Chronic Exposure to Aluminum Impairs Neuronal Glutamate-Nitric Oxide-Cyclic GMP Pathway

Abstract: Humans are exposed to aluminum from environmental sources and therapeutic treatments. However, aluminum is neurotoxic and is considered a possible etiologic factor in Alzheimer's disease and other neurological disorders. The molecular mechanism of aluminum neurotoxicity is not understood. We tested the effects of aluminum on the glutamate-nitric oxide-cyclic GMP pathway in cultured neurons. Neurons were exposed to 50 µ M aluminum in culture medium for short-term (4 h) or long-term (8–14 days) periods, or rats were prenatally exposed, i.e., 3.7% aluminum sulfate in the drinking water, during gestation. Chronic (but not short-term) exposure of neurons to aluminum decreased glutamate-induced activation of nitric oxide synthase by 38% and the formation of cyclic GMP by 77%. The formation of cyclic GMP induced by the nitric oxide-generating agent S -nitroso- N -acetylpenicillamine was reduced by 33%. In neurons from rats prenatally exposed to aluminum but not exposed to it during culture, glutamate-induced formation of cyclic GMP was inhibited by 81%, and activation of nitric oxide synthase was decreased by 85%. The formation of cyclic GMP induced by S -nitroso- N -acetylpenicillamine was not affected. These results indicate that chronic exposure to aluminum impairs glutamate-induced activation of nitric oxide synthase and nitric oxide-induced activation of guanylate cyclase. Impairment of the glutamate-nitric oxide-cyclic GMP pathway in neurons may contribute to aluminum neurotoxicity.     

Arginine Availability Controls the N-Methyl-d-Aspartate-Induced Nitric Oxide Synthesis: Involvement of a Glial-Neuronal Arginine Transfer

Abstract: The neuronal nitric oxide (NO) synthase generates NO from arginine. NO mediates its physiological effects mainly by stimulating the synthesis of cyclic GMP. We have investigated the role of the arginine availability on the NMDA-induced cyclic GMP accumulation in immature rat brain slices. The effect of NMDA was blocked by the inhibitor of the NO synthase, N ^G-nitro- l -arginine, and by the antagonist of ionotropic non-NMDA receptors, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). This inhibition was not due to a direct interaction of CNQX with the NMDA receptor, and it was overcome by the presence of exogenously applied arginine. CNQX also blocked the NMDA-evoked release of [³H]arginine from cerebellar slices. Moreover, the arginine uptake inhibitor l -lysine reduced the cyclic GMP response to NMDA significantly. Therefore, the extracellular arginine availability, which is dependent on the activation of ionotropic non-NMDA receptors, determines the rate of the NO biosynthesis by the neuronal NO synthase. Together with the reported release of arginine from glial cells upon activation of glial ionotropic non-NMDA receptors and the predominant glial localization of arginine, these data provide the first evidence of an essential role of the arginine transfer from glial cells to neurons for the biosynthesis of NO.     

Can nitric oxide be spin trapped by nitrone and nitroso compounds?

Increasing interest in the study of nitric oxide (NO^·) in may facets of biological research necessitates a search for accurate techniques to directly identify the free radical. One recently employed strategy for NO^· detection is the method of electron spin resonance (ESR) used in combination with nitrone and nitroso spin traps. Applying this technique to our studies with nitric oxide synthase (NOS), we found that NO^· generated directly from the enzyme system could not be detected. Further investigation revealed that 3,5-dibromo-4-nitrosobenzenesulfonic acid (DBNBS) inhibited NO^· generation by NOS at concentrations used fro spin trapping. Reexamining the ability of various nitrones and DBNBS to spin trap authentic NO^· dissolved in buffer, we obtained ESR spectra similar to those previously reported for the spin trap DBNBS. However, continuing our studies with ¹⁵NO^· and N-hydroxylamine, we found these spectra to be artifactual. Our results emphasize the need to synthesized new spin traps, since currently available compounds are not capable of spin trapping NO^· generated by NOS.     

