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.     

Prenatal Exposure to Aluminum Reduces Expression of Neuronal Nitric Oxide Synthase and of Soluble Guanylate Cyclase and Impairs Glutamatergic Neurotransmission in Rat Cerebellum

Exposure to aluminum (Al) produces neurotoxic effects in humans. However, the molecular mechanism of Al neurotoxicity remains unknown. Al interferes with glutamatergic neurotransmission and impairs the neuronal glutamate-nitric oxide-cyclic GMP (cGMP) pathway, especially in rats prenatally exposed to Al. The aim of this work was to assess whether Al interferes with processes associated with activation of NMDA receptors and to study the molecular basis for the Al-induced impairment of the glutamate-nitric oxide-cGMP pathway. We used primary cultures of cerebellar neurons prepared from control rats or from rats prenatally exposed to Al. Prenatal exposure to Al prevented glutamate-induced proteolysis of the microtubule-associated protein-2, disaggregation of microtubules, and neuronal death, indicating an impairment of NMDA receptor-associated signal transduction pathways. Prenatal exposure to Al reduced significantly the content of nitric oxide synthase and guanylate cyclase and increased the content of calmodulin both in cultured neurons and in the whole cerebellum. This effect was selective for proteins of the glutamate-nitric oxide-cGMP pathway as the content of mitogen-activated protein kinase and the synthesis of most proteins were not affected by prenatal exposure to Al. The alterations in the expression of proteins of the glutamate-nitric oxide-cGMP pathway could be responsible for some of the neurotoxic effects of Al.     

Serotonin Inhibition of the NMDA Receptor/Nitric Oxide/Cyclic GMP Pathway in Rat Cerebellum: Involvement of 5-Hydroxytryptamine2C Receptors

Abstract: Previous studies have shown that 5-hydroxytryptamine (5-HT) can potently inhibit glutamatergic transmission in rat cerebellum through the activation of multiple 5-HT receptors. The aim of this study was to subclassify the 5-HT₂ receptor mediating inhibition of the cyclic GMP response elicited by N -methyl- d -aspartate in adult rat cerebellar slices. Seven receptor antagonists, endowed with relative selectivities for the 5-HT_2A, 5-HT_2B, and 5-HT_2C subtypes, differentially affected the inhibition by (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane of the cyclic GMP response, suggesting that the receptor involved belongs to the 5-HT_2C subtype.     

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.     

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.     

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.     

Regulation of Neuronal Nitric Oxide and Cyclic GMP Formation by Ca2+

Nitric oxide (NO) acts as a messenger molecule in the CNS by activating soluble guanylyl cyclase. Rat brain synaptosomal NO synthase was stimulated by Ca2+ in a concentration-dependent manner with half-maximal effects observed at 0.3 microM and 0.2 microM when its activity was assayed as formation of NO and L-citrulline, respectively. Cyclic GMP formation was apparently inhibited, however, at Ca2+ concentrations required for the activation of NO synthase, indicating a down-regulation of the signal in NO-producing cells. Purified synaptosomal guanylyl cyclase was not inhibited directly by Ca2+, and the effect was not mediated by a protein binding to guanylyl cyclase at low or high Ca2+ concentrations. In cytosolic fractions, the breakdown of cyclic GMP, but not that of cyclic AMP, was highly stimulated by Ca2+, and 3-isobutyl-1-methylxanthine did not block this reaction effectively. The effects of Ca2+ on cyclic GMP hydrolysis and on apparent guanylyl cyclase activities were abolished almost completely in the presence of the calmodulin antagonist calmidazolium, whose effect was attenuated by added calmodulin. Thus, a Ca2+/calmodulin-dependent cyclic GMP phosphodiesterase is highly active in synaptic areas of the brain and may prevent elevations of intracellular cyclic GMP levels in activated, NO-producing neurons.     

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.     

Chronic exposure to 2,5-hexanedione impairs the glutamate-nitric oxide-cyclic GMP pathway in cerebellar neurons in culture and in rat brain in vivo

2,5-Hexanedione is a neurotoxic metabolite of hexane. The mechanisms of its neurotoxicity remain unclear. We assessed whether chronic exposure to 2,5-hexanedione affects the glutamate-nitric oxide-cGMP pathway in primary cultures of cerebellar neurons and/or in the cerebellum of rats.

