Oxidation chemistry of catecholamines and neuronal degeneration: an update.

Aberrant oxidative pathways of catecholamine neurotransmitters, i.e. dopamine and norepinephrine, are an important biochemical correlate of catecholaminergic neuron loss in some disabling neurodegenerative diseases of the elderly, notably Parkinson's disease. In an oxidative stress setting, under conditions of elevated lipid peroxidation, iron accumulation, impaired mitochondrial functioning and antioxidant depletion, catecholamines are oxidatively converted to the corresponding o-quinones, which may initiate a cascade of spontaneous reactions, including intramolecular cyclization, aminoethyl side chain fission and interaction with molecular targets. The overall outcome of the competing pathways may vary depending on contingent factors and the biochemical environment, and may include formation of nitrated derivatives, neuromelanin deposition, generation of chain fission products, conjugation with L-cysteine leading eventually to cytotoxic responses and altered cellular function. In addition, catecholamines may interact with products of lipid peroxidation and other species derived from oxidative breakdown of biomolecules, notably glyoxal and other aldehydes, leading e.g. to tetrahydroisoquinolines via Pictet-Spengler chemistry. After a brief introductory remark on oxidative stress biochemistry, the bulk of this review will deal with an overview of the basic chemical pathways of catecholamine oxidation, with special emphasis on the analogies and differences between the central neurotransmitters dopamine and norepinephrine. This chemistry will form the basis for a concise discussion of the latest advances in the mechanisms of catecholamine-associated neurotoxicity in neuronal degeneration.

Role of solvent, pH, and molecular size in excited-state deactivation of key eumelanin building blocks: implications for melanin pigment photostability.

Ultrafast time-resolved fluorescence spectroscopy has been used to investigate the excited-state dynamics of the basic eumelanin building block 5,6-dihydroxyindole-2-carboxylic acid (DHICA), its acetylated, methylated, and carboxylic ester derivatives, and two oligomers, a dimer and a trimer in the O-acetylated forms. The results show that (1) excited-state decays are faster for the trimer relative to the monomer; (2) for parent DHICA, excited-state lifetimes are much shorter in aqueous acidic medium (380 ps) as compared to organic solvent (acetonitrile, 2.6 ns); and (3) variation of fluorescence spectra and excited-state dynamics can be understood as a result of excited-state intramolecular proton transfer (ESIPT). The dependence on the DHICA oligomer size of the excited-state deactivation and its ESIPT mechanism provides important insight into the photostability and the photoprotective function of eumelanin. Mechanistic analogies with the corresponding processes in DNA and other biomolecules are recognized.

Melanosis of the urinary bladder in a cow.

Melanosis of the urinary bladder is a very rare condition characterized by an abnormal black or brownish-black pigmentation of the organ. The pigmentary disorder can involve both the urothelial cell layers and/or the submucosa. The biologic potential of the melanosis of urinary bladder remains unknown because only a few cases have been reported in medical literature. So far melanosis of the urinary bladder is not known to occur in cattle. Here we describe the first case of melanosis of the urinary bladder in an inbred red-spotted, 7-year-old cow. Light, ultrastructural, and immunohistochemic investigations demonstrated melanin pigment in the submucosa and lamina propria but not the urothelium of the bladder. In addition, biochemical characterization of the pigment-laden cells demonstrated that the pigment of this disorder consisted mainly of eumelanin, thus corroborating the morphologic studies. Finally, virologic examination revealed the presence of bovine papillomavirus type 2.

The chemical basis of the antinitrosating action of polyphenolic cancer chemopreventive agents.

A regular intake of polyphenolic agents widely found in fruits and vegetables is believed to decrease the incidence of certain forms of cancer, due in part to their ability to act as antinitrosating agents capable of lowering the impact of toxic nitrosation processes and carcinogenic nitrosamine formation within the acidic environment of the stomach. As a result, the study of the interactions between reactive nitrogen species and phenolic antioxidants has emerged as an area of great promise for delineating innovative strategies in cancer chemoprevention. The burst of interest in (poly)phenolic cancer chemopreventive agents of dietary origin is exemplified by the exponential growth of scientific literature on green tea catechins, as well as on hydroxycinnamates, hydroxytyrosol, flavonoids and other phenolic compounds of the Mediterranean diet, currently regarded as a cultural model for dietary improvement. However, as is often the case with rapidly growing fields, most of these advances have not yet been assessed nor properly integrated into a well defined conceptual framework, whereby several aspects of the chemistry underlying their mechanism of action have remained either obscure or have been taken for granted without sufficient experimental support. The objective of this paper is to provide an account of the chemical mechanisms through which polyphenolic compounds of dietary origin may react with nitrite-derived nitrosating species under conditions that model those occurring in the stomach and other acidic biological compartments. The relevance of this chemistry to the actual role of these substances in DNA protection and cancer prevention remains a critical goal for future studies.