Nitric Oxide-Induced Release of Acetylcholine in the Nucleus Accumbens: Role of Cyclic GMP, Glutamate, and GABA

Abstract: We have previously shown that the basal acetylcholine release in the ventral striatum is under the enhancing influence of endogenous nitric oxide (NO) and that NO donors cause pronounced increases in the acetylcholine release rate. To investigate the role of cyclic GMP, glutamate, and GABA in the NO-induced acetylcholine release, we superfused the nucleus accumbens, (Nac) of the anesthetized rat with various compounds through a push-pull cannula and determined the neurotransmitter released in the perfusate. Superfusion of the Nac with the NO donors diethylamine/NO (DEANO; 100 µmol/L), S-nitroso-N-acetylpenicillamine (SNAP; 200 µmol/L), or 3-morpholinosydnonimine (SIN-1; 200 µmol/L) enhanced the acetylcholine release rate. The guanylyl cyclase inhibitor 1H-(1,2,4)-oxodiazolo(4,3-a)quinoxalin-1-one (ODQ; 10 µmol/L) abolished the effects of DEANO and SIN-1. 6-(Phenylamino)-5,8-quinolinedione (LY-83583; 100 µmol/L), which also inhibits cyclic GMP synthesis, inhibited the releasing effects of DEANO and of SNAP, whereas the effect of SIN-1 on acetylcholine release was not influenced. The DEANO-induced release of acetylcholine was also abolished in the presence of 20 µmol/L 6,6-dinitroquinoxaline-2,3-dione (DNQX) and 10 µmol/L (±)-2-amino-5-phosphonopentanoic acid (AP-5). Simultaneous superfusion with 50 µmol/L quinpirole and 10 µmol/L 7-bromo-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SKF 83566) was ineffective. Superfusion with 500 µmol/L DEANO decreased the release of acetylcholine. The inhibitory effect of 500 µmol/L DEANO was reversed to an enhanced release on superfusion with 20 µmol/L bicuculline. Bicuculline also enhanced the basal release rate. These findings indicate that cyclic GMP mediates the NO-induced release of acetylcholine by enhancing the outflow of glutamate. Dopamine is not involved in this process. Only high concentrations of NO increase the output of GABA, which in turn decreases acetylcholine release. Our results suggest that cells that are able to release glutamate, such as glutamatergic neurons, are the main target of NO in the Nac.     

Glutamate Dehydrogenase in Human Brain: Regional Distribution and Properties

Glutamate dehydrogenase (GDH) activity was studied in 17 regions of six human brains. Duration and conditions of the postmortem period did not affect enzyme activity. Specific activity ranged between 103 and 377 nmoles/min/mg protein at 25 degrees C and it was 10-fold higher than that found in leukocytes. Apart from exclusively white matter regions (corpus callosum and centrum ovale), there was a moderate regional distribution (2.5-fold variation), with highest values in the inferior olive and hypothalamus, and lowest in the cerebellum and lenticular nucleus. With alpha-ketoglutarate (alpha-KG), NADH, or NH4+ as variable substrate, the apparent Km values in human brain were Km alpha-KG = 1.9 X 10(-3) M, KmNADH = 0.21 X 10(-3) M, and KmNH4+ = 28 X 10(-3) M, and in leukocytes they were Km alpha-KG = 1.7 X 10(-3) M, KmNADH = 0.24 X 10(-3) M, and KmNH4+ = 28 X 10(-3) M. The effects of cofactors, inhibitor, and pH were similar in brain and leukocyte GDH.     

Intracellular polyamine levels are involved in NMDA-evoked nitric oxide production in chick retina cells

The NMDA-sensitive glutamate receptor complex can be modulated by numerous drugs and endogenous substances such as polyamines. We studied the pathway of arginine/nitric oxide/cyclic GMP in cultured chick retina cells through NMDA receptor activation, seen as a function of both differentiation stages of culture and intracellular polyamine levels. In our experimental conditions, the nitric oxide synthase activity was stimulated by NMDA from three to four times between embryonic day (E) 8 plus 5 days in vitro (C) and E8C7. The NMDA response was blocked by MK-801 (10 microM) by >60% at stage E8C5. During culture differentiation, the NMDA-induced increase in nitric oxide synthase activity at the E8C5 stage was blocked by preliminary incubation (24 h) of the cells with alpha-difluoromethylornithine, the inhibitor of polyamine biosynthesis. This effect was assessed by a reduction of NMDA-evoked cyclic GMP formation in polyamine-depleted retina cells. Thus, intracellular polyamine levels are involved in NMDA-evoked nitric oxide production. Our results indicate that (a) the developmental pattern of polyamine levels can be associated with the modulation of NMDA-evoked events and (b) the NMDA-mediated effects have been reduced in alpha-difluoromethylornithine-treated cell cultures. These observations provide evidence for a physiological interaction between polyamines and NMDA-sensitive glutamate receptors during differentiation stages of cultured chick retina cells.     

