, which is called an early stage melanosome. The early stage melanosomes are more or less irregular in shape with a size up to 1.5 μm and contain membranous, granular, or vesicular material. They seem to originate from Golgi bodies and/or endoplasmic reticulum. Membranes that frequently are present in the early stage melanosomes may originate from fusion of vesicles or from incorporation of Golgi membranes into early stage melanosomes. Free cytoplasmic material or mitochondria probably are also incorporated into the early stage melanosomes or melanosomes. Therefore, the origin of the early stage melanosomes seems to be similar to that of autophagosomes. The early stage melanosomes mature to melanosomes in which several dozen melanin granules are formed. These melanosomes, at last, release the melanin granules together with other cellular material, including early stage melanosomes, into the lumen of the ink gland. This finding confirms the earlier postulated holocrine character of the release. Active tyrosinase was localized in the lumen of the ink sac as already shown by biochemical methods. There was also additional evidence that most of the material of broken down cells inside the lumen of the ink sac seems to be converted into melanin granules.     

Toxicity of melanin-free ink of Sepia officinalis to transformed cell lines: identification of the active factor as tyrosinase

The melanin-free ink of the cephalopod Sepia officinalis is shown to contain a heat labile proteinaceous component toxic to a variety of cell lines, including PC12 cells. Gel filtration chromatography indicated that the toxic component was concentrated in those fractions eluted at a molecular weight higher than 100 kDa and exhibiting the highest tyrosinase activity. SDS-PAGE analysis of the active fractions displayed a single major band migrating at an approximate molecular weight of 100 kDa, identical with that of the single tyrosinase band in the melanin-free ink. These data unambiguously demonstrated the identity of the toxic component with tyrosinase. Treatment of purified Sepia as well as of mushroom tyrosinase with an immobilized version of proteinase K resulted in a parallel loss of tyrosinase activity and cytotoxicity. Sepia apotyrosinase was ineffective in inducing cytotoxicity in PC12 cells. Purified Sepia tyrosinase was found to induce a significant increase in caspase 3 activity in PC12 cells, leading eventually to an irreversible apoptotic process. Overall, these results disclose a hitherto unrecognized property of tyrosinase that may lead to a reappraisal of its biological significance beyond that of a mere pigment producing enzyme.     

Melanin standard method: empirical formula.

Melanin isolated from the ink sac of cuttle fish (Sepia melanin) is a proposed standard for natural eumelanin. Sepia melanin isolated by a standard protocol was submitted for both elemental analysis and quantitative amino acid analysis. The contribution of the detected amino acids to the elemental composition is subtracted from the total elemental analysis, and the resultant elemental composition reflects the composition of the Sepia melanin backbone chromophore. The assumption is made that, for eumelanins, there is only one nitrogen atom per monomeric unit, and thus, the empirical formula for the average monomeric Sepia melanin backbone chromophore was determined. Three key parameters can be determined for any melanin sample; namely, the molar C/N for the average monomeric unit, the formula weight of the average monomeric unit, and the total percent composition of amino acid residues. Three commonly used melanin preparations, namely, natural Sepia melanin, melanin prepared by the in vitro tyrosinase catalyzed polymerization of tyrosine (tyrosine-enzymatic melanin), and a polymer synthesized by the peroxide oxidative polymerization of tyrosine (tyrosine-chemical melanin), have been subjected to this standard method of characterization. Tyrosine-enzymatic and Sepia melanin are quite similar and tyrosine-chemical melanin is fundamentally different from the other two melanins.     

The effect of preparation procedures on the morphology of melanin from the ink sac of Sepia officinalis