Dopaquinone redox exchange with dihydroxyindole and dihydroxyindole carboxylic acid.

A pulse radiolytic investigation has been conducted to establish whether a redox reaction takes place between dopaquinone and 5,6-dihydroxyindole (DHI) and its 2-carboxylic acid (DHICA) and to measure the rate constants of the interactions. To obviate possible confounding reactions, such as nucleophilic addition, the method employed to generate dopaquinone used the dibromide radical anion acting on dopa to form the semiquinone which rapidly disproportionates to dopaquinone. In the presence of DHI the corresponding indole-5,6-quinone (and/or tautomers) was also formed directly but, by judicious selection of suitable relative concentrations of initial reactants, we were able to detect the formation of additional indolequinone from the redox exchange reaction of DHI with dopaquinone which exhibited a linear dependency on the concentration of DHI. Computer simulation of the experimental time profiles of the absorption changes showed that, under the conditions chosen, redox exchange does proceed but not quite to completion, a forward rate constant of 1.4 x 10(6)/M/s being obtained. This is in the same range as the rate constants previously established for reactions of dopaquinone with cyclodopa and cysteinyldopa. In similar experiments carried out with DHICA, the reaction more obviously does not go to completion and is much slower, k (forward) =1.6 x 10(5)/M/s. We conclude that, in the eumelanogenic pathway, DHI oxidation may take place by redox exchange with dopaquinone, although such a reaction is likely to be less efficient for DHICA.

Free radical oxidation of 15-(S)-hydroxyeicosatetraenoic acid with the Fenton reagent: characterization of an epoxy-alcohol and cytotoxic 4-hydroxy-2E-nonenal from the heptatrienyl radical pathway.

The oxidation of (5Z,8Z,11Z,13E,15S)-15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-(S)-HETE, 1a) with the Fenton reagent (Fe2+/EDTA/H2O2) was investigated. In phosphate buffer, pH 7.4, the reaction proceeded with 75% substrate consumption after 1 h to give a mixture of products, one of which was identified as (2E,4S)-4-hydroxy-2-nonenal (3a, 18% yield). Methylation of the mixture with diazomethane allowed isolation of another main product which could be identified as methyl (5Z,8Z,13E)-11,12-trans-epoxy-15-hydroxy-5,8,13-eicosatrienoate (2a methyl ester, 8% yield). A similar oxidation carried out on (15-(2)H)-15-HETE (1b) indicated complete retention of the label in 2b methyl ester and 3b, consistent with an oxidation pathway involving as the primary event H-atom abstraction at C-10. Overall, these results support the recently proposed role of 1a as a potential precursor of the cytotoxic gamma-hydroxyalkenal 3a and disclose a hitherto unrecognized interconnection between 1a and the epoxy-alcohol 2a, previously implicated only in the metabolic transformations of the 15-hydroperoxy derivative of arachidonic acid.

Free radical oxidation of coriolic acid (13-(S)-hydroxy-9Z,11E-octadecadienoic acid).

The reaction of (13S,9Z,11E)-13-hydroxy-9,11-octadecadienoic acid (1a), one of the major peroxidation products of linoleic acid and an important physiological mediator, with the Fenton reagent (Fe(2+)/EDTA/H(2)O(2)) was investigated. In phosphate buffer, pH 7.4, the reaction proceeded with >80% substrate consumption after 4h to give a defined pattern of products, the major of which were isolated as methyl esters and were subjected to complete spectral characterization. The less polar product was identified as (9Z,11E)-13-oxo-9,11-octadecadienoate (2) methyl ester (40% yield). Based on 2D NMR analysis the other two major products were formulated as (11E)-9,10-epoxy-13-hydroxy-11-octadecenoate (3) methyl ester (15% yield) and (10E)-9-hydroxy-13-oxo-10-octadecenoate (4) methyl ester (10% yield). Mechanistic experiments, including deuterium labeling, were consistent with a free radical oxidation pathway involving as the primary event H-atom abstraction at C-13, as inferred from loss of the original S configuration in the reaction products. Overall, these results provide the first insight into the products formed by oxidation of 1a with the Fenton reagent, and hint at novel formation pathways of the hydroxyepoxide 3 and hydroxyketone 4 of potential (patho)physiological relevance in settings of oxidative stress.