Nitric oxide has dual opposite roles during early and late phases of apoptosis in cerebellar granule neurons

The involvement and the role of nitric oxide (NO) as a signaling molecule in the course of neuronal apoptosis, whether unique or modulated during the progression of the apoptotic program, has been investigated in a cellular system consisting of cerebellar granule cells (CGCs) where apoptosis can be induced by lowering extracellular potassium. Several parameters involved in NO signaling pathway, such as NO production, neuronal nitric oxide synthase (nNOS) expression, and cyclic GMP (cGMP) production were examined in the presence or absence of different inhibitors. We provide evidence that nitric oxide has dual and opposite effects depending on time after induction of apoptosis. In an early phase, up to 3 h of apoptosis, nitric oxide supports survival of CGCs through a cGMP-dependent mechanism. After 3 h, nNOS expression and activity decreased resulting in shut down of NO and cGMP production. Residual NO then contributes to the apoptotic process by reacting with rising superoxide anions leading to peroxynitrite production and protein inactivation. We conclude that whilst NO over-production protects neurons from death in the early phase of neuronal damage, its subsequent reduction may contribute to neuronal degeneration and ultimate cell death.     

Nitric oxide can differentially modulate striatal neurotransmitter concentrations via soluble guanylate cyclase and peroxynitrite formation

In vivo microdialysis was used to investigate whether nitric oxide (NO) modulates striatal neurotransmitter release in the rat through inducing cyclic GMP formation via soluble guanylate cyclase or formation of peroxynitrite (ONOO(-)). When NO donors, S-nitroso-N-acetyl-DL-penicillamine (SNAP; 1 mM) or (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1- ium-1, 2-diolate (NOC-18; 1 mM), were retrodialysed for 15 min, acetylcholine (ACh), serotonin (5-HT), glutamate (Glu), gamma-aminobutyric acid (GABA), and taurine levels were significantly increased, whereas those of dopamine (DA), dihydroxyphenylacetic acid (DOPAC), and 5-hydroxyindoleacetic acid (5-HIAA) were decreased. Only effects on ACh, 5-HT, and GABA showed calcium dependency. Inhibition of soluble guanylate cyclase by 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ; 100 and 200 microM) dose-dependently reduced NO donor-evoked increases in ACh, 5-HT, Glu, and GABA levels. Coperfusion of SNAP or NOC-18 with an ONOO(-) scavenger, L-cysteine (10 mM) resulted in enhanced concentrations of Glu and GABA. On the other hand, DA concentrations increased rather than decreased, and no reductions in DOPAC and 5-HIAA occurred. This increase in DA and the potentiation of Glu and GABA were calcium-dependent and prevented by ODQ. Similar to NO, infusions of ONOO(-) (10 or 100 microM) decreased DA, DOPAC, and 5-HIAA. Overall, these results demonstrate that NO increases ACh, 5-HT, Glu, and GABA levels primarily through a cyclic GMP-dependent mechanism. For DA, DOPAC, and 5-HIAA, effects are determined by levels of ONOO(-) stimulated by NO donors. When these are high, they effectively reduce extracellular concentrations through oxidation. When they are low, DA concentrations are increased in a cyclic GMP-dependent manner and may act to facilitate Glu and GABA release further. Thus, changes in brain levels of antioxidants, and the altered ability of NO to stimulate cyclic GMP formation during ageing, or neurodegenerative pathologies, may particularly impact on the functional consequences of NO on striatal dopaminergic and glutamatergic function.     