Chronic exposure of cultured cerebellar neurons to 2,5-hexanedione (200 μM) reduced by ≈50% NMDA-induced formation of cGMP. Activation of soluble guanylate cyclase by nitric oxide was reduced by 46%. This treatment reduced the content of neuronal nitric oxide synthase and soluble guanylate cyclase in neurons by 23 and 20%, respectively.

In the cerebellum of rats chronically exposed to 2,5-hexanedione (in the drinking water) NMDA-induced formation of cGMP was reduced by 55% as determined by in vivo brain microdialysis. Activation of soluble guanylate cyclase by nitric oxide was reduced by 65%. The content of neuronal nitric oxide synthase and of soluble guanylate cyclase was reduced by 25 and 21%, respectively, in the cerebellum of these rats.

The effects are the same in both systems, indicating that cultured neurons are a good model to study the mechanisms of neurotoxicity of 2,5-hexanedione.

These results indicate that chronic exposure to 2,5-hexanedione affects the glutamate-nitric oxide-cGMP pathway at different steps both in cultured neurons and in cerebellum of the animal in vivo. The alteration of this pathway may contribute to the neurotoxic effects of 2,5-hexanedione.     

Nitric oxide-cyclic GMP signaling in stem cell differentiation

The nitric oxide–cyclic GMP (NO–cGMP) pathway mediates important physiological functions associated with various integrative body systems including the cardiovascular and nervous systems. Furthermore, NO regulates cell growth, survival, apoptosis, proliferation, and differentiation at the cellular level. To understand the significance of the NO–cGMP pathway in development and differentiation, studies have been conducted both in developing embryos and in stem cells. Manipulation of the NO–cGMP pathway, by employing activators and inhibitors as pharmacological probes, and genetic manipulation of NO signaling components have implicated the involvement of this pathway in the regulation of stem cell differentiation. This review focuses on some of the work pertaining to the role of NO–cGMP in the differentiation of stem cells into cells of various lineages, particularly into myocardial cells, and in stem cell-based therapy.     

Effects of Physostigmine and Some Nitric Oxide-Cyclic GMP-Related Compounds on Muscarinic Receptor-Mediated Autoinhibition of Hippocampal Acetylcholine Release

Abstract: We have investigated the effects of (a) the cholinesterase inhibitor physostigmine and (b) drugs that are known to change intracellular cyclic GMP levels on the autoinhibition of acetylcholine release from rat hippocampal slices. Autoinhibition was triggered by submaximal electrical stimulation in both the absence and presence of physostigmine. The results obtained indicate that an unusual increase in the extracellular acetylcholine content, such as that induced by cholinesterase inhibition, is not essential for autoinhibition triggering. Dibutyryl cyclic GMP reduced significantly the stimulation-evoked acetylcholine release in the presence, but not in the absence, of atropine. Neither sodium nitroprusside nor glyceryl trinitrate exerted a dibutyryl cyclic GMP-like effect. N ^G-Nitro-L-arginine did not lessen the autoinhibition. These results indicate that an increase in the intracellular cyclic GMP level reduces acetylcholine release, and that the muscarinic receptor stimulation-nitric oxide synthesis-(soluble) guanylyl cyclase activation pathway is not involved in the cholinergic autoinhibition process.     

Stimulation of the brain NO/cyclic GMP pathway by peripheral administration of tetrahydrobiopterin in the hph-1 mouse

Mutations in GTP-cyclohydrolase I (GTP-CH) have been identified as causing a range of inborn errors of metabolism, including dopa-responsive dystonia. GTP-CH catalyses the first step in the biosynthesis of tetrahydrobiopterin (BH4), a cofactor necessary for the synthesis of catecholamines and serotonin. Current therapy based on monoamine neurotransmitter replacement may be only partially successful in correcting the neurological deficits. The reason might be that BH4 is also a cofactor for nitric oxide synthase. Using a strain of mutant GTP-CH-deficient (hph-1) mice, we demonstrate that in addition to impaired monoamine metabolism, BH4 deficiency is also associated with diminished nitric oxide synthesis in the brain (as evaluated by measuring the levels of cyclic GMP), when compared with wild-type animals. We have found a decline in the levels of BH4 with age in all animals, but no gender-related differences. We found a strong association between the levels of BH4 and cyclic GMP in hph-1 mice but not in wild-type animals. We also demonstrate that acute peripheral administration of BH4 (100 micromol/kg s.c.) in hph-1 mice significantly elevated the brain BH4 concentration and subsequently cyclic GMP levels in cerebellum, with peaks at 2 and 3 h, respectively. We suggest that BH4 administration should be considered in BH4 deficiency states in addition to monoamine replacement therapy.     