Oxidative conversion of 6-nitrocatecholamines to nitrosating products: a possible contributory factor in nitric oxide and catecholamine neurotoxicity associated with oxidative stress and acidosis.

Oxidation of 6-nitrodopamine (1) and 6-nitronorepinephrine (2), as well as of the model compounds 4-nitrocatechol and 4-methyl-5-nitrocatechol, with horseradish peroxidase (HRP)/H(2)O(2), lactoperoxidase (LPO)/H(2)O(2), Fe(2+)/H(2)O(2), Fe(2+)-EDTA/H(2)O(2) (Fenton reagent), HRP or Fe(2+)/EDTA in combination with D-glucose-glucose oxidase, or Fe(2+)/O(2), resulted in the smooth formation of yellowish-brown pigments positive to the Griess assay. In the case of 1, formation of the Griess positive pigment (GPP-1) promoted by HRP/H(2)O(2) proceeded through the intermediacy of two main dimeric species that could be isolated and identified as 3 and the isomer 4, featuring the 4-nitro-6,7-dihydroxyindole system linked to a unit of 1 through ether bonds. Spectroscopic (FAB-MS, (1)H NMR) and chemical analysis of GPP-1 indicated a mixture of oligomeric species related to 3 and 4 in which oxidative modification of the nitrocatechol moiety of 1 led to the generation of reactive nitro groups supposedly linked to sp(3) hybridized carbons. In the pH range 3-6, GPP-1 induced concentration- and pH-dependent nitrosation of 2,3-diaminonaphthalene, but very poor (up to 2%) nitration of 600 microM tyrosine. At pH 7.4, 1 exerted significant toxicity to PC12 cells, while GPP-1 proved virtually innocuous. By contrast, when assayed on Lactobacillus bulgaricus cells at pH 3.5, 1 was inactive whereas GGP-1 caused about 70% inhibition of cell growth. Overall, these results hint at novel pH-dependent mechanisms of nitrocatecholamine-induced cytotoxicity of possible relevance to ischemia- or inflammation-induced catecholaminergic neuron damage.

Ni(2+), a double-acting inhibitor of neuronal nitric oxide synthase interfering with L-arginine binding and Ca(2+)/calmodulin-dependent enzyme activation.

Ni(2+), a toxic and carcinogenic pollutant and one of the leading causes of contact dermatitis, is shown to inhibit neuronal nitric oxide synthase (nNOS) in a competitive, reversible manner with respect to the substrate l-arginine (K(i) = 30 +/- 4 microM). The IC(50) values were dependent on calmodulin (CaM) concentration, but proved independent of Ca(2+), tetrahydrobiopterin (BH(4)) and other essential cofactors. Ni(2+) also inhibited CaM-dependent cytochrome c reduction, NADPH oxidation, and H(2)O(2) production by nNOS. Overall, the action profile of Ni(2+) was suggestive of an unusual, double-acting inhibitor of nNOS affecting l-arginine-binding and Ca(2+)/CaM-dependent enzyme activation.

An unusual decarboxylative Maillard reaction between L-DOPA and D-glucose under biomimetic conditions: factors governing competition with Pictet-Spengler condensation.

In 0.1 M phosphate buffer at pH 7.4 and 37 degrees C, the tyrosine metabolite L-3,4-dihydroxyphenylalanine (L-DOPA) reacts smoothly with D-glucose to afford, besides diastereoisomeric tetrahydroisoquinolines 1 and 2 by Pictet-Spengler condensation, a main product shown to be the unexpected decarboxylated Amadori compound N-(1-deoxy-D-fructos-1-yl)-dopamine (3). Under similar conditions, dopamine gave only tetrahydroisoquinoline products 4 and 5, whereas L-tyrosine gave exclusively the typical Amadori compound 6. Fe(3+) and Cu(2+) ions, which accumulate in relatively high levels in parkinsonian substantia nigra, both inhibited the formation of 3. Cu(2+) ions also inhibited the formation of 1 and 2 to a similar degree, whereas Fe(3+) ions increased the yields of 1 and 2. Apparently, the formation of 3 would not be compatible with a simple decarboxylation of the initial Schiff base adduct, but would rather involve the decarboxylative decomposition of a putative oxazolidine-5-one intermediate assisted by the catechol ring. These results report the first decarboxylative Maillard reaction between an amino acid and a carbohydrate under biomimetic conditions and highlight the critical role of transition metal ions in the competition with Pictet-Spengler condensation.