Nitric Oxide Regulates Cyclic GMP-Dependent Protein Kinase Phosphorylation in Rat Brain

Abstract: Nitric oxide (NO) acts via soluble guanylyl cyclase to increase cyclic GMP (cGMP), which can regulate various targets including protein kinases. Western blotting showed that type II cGMP-dependent protein kinase (cGK II) is widely expressed in various brain regions, especially in the thalamus. In thalamic extracts, the phosphorylation of several proteins, including cGK II, was increased by exogenous NO or cGMP. In vivo pretreatment with a NO synthase inhibitor reduced the phosphorylation of cGK II, and this could be reversed by exogenous NO or cGMP. Conversely, brainstem electrical stimulation, which enhances thalamic NO release, caused a NO synthase-dependent increase in the phosphorylation of thalamic cGK II. These results indicate that endogenous NO regulates cGMP-dependent protein phosphorylation in the thalamus. The activation of cGKII by NO may play a role in thalamic mechanisms underlying arousal.     

Excitatory Amino Acid Receptors Coupled to the Nitric Oxide/Cyclic GMP Pathway in Rat Cerebellum During Development

The coupling of excitatory amino acid receptors to the formation of nitric oxide (NO) from arginine during the postnatal development of rat cerebellum was assayed in slice preparations by measuring cyclic GMP accumulation. In the immature tissue, N-methyl-D-aspartate (NMDA) and glutamate were highly efficacious agonists, whereas alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and quisqualate evoked only small responses. The effect of glutamate at all concentrations tested (up to 10 mM) was abolished by the NMDA antagonist, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801). In adult slices, AMPA and quisqualate were much more effective and their effects were inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione, an antagonist for ionotropic non-NMDA receptors, whereas the apparent efficacy of NMDA was greatly reduced. The major changes took place between 8 and 14 days postnatum and, in the case of NMDA, part of the loss of sensitivity appeared to reflect a decline in the ambient levels of glycine with age. Moreover, a component of the response to glutamate in the adult was resistant to MK-801. Cyclic GMP accumulations induced by NMDA and non-NMDA agonists alike were Ca(2+)-dependent and could be antagonized by competitive NO synthase inhibitors in an arginine-sensitive manner, indicating that they are all mediated by NO formation. With one of the inhibitors, L-NG-nitroarginine, a highly potent component (IC50 = 6 nM) evident in slices from rats of up to 8 days old was lost during maturation, indicating that there may be a NO synthase isoform which is prominent only in the immature tissue. Cyclic GMP levels in adult slices under "basal" conditions were reduced markedly by blocking NMDA receptors, by inhibiting action potentials with tetrodotoxin, or by NO synthase inhibition, suggesting that the endogenous transmitter released during spontaneous synaptic activity acts mainly through NMDA receptors to trigger NO formation.     

Metallothionein-III prevents glutamate and nitric oxide neurotoxicity in primary cultures of cerebellar neurons

Metallothionein (MT)-III, a member of the MT family of metal-binding proteins, is mainly expressed in the CNS and is abundant in glutamatergic neurons. Results in genetically altered mice indicate that MT-III may play neuroprotective roles in the brain, but the mechanisms through which this protein functions have not been elucidated. The aim of this work was to assess whether MT-III is able to prevent glutamate neurotoxicity and to identify the step of the neurotoxic process interfered with by MT-III. Glutamate neurotoxicity in cerebellar neurons in culture is mediated by excessive activation of glutamate receptors, increased intracellular calcium, and increased nitric oxide. It is shown that MT-III prevented glutamate- and nitric oxide-induced neurotoxicity in a dose-dependent manner, with nearly complete protection at 0.3-1 microgram/ml. MT-III did not prevent the glutamate-induced rise of intracellular calcium level but reduced significantly the nitric oxide-induced formation of cyclic GMP. Circular dichroism analysis revealed that nitric oxide triggers the release of the metals coordinated to the cysteine residues of MT-III, indicative of the S(Cys)-nitrosylation of the protein. Therefore, the present results indicate that MT-III can quench pathological levels of nitric oxide, thus preventing glutamate and nitric oxide neurotoxicity.     