The structure of melanin extracted from the ink sac of the cuttlefish Sepia officinalis was examined for different methods of isolation and purification of the pigment. Scanning electron microscopy (SEM) images of Sepia eumelanin prepared by different procedures establish that multi-microm-sized aggregates reported by previous workers are generated by their sample preparation, and that the dominant constituents of Sepia melanin are approximately 150 nm spherical granules. Brunauer-Emmett-Teller (BET) measurements reveal that Sepia eumelanin from Sigma (prepared by spray drying the pigment) has a surface area of 14.3 m2/g. Pigment extracted directly from the fresh ink sac and then freeze-dried has a surface area of 21.5 m2/g, while CO2-supercritically dried has a surface area of 37.5 m2/g. This is consistent with SEM images showing that the process of freeze-drying produces aggregates, but to a lesser extent than spray drying. Supercritical drying of the sample produces suspensions of the individual approximately 150 nm granule, which is more reflective of the natural pigment. Brunauer-Emmett-Teller surface area analysis and Barrett-Joyner-Halenda (BJH) pore volume analysis indicate that the surface of the granules is not smooth and the interior of the granules is not porous, but rather the aggregates of granules are porous. Ultra-high resolution SEM and atomic force microscopy (AFM) images show the granules are easily deformed and are comprised of smaller constituents. De-aggregation of the granules by sonication and ultra-filtration reveal a range of structures depending on the pore size of the membrane used. The implications of these results on quantifying photochemical properties and kinetic reaction rate constants of melanin are discussed.     

Metal-ion interactions and the structural organization of Sepia eumelanin

The structural organization of melanin granules isolated from ink sacs of Sepia officinalis was examined as a function of metal ion content by scanning electron microscopy and atomic force microscopy. Exposing Sepia melanin granules to ethelenediaminetetraacetic acid (EDTA) solution or to metal salt solutions changed the metal content in the melanin, but did not alter granular morphology. Thus ionic forces between the organic components and metal ions in melanin are not required to sustain the natural morphology once the granule is assembled. However, when aqueous suspensions of Sepia melanin granules of varying metal content are ultra-sonicated, EDTA-washed and Fe-saturated melanin samples lose material to the solution more readily than the corresponding Ca(II) and Mg(II)-loaded samples. The solubilized components are found to be 5,6-dihydroxyindole-2-carboxylic acid (DHICA)-rich constituents. Associated with different metal ions, Na(I), Ca(II) and Mg(II) or Fe(III), these DHICA-rich entities form distinct two-dimensional aggregation structures when dried on the flat surface of mica. The data suggest multiply-charged ions play an important role in assisting or templating the assembly of the metal-free organic components to form the three-dimensional substructure distributed along the protein scaffold within the granule.     

Control of GnRH expression in the olfactory lobe of Octopus vulgaris

In the cephalopod mollusk Octopus vulgaris, the gonadotropic hormone released by the optic gland controls sexual maturity. Several lobes of the central nervous system control the activity of this gland. In one of these lobes, the olfactory lobe, a gonadotropin releasing hormone (GnRH) neuronal system has been described. We assume that several inputs converge on the olfactory lobes in order to activate GnRH neurons and that a glutamatergic system mediates the integration of stimuli on these neuropeptidergic neurons. The presence of N-methyl-d-aspartate (NMDA) receptor immunoreactivity in the neuropil of olfactory lobes and in the fibers of the optic gland nerve, along with the GnRH nerve endings strongly supports this hypothesis. A distinctive role in the control of GnRH secretion has also been attributed, in vertebrates, to nitric oxide (NO). The lobes and nerves involved in the nervous control of reproduction in Octopus contain nitric oxide synthase (NOS). Using a set of experiments aimed at manipulate a putative l-glutamate/NMDA/NO signal transduction pathway, we have demonstrated, by quantitative real-time PCR, that NMDA enhances the expression of GnRH mRNA in a dose-response manner. The reverting effect of a selective antagonist of NMDA receptors (NMDARs), 2-amino-5-phosphopentanoic acid (D-APV), confirms that such an enhancing action is a NMDA receptor-mediated response. Nitric oxide and calcium also play a positive role on GnRH mRNA expression. The results suggest that in Octopusl-glutamate could be a key molecule in the nervous control of sexual maturation.     

Melanin standard method: particle description.