Thiouracil antithyroid drugs as a new class of neuronal nitric oxide synthase inhibitors.

Two established antithyroid drugs, 6-propyl-2-thiouracil and 6-methyl-2-thiouracil, as well as S-methylthiouracil, are shown to be competitive inhibitors of nitric oxide synthase (NOS) (K(I) values ranging from 14 to 60 microM), with moderate selectivity for the neuronal isoform. Other thioureylene and thioamide-containing heterocyclic systems proved virtually ineffective as NOS inhibitors. Besides offering novel useful leads for inhibitor design as well as to probe the active site of neuronal NOS, the results of this study may have interesting implications in relation to the antithyroid activity of thiouracils and their possible neurological effects.

Inhibition of neuronal nitric oxide synthase by 6-nitrocatecholamines, putative reaction products of nitric oxide with catecholamines under oxidative stress conditions.

6-Nitrodopamine and 6-nitronoradrenaline (6-nitronorepinephrine), putative products of the nitric oxide (NO)-dependent nitration of dopamine and noradrenaline, are reported to be reversible, competitive inhibitors of neuronal nitric oxide synthase (nNOS) with K(i) values of 45 and 52 microM respectively. The nitrocatecholamines inhibited H(2)O(2) production in the absence of L-arginine and tetrahydrobiopterin (BH(4)) (the IC(50) values for 6-nitrodopamine and 6-nitronoradrenaline were 85 and 55 microM respectively) but without affecting cytochrome c reduction. The apparent K(i) values for nitrocatecholamine inhibition of enzyme activation by BH(4) were 18 microM for 6-nitrodopamine and 40 microM for 6-nitronoradrenaline. Both nitrocatecholamines antagonized the dimerization of nNOS induced by BH(4) and by L-arginine, the effect being reversed by BH(4) (more than 10 microM) and L-arginine (e.g. 100 microM). Overall, these results suggest that nitrocatecholamines interfere with nNOS activity by binding to the enzyme in the proximity of the substrate and BH(4)-binding sites near the haem group.

Nitric oxide synthase (NOS) in the brain of the cephalopod Sepia officinalis.

Nitric oxide synthase-like protein (NOS) is shown to be present in specific regions of the central nervous system (CNS) of the cephalopod mollusc Sepia officinalis (cuttlefish). NOS activity, which is Ca(2+)/calmodulin-dependent, was determined by measuring the conversion of L-[(14)C]arginine in L-[(14)C]citrulline. The partially purified NOS from brain and optic lobes exhibited on SDS-PAGE a band at 150 kDa that was immunolabelled by antibodies raised against the synthetic peptide corresponding to the amino acids 1,414-1,429 of the C-terminus of rat nNOS. This same antibody was then used for immunohistochemical staining of serial sections of the cuttlefish CNS to reveal localized specific staining of cell bodies and fibers in several lobes of the brain. Staining was found in many lower motor centers, including cells and fibers of the inferior and superior buccal lobes (feeding centers); in some higher motor centers (anterior basal and peduncle lobes); in learning centers (vertical, subvertical, and superior frontal lobes); and in the visual system [retina and deep retina (optic lobe)]. Immunopositivity was also found in the olfactory lobe and organ and in the sucker epithelium. These findings suggest that nitric oxide (NO) may be involved as a signaling molecule in feeding, motor, learning, visual, and olfactory systems in the cuttlefish brain. The presence of NOS in the cephalopod "cerebellum" and learning centers is discussed in the context of the vertebrate CNS.

2-thiouracil is a selective inhibitor of neuronal nitric oxide synthase antagonising tetrahydrobiopterin-dependent enzyme activation and dimerisation.

2-thiouracil (TU), an established antithyroid drug and melanoma-seeker, was found to selectively inhibit neuronal nitric oxide synthase (nNOS) in a competitive manner (K(i)=20 microM), being inactive on the other NOS isoforms. The drug apparently interfered with the substrate- and tetrahydrobiopterin (BH(4))-binding to the enzyme. It caused a 60% inhibition of H(2)O(2) production in the absence of L-arginine and BH(4), and antagonised BH(4)-induced dimerisation of nNOS, but did not affect cytochrome c reduction. These results open new perspectives in the understanding of the antithyroid action of TU and provide a new lead structure for the development of selective nNOS inhibitors to elucidate the interdependence of the substrate and pteridine sites and to modulate pathologically aberrant NO formation.