Ornithine Decarboxylase in Human Brain: Influence of Aging, Regional Distribution, and Alzheimer's Disease

Abstract: Although experimental animal data have implicated ornithine decarboxylase, a key regulatory enzyme of polyamine biosynthesis, in brain development and function, little information is available on this enzyme in normal or abnormal human brain. We examined the influence, in autopsied human brain, of postnatal development and aging, regional distribution, and Alzheimer's disease on the activity of ornithine decarboxylase. Consistent with animal data, human brain ornithine decarboxylase activity was highest in the perinatal period, declining sharply (by ∼60%) during the first year of life to values that remained generally unchanged up to senescence. In adult brain, a moderately heterogeneous regional distribution of enzyme activity was observed, with high levels in the thalamus and occipital cortex and low levels in cerebellar cortex and putamen. In the Alzheimer's disease group, mean ornithine decarboxylase activity was significantly increased in the temporal cortex (+76%), reduced in occipital cortex (−70%), and unchanged in hippocampus and putamen. In contrast, brain enzyme activity was normal in patients with the neurodegenerative disorder spinocerebellar ataxia type I. Our demonstration of ornithine decarboxylase activity in neonatal and adult human brain suggests roles for ornithine decarboxylase in both developing and mature brain function, and we provide further evidence for the involvement of abnormal polyamine system activity in Alzheimer's disease.     

A Kainate Receptor Linked to Nitric Oxide Synthesis from Arginine

In slices of young rat cerebellum, the glutamate analogue kainate induced a large accumulation of cyclic GMP, which was inhibited by non-N-methyl-D-aspartate antagonists. Quisqualate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate evoked only small cyclic GMP responses and inhibited the effect of kainate. When tested in cerebellar cell suspensions, glutamate was also a potent antagonist of the cyclic GMP response to kainate. Superoxide dismutase enhanced the response in the isolated cells, whereas haemoglobin and methylene blue were inhibitory. The response in slices was Ca2+ dependent, augmented by arginine, and inhibited by L-NG-monomethylarginine in a manner that could be reversed by additional arginine. It is concluded that stimulation of kainate receptors leads to activation of the enzyme that synthesizes nitric oxide from arginine and that activation of soluble guanylate cyclase by the released nitric oxide accounts for the cyclic GMP generation.     

Characterization of Neuronal Amino Acid Transporters: Uptake of Nitric Oxide Synthase Inhibitors and Implication for Their Biological Effects

Abstract: In the present study we investigated uptake of the nitric oxide (NO) synthase inhibitors N ^G-methyl- l -arginine and N ^G-nitro- l -arginine by the mouse neuroblastoma × rat glioma hybrid cell line NG108-15. Uptake of N ^G-methyl- l -arginine was characterized by biphasic kinetics ( K _m1 = 8 µmol/L, V _max1 = 0.09 nmol × mg⁻¹× min⁻¹; K _m2 = 229 µmol/L, V _max2 = 2.9 nmol × mg⁻¹× min⁻¹) and was inhibited by basic but not by neutral amino acids. Uptake of N ^G-nitro- l -arginine followed Michaelis-Menten kinetics ( K _m = 265 µmol/L, V _max = 12.8 ± 0.86 nmol × mg⁻¹× min⁻¹) and was selectively inhibited by aromatic and branched chain amino acids. Further characterization of the transport systems revealed that uptake of N ^G-methyl- l -arginine is mediated by system y⁺, whereas systems L and T account for the transport of N ^G-nitro- l -arginine. In agreement with these data on uptake of the inhibitors, l -lysine and l -ornithine antagonized the inhibitory effects of N ^G-methyl- l -arginine on bradykinin-induced intracellular cyclic GMP accumulation, whereas l -tryptophan, l -phenylalanine, and l -leucine interfered with the effects of N ^G-nitro- l -arginine. These data suggest that rates of uptake are limiting for the biological effects of NO synthase inhibitors.     

Nitric Oxide Implication in the Control of Neurosecretion by Chromaffin Cells

Abstract: In this work, we have studied the effects of pure nitric oxide (NO) on the regulation of catecholamine (CA) secretion by chromaffin cells, as well as the possible presence of its synthesizing enzyme l -arginine:NO synthase (NOS) in these cells. Our results show that NO produces a large stimulation of basal CA secretion. This effect was calcium- and concentration-dependent (EC₅₀ = 64 ± 8 µ M ) and was not due to nonspecific damage of the tissue by NO. NO also modulates the CA secretion evoked by nicotine in a dose-dependent manner. Although it has a stimulatory effect on the CA secretion evoked by low doses of nicotine (<3 µ M ; EC₅₀ = 16 ± 3 µ M ), it produces a dose-dependent inhibition of the CA secretion induced by high doses of nicotine (≥30 µ M ; IC₅₀ = 52 ± 6 µ M ). The mechanism by which NO modulates CA secretion seems to be through the increase in the cyclic GMP levels, because there was a close correlation between the CA secretion and the cyclic GMP levels. The presence of a specific activity of NOS in chromaffin cells has been demonstrated by two independent methods: release of [¹⁴C]citruiline from [¹⁴C]arginine and formation of an NO-hemoglobin complex. NOS activity was about 0.5 pmol/min/mg of protein. It was calcium- and mainly calmodulin-dependent and could be specifically blocked by the NOS inhibitor N -methyl- l -arginine. These results suggest that NO could be an important intracellular messenger in the regulation of neurosecretion in chromaffin cells.     