Melanin isolated from the ink sac of Sepia officinalis (Sepia melanin) has been proposed as a standard for natural eumelanin. There are no standard methods for the isolation, purification, and storage of melanins. Mild methods designed to preserve the native composition and structure of melanin are needed. The specific aim of the present work, using Sepia melanin, was to develop a mild and generally applicable protocol for the isolation and purification of melanins. It is well established that melanin polymers contain a large number of free carboxylic acid residues. These anionic residues are responsible for the cation exchange properties observed for melanins. Heating melanins with hydrochloric acid at reflux has been demonstrated to lead to extensive decarboxylation. Indeed, heat alone has been shown to cause decarboxylation, and care must be exercised to avoid such conditions. By analogy with cation exchange resins, melanins should be isolated and named according to the associated counterion (e.g., Sepia melanin--K+ form). The method reported here avoided extremes in pH and temperature, and was designed to yield melanin in the K+ form. Physical disaggregation of particulate melanin using a wet milling step was also found to facilitate removal of significant quantities of adsorbed protein. The following physical parameters were used to monitor the purification and to characterize the resultant melanin: pH, conductance, particle size, and diffuse reflectance spectroscopy.     

In Vivo NO/cGMP Signalling in the Hippocampus

In the hippocampus of freely-moving rats, basal extracellular levels of cGMP are inhibited by L-NARG or ODQ whereas they are increased by NO donors or phosphodiesterase inhibitors. Activation of NMDA receptors also augments cGMP dialysate levels in a MK-801 and L-NARG sensitive manner, an effect dramatically diminished during ageing. Experiments with AMPA, AMPA receptor antagonists and cyclothiazide revealed complex relationships with GABAergic circuits that potently control the NO/cGMP pathway. Furthermore, the activity of this neurochemical cascade is also modulated by hippocampal nicotinic receptors via enhancement of endogenous glutamate release and stimulation of NMDA receptors. From a behavioural point of view, increased hippocampal excitation leads to the appearance of epileptic-like manifestations that, however, seem unrelated to the increase of NO/cGMP formation.     

A Calcium/Calmodulin-Dependent Nitric Oxide Synthase, NMDAR2/3 Receptor Subunits, and Glutamate in the CNS of the Cuttlefish Sepia officinalis

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 N(G)-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 micromol of glutamate/mg of protein; optic lobes: 0.22 +/- 0.04; vertical complex: 0.65 +/- 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.     

Nitric oxide: a vasodilatatory mediator in the cephalic aorta of Sepia officinalis (L.) (Cephalopoda)

Evidence is presented that nitric oxide (NO) may regulate blood pressure in cephalopod molluscs. In vitro tests performed on the cephalic aorta of Sepia officinalis (L.) (Cephalopoda) showed that the NO releasers (glyceroltrinitrate, sodium nitroprusside, 3-morpholinylsydnoneimine chloride and KNO(2)) induced concentration-dependent vasodilatation of vessel segments (without the tunica adventitia/periadventitia) precontracted by dopamine. These vasodilatatory actions could be totally blocked by oxadiazolo[4,3-a] quinoxalin-1-one, an inhibitor of the NO-sensitive guanylyl cyclase, and partially mimicked by the cyclic guanosine monophosphate (cGMP) analogue 8-bromo cGMP and by the phosphodiesterase inhibitor, zaprinast. The NO-precursor, L-arginine, showed vasodilatatory effects only on segments of the aorta in which the layers containing nerves (tunica adventitia/periadventitia) had been left intact, suggesting that NO synthase may be located within peripheral nerves.     

NMDA receptor stimulation induces temporary alpha-tubulin degradation signaled by nitric oxide-mediated tyrosine nitration in the nervous system of Sepia officinalis

Biochemical and immunohistochemical evidence is reported, showing basal protein nitration in specific regions of the optic lobes of Sepia officinalis, mainly in the fiber layers of the plexiform zone. SDS-PAGE analysis of optic lobe extracts revealed an intense 3-nitrotyrosine immunoreactive band identified as alpha-tubulin by immunoprecipitation and partial purification. Stimulation of NMDA receptors resulted in a selective decrease in alpha-tubulin levels within 30 min with partial recovery after 4 h. The effect was suppressed by the NO synthase (NOS) inhibitor L-nitroarginine. Incubation of optic lobes with free 3-nitrotyrosine resulted likewise in a selective loss of alpha-tubulin, due apparently to incorporation of the amino acid into the C-terminus of detyrosinated alpha-tubulin to give the nitrated protein purportedly more susceptible to degradation. Overall, these results point to a novel potential physiologic role of NO and free 3-nitrotyrosine in the control of the alpha-tubulin tyrosination/detyrosination cycle and turnover in Sepia nervous tissue.     

Optic gland implants and their effects on the gonads of Octopus.

1. Optic glands transplanted from one Octopus vulgaris into another cause enlargement of the gonads and ducts of the recipient. 2. Enlargement occurs whether or not the gland was secreting when implanted and regardless of the sex of the donor or recipient. 3. Glands derived from Eledone moschata or Octopus macropus implanted into O. vulgaris are as effective as glands derived from O. vulgaris. 4. Implants derived from Sepia officinalis or Loligo vulgaris appear to be ineffective.     

Effect of nitric oxide production on the redox modulatory site of the NMDA receptor-channel complex.

Nitric oxide (NO) is an important messenger both systemically and in the CNS. In digital Ca2+ imaging and patch-clamp experiments, clinically available nitroso compounds that generate NO are shown to inhibit responses mediated by the NMDA subtype of the glutamate receptor on rat cortical neurons in vitro. A mechanism of action for this effect was investigated by using the specific NO-generating agent S-nitrosocysteine. We propose that free sulfhydryl groups on the NMDA receptor-channel complex react to form one or more S-nitrosothiols in the presence of NO. If vicinal thiol groups react in this manner, they can form a disulfide bond(s), which is thought to constitute the redox modulatory site of the receptor, resulting in a relatively persistent blockade of NMDA responses. These reactions with NO can afford protection from NMDA receptor-mediated neurotoxicity. Our results demonstrate a new pathway for NO regulation of physiological function that is not via cGMP, but instead involves reactions with membrane-bound thiol groups on the NMDA receptor-channel complex.     

Cyclic GMP and Nitric Oxide Synthase in Aging and Alzheimer's Disease

Cyclic guanosine monophosphate (cGMP) is an important secondary messenger synthesized by the guanylyl cyclases which are found in the soluble (sGC) and particular isoforms. In the central nervous system, the nitric oxide (NO)-sensitive sGC isoform is the major enzyme responsible for cGMP synthesis. Phosphodiesterases (PDEs) are enzymes for hydrolysis of cGMP in the brain, and they are mainly isoforms 2, 5, and 9. The NO/cGMP signaling pathway has been shown to play an important role in the process underlying learning and memory. Aging is associated with an increase in PDE expression and activity and a decrease in cGMP concentration. In addition, aging is also associated with an enhancement of neuronal NO synthase, a lowering of endothelial, and no alteration in inducible activity. The observed changes in NMDA receptor density along with the Ca²⁺/NO/cGMP pathway underscore the lower synaptic plasticity and cognitive performance during aging. This notion is in agreement with last data indicating that inhibitors of PDE2 and PDE9 improve learning and memory in older rats. In this review, we focus on recent studies supporting the role of Ca²⁺/NO/cGMP pathway in aging and Alzheimer's disease.     

Up-regulation of tyrosinase gene by nitric oxide in human melanocytes

Ultraviolet light (UV) radiation causes skin-tanning, which is thought to be mediated by stimulating the release of melanogenic factors from keratinocytes as well as other cells. Nitric oxide (NO) has been reported to be generated after UV radiation and to stimulate melanocytes as one of the melanogens. In a previous experiment by another group on melanogenesis induced by NO, increases in both tyrosinase activity and tyrosinase protein levels were observed after daily stimulation of NO for 4 days. In the present study, we investigated tyrosinase gene expression within the first 24 hr of NO-induced melanogenesis. Tyrosinase mRNA expression was found to be induced 2 hr after a single treatment with S-nitroso-N-acetyl-L-arginine. An increase of tyrosinase activity was also detected time-dependently within the 24-hr period, accompanied by an increase of tyrosinase protein levels. The induction of mRNA expression was suppressed by a cyclic guanosine 3',5'-monophosphate (cGMP)-dependent protein kinase (cGMP/PKG) inhibitor. These results suggest that the enhancement of tyrosinase gene expression via the cGMP pathway may be a primary mechanism for NO-induced melanogenesis.