Acid-promoted reactions of ethyl linoleate with nitrite ions: formation and structural characterization of isomeric nitroalkene, nitrohydroxy, and novel 3-nitro-1,5-hexadiene and 1,5-dinitro-1, 3-pentadiene products.

The reaction of ethyl linoleate (1) with NO(2)(-) in different air-equilibrated acidic media resulted in the formation of complex patterns of products, some of which could be isolated by repeated TLC fractionation and were formulated as the nitroalkenes 2-5, the novel (1E, 5Z)-3-nitro-1,5-hexadienes 6/7, the novel (E,E)-1, 5-dinitro-1,3-pentadiene derivatives 8 and 9, and the nitro alcohols 10/11 and 12/13 by extensive GC-MS and 2D NMR analysis, as aided by 1D Hartmann-Hahn proton mapping experiments. Similar reaction of methyl oleate gave mainly nitroalkene (14/15) and allylic nitro derivatives (16/17). Formation of 2-13 may be envisaged in terms of HNO(2)-mediated nitration pathways in which regioisomeric beta-nitroalkyl radical intermediates derived from attack of NO(2) to the 1,4-pentadiene moiety of 1 evolve through competitive H-atom abstraction and free radical combination routes.

A novel octahydropyridobenzothiazepine metabolite in human urine: biomimetic formation from the melanogen 5-S-cysteinyldopa and formaldehyde via a peculiar sulfur-controlled double Pictet-Spengler condensation.

HPLC evidence is reported demonstrating the occurrence in some human urine samples of a novel catecholic metabolite, (3R,7S)-3, 7-dicarboxy-10,11-dihydroxy-2,3,4,5,6,7,8,9-octahydropyrido[ 4,3-g][1, 4]benzothiazepine (2). The compound was shown to arise by a double Pictet-Spengler condensation of the urinary melanogen 5-S-cysteinyldopa (1) with formaldehyde, in which regioselective formation of the six-membered ring ortho to the activating hydroxyl group lends assistance to the subsequent closure of the seven-membered 1,4-thiazepine moiety. Under physiologically relevant conditions, i.e., in 0.1 M phosphate buffer pH 7.4 and at 37 degrees C, the 7,8-tetrahydroisoquinoline 5 was the sole detectable intermediate in the formation of 2. N-Acetylcysteinyldopa (4) reacted likewise with formaldehyde to give the 7, 8-dihydroxytetrahydroisoquinoline 6. The anomalous regiochemistry underlying formation of 5 and 6 was rationalized with the aid of AM1/PM3 calculations on the model alkylthiocatechol 10, predicting a higher HOMO-controlled reactivity on the position ortho rather than para to the activating hydroxyl group. The potential of the reported chemistry as a convenient synthetic access to the 2,3,4, 5-tetrahydro[1,4]benzothiazepine ring system is suggested by the efficient conversion of the cysteinylcatechol 3 to 8 in the presence of formaldehyde.

N-Methyl-D-aspartate receptor stimulation activates tyrosinase and promotes melanin synthesis in the ink gland of the cuttlefish Sepia officinalis through the nitric Oxide/cGMP signal transduction pathway. A novel possible role for glutamate as physiologic activator of melanogenesis.

The tyrosinase-catalyzed conversion of l-tyrosine to melanin represents the most distinctive biochemical pathway in the ink gland of the cuttlefish Sepia officinalis; however, the molecular mechanisms underlying its activation have remained so far largely uncharted. In this paper we demonstrate for the first time that l-glutamate can stimulate tyrosinase activity and promote melanin synthesis in Sepia ink gland via the N-methyl-d-aspartate (NMDA) receptor/NO/cGMP signal transduction pathway. Incubation of intact ink glands with either l-glutamate or NMDA resulted in an up to 18-fold increase of tyrosinase activity and a more than 6-fold elevation of cGMP levels. Comparable stimulation of tyrosinase was induced by an NO donor and by 8-bromo-cGMP. An NMDA receptor antagonist, NO synthase (NOS) inhibitors, and a guanylate cyclase blocker suppressed NMDA-induced effects. Immunohistochemical evidence indicated that enhanced cGMP production was localized largely in the mature part of the ink gland. Increased de novo synthesis of melanin was demonstrated in NMDA- and NO-stimulated ink glands by a combined microanalytical approach based on spectrophotometric determination of pigment levels and high performance liquid chromatography quantitation of pyrrole-2,3, 5-tricarboxylic acid, a specific melanin marker, in melanosome-containing fractions. These results fill a longstanding gap in the understanding of the complex biochemical mechanisms underlying activation of melanogenesis in the mature ink gland cells of S. officinalis and disclose a novel physiologic role of the excitatory neurotransmitter glutamate mediated by the NMDA receptor/NO/cGMP signaling pathway.