Anomalous Increase in Nitric Oxide Synthase Activity by Certain Nitric Oxide-Generating Compounds in Intact Neuronal Cells

Abstract: It has been shown that nitric oxide (NO) regulates NO synthase (NOS) activity through negative feedback in cytosolic enzyme preparations in various cell types. We compared the effects of the NO-generating compounds S-nitroso-N-acetylpenicillamine (SNAP), 3-morpholinosydnonimine (SIN-1), and sodium nitroprusside (SNP) on NOS activity in intact neuroblastoma N1E-115 cells and in the cytosol obtained from the same cells. Enzyme activity was measured by the conversion of l -[³H]arginine into l -[³H]citrulline. At concentrations that elicit almost complete inhibition of NOS activity in cytosolic enzyme preparations of these cells, SIN-1 and SNP did not cause significant attenuation of enzyme activity measured at 45 min in intact cells. It is surprising that SIN-1 and SNP markedly stimulated l -[³H]citrulline formation in a time- and concentration-dependent manner when cells were incubated with the compounds for >1.5 h. Neither inhibitory nor stimulatory effects of SNAP on NOS were observed in intact N1E-115 cells. This is in contrast to the inhibitory effects of SNAP in cytosolic preparations of the enzyme. The increased NOS activity by SIN-1 or SNP in intact cells was dependent on the presence of extracellular Ca²⁺, suggesting that it might be due to increased Ca²⁺ influx. On the other hand, measurements of the activity of lactate dehydrogenase showed that there was no generalized increase in cell permeability in response to SIN-1 or SNP. There was no agreement in the rank order of potencies of these compounds in activating guanylate cyclase and in affecting NOS activity, both in broken-cell preparations and in intact cells. Thus, modulation of NOS activity by NO-releasing compounds is not dependent on cyclic GMP formation and might not be related in a simple fashion to NO generation. Alternatively, activation of guanylate cyclase and stimulation of NOS activity might require different redox species of NO. Our present findings might be of clinical relevance in relation to long-term use of NO-generating compounds as therapeutic agents.     

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.     

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 KNO₂) 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. Accepted: 11 August 1998     

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 Regulates Substance P Release from Rat Spinal Cord Synaptosomes

Abstract: In order to determine whether nitric oxide (NO) acts directly upon nerve terminals to regulate the synaptic transmission at the level of spinal cord, effects of NO-donors on release of substance P (SP) and glutamic acid (Glu) were investigated by superfusion of synaptosomes prepared from the rat spinal cord. Basal levels of endogenous SP and Glu release were 5.99 ± 2.50 fmol/min/mg of protein and 26.2 ± 4.8 pmol/min/mg of protein, respectively. Exposure to a depolarizing concentration of KCI evoked 2.7- and 3.8-fold increases in SP and Glu release in a calcium-dependent manner, respectively. Sodium nitroprusside (NP) caused a reduction in the depolarization-evoked overflow of SP in a concentration-dependent manner without affecting its basal release, although it failed to affect either basal or evoked release of Glu. The reduction in SP overflow was also observed by the perfusion with S -nitroso- N -acetyl-penicillamine or membrane-permeable cyclic GMP, but not with cyclic AMP. NP caused the concentration-dependent increases in cyclic GMP levels in synaptosomes. Together with reports that excitatory amino acids stimulate NO synthase and release NO in the spinal cord, these data suggest that there may be an interaction between nerve terminals containing Glu and SP, and that NO may directly participate in the regulation of synaptic transmission in SP-containing nerve terminals, which may be mediated through the activation of guanylate cyclase and the increase in cyclic GMP levels.