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.     

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.     

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.     

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.     

Stimulation of Cyclic GMP Production via a Nitrosyl Factor in Sensory Neuronal Cultures by Algesic or Inflammatory Agents

Abstract: Capsaicin stimulates cyclic GMP production via nitric oxide (NO) (or another nitrosyl factor) in dorsal root ganglion (DRG) neurons maintained in culture. The purpose of the present study was to characterize further capsaicin stimulation of cyclic GMP production in DRG cells maintained in culture, investigate other algesic and/or inflammatory agents for effects on cyclic GMP production, and examine cells responsible for NO production and cyclic GMP production. Capsaicin stimulation of cyclic GMP production in DRG cells was dose dependent, receptor mediated, and attenuated by hemoglobin. Prostaglandin E₂, substance P, and calcitonin gene-related peptide did not affect basal, capsaicin-stimulated, or bradykinin-stimulated cyclic GMP production. Other inflammatory or algesic agents, including serotonin, histamine, ATP, glutamate, aspartate, and NMDA, did not affect cyclic GMP production. Pretreatment of DRG cells with lipopolysaccharide increased basal cyclic GMP production in neuronal but not in nonneuronal cultures and facilitated stimulation of cyclic GMP production by l -arginine. Capsaicin pretreatment of neuronal DRG cultures, which destroys capsaicin-sensitive (small diameter) afferent neurons, attenuated capsaicin- and bradykinin-stimulated cyclic GMP production but did not affect basal or sodium nitroprusside-stimulated cyclic GMP production. These results indicate that capsaicin elicits production of a nitrosyl factor via capsaicin-sensitive (small diameter) neurons. Capsaicin evoked cyclic GMP production in nonneuronal DRG cultures in the presence but not in the absence of apposed neuronal DRG cultures. Overall, these findings suggest that specific exogenous (or endogenous) substances may stimulate production of a nitrosyl factor(s) by a subset of DRG neurons, and nitrosyl factors produced by these neurons may affect cyclic GMP production in neighboring neuronal or non-neuronal cells.     

Decreased Nitric Oxide Production in the Rat Brain after Chronic Arsenic Exposure

Chronic arsenic exposure is associated with nervous system damage, vascular disease, hepatic and renal damage as well as different types of cancer. Alterations of nitric oxide (NO) in the periphery have been detected after arsenic exposure, and we explored here NO production in the brain. Female Wistar rats were exposed to arsenite in drinking water (4–5 mg/kg/day) from gestation, lactation and until 4 months of age. NOS activity, NO metabolites content, reactive oxygen species production (ROS) and lipid peroxidation (LPx) were determined in vitro in the striatum, and NO production was estimated in vivo measuring citrulline by microdialysis. Exposed animals showed a significantly lower response to NMDA receptor stimulation, reduction of NOS activity and decreased levels of nitrites and nitrates in striatum. These markers of NO function were accompanied by significantly higher levels of LPx and ROS production. These results provide evidence of NO dysfunction in the rat brain associated with arsenic exposure.     

Chronic nitric oxide synthase blockade desensitizes the heart to the negative metabolic effects of nitric oxide

The consequences of chronic nitric oxide synthase (NOS) blockade on the myocardial metabolic and guanylyl cyclase stimulatory effects of exogenous nitric oxide (NO) were determined. Thirty-three anesthetized open-chest rabbits were randomized into four groups: control, NO donor S-nitroso-N-acetyl-penicillamine (SNAP, 10(-4 )M), NOS blocking agent N(G)-nitro-L-arginine methyl ester (L-NAME, 20 mg/kg/day) for 10 days followed by a 24 hour washout and L-NAME for 10 days followed by a 24 hour washout plus SNAP. Myocardial O(2) consumption was determined from coronary flow (microspheres) and O(2) extraction (microspectrophotometry). Cyclic GMP and guanylyl cyclase activity were determined by radioimmunoassay. There were no baseline metabolic, functional or hemodynamic differences between control and L-NAME treated rabbits. SNAP in controls caused a reduction in O(2) consumption (SNAP 5.9+/-0.6 vs. control 8.4+/-0.8 ml O(2)/min/100 g) and a rise in cyclic GMP (SNAP 18.3+/-3.8 vs. control 10.4+/-0.9 pmol/g). After chronic L-NAME treatment, SNAP