Interactions of nitric oxide with lipid peroxidation products under aerobic conditions: inhibitory effects on the formation of malondialdehyde and related thiobarbituric acid-reactive substances.

Under aerobic conditions, exposure of peroxidized lipids to nitric oxide (NO) was found to result in a rapid decrease in the levels of thiobarbituric acid-reactive substances (TBARS). Addition of 10-100 microM NO to rat brain homogenates preincubated for 2 h at 37 degrees C caused up to a 20% decrease in the levels of TBARS compared to controls. A similar inhibitory effect was observed on TBARS produced by Fe(2+)-induced decomposition of 15-hydroperoxyeicosatetraenoic acid (15-HPETE), due apparently to NO-induced decomposition of the hydroperoxide (ferrous oxidation/xylenol orange assay). Prostaglandin G(2) (PGG(2), 35 microM), as a model bicyclic endoperoxide, and malondialdehyde (MDA, 20 microM), the main component of TBARS, proved also susceptible to degradation by NO or NO donors (diethylamine NONOate, DEA/NO) at concentrations of 100 microM or higher in 0.05 M phosphate buffer, pH 7.4, and at 37 degrees C, as indicated by the reduced response to the TBA assay. No significant effect on TBARS determination was caused by nitrite ions. These and other data indicate that NO can inhibit TBARS formation by decomposing primary lipid peroxidation products, chiefly 15-HPETE and related hydroperoxides, and, to a lesser extent, later stage TBARS precursors, including bicyclic endoperoxides and MDA, via nitrosation and other oxidative routes, without however affecting chromogenic reactions during the assay.

Nitrite- and peroxide-dependent oxidation pathways of dopamine: 6-nitrodopamine and 6-hydroxydopamine formation as potential contributory mechanisms of oxidative stress- and nitric oxide-induced neurotoxicity in neuronal degeneration.

In the presence of nitrite ions (NO(2)(-)) in phosphate buffer (pH 7. 4) and at 37 degrees C, dopamine was oxidized by a variety of hydrogen peroxide (H(2)O(2))-dependent enzymatic and chemical systems to give, in addition to black melanin-like pigments via 5, 6-dihydroxyindoles, small amounts of the potent neurotoxin 6-hydroxydopamine (1) and of 6-nitrodopamine (2), a putative reaction product of dopamine with NO-derived species. Treatment of 0. 5 or 1 mM dopamine with horseradish peroxidase (HRP) or lactoperoxidase (LPO) in the presence of 1 or 2 mM H(2)O(2) with NO(2)(-) at a concentration of 0.5-10 mM resulted in the formation of 1 and 2 in up to 8 and 2 microM yields, respectively, depending on the substrate concentration and the NO(2)(-):H(2)O(2) ratio. Nitration and hydroxylation of 0.1 mM dopamine was observed with 1 mM NO(2)(-) using HRP and the D-glucose/glucose oxidase system to generate H(2)O(2) in situ. In the presence of NO(2)(-)-, Fe(2+)-, or Fe(2+)/EDTA-promoted oxidations of dopamine with H(2)O(2) also led to the formation of 1 and 2, the apparent product ratios varying with peroxide concentration and the partitioning of the metal between EDTA and catecholamine chelates. In the presence of NO(2)(-), Fe(2+)-promoted autoxidation of dopamine gave 2 but no detectable 1. When injected into the brains of laboratory rats, 2 caused sporadic behavioral changes, indicating that it could elicit a neurotoxic response, albeit to a lower extent than 1. Model experiments using tyrosinase as an oxidizing system and mechanistic considerations suggested that formation of 2 does not involve reactive nitrogen radicals but results mainly from nucleophilic attack of NO(2)(-) to dopamine quinone. Generation of 1, on the other hand, may be derives from different H(2)O(2)-dependent pathways. Collectively, these results outline a complex interplay of NO(2)(-)- and peroxide-dependent oxidation pathways of dopamine, which may contribute to impair dopaminergic neurotransmission and induce cytotoxic processes in neurodegenerative disorders.

A calcium/calmodulin-dependent nitric oxide synthase, NMDAR2/3 receptor subunits, and glutamate in the CNS of the cuttlefish Sepia officinalis: localization in specific neural pathways controlling the inking system.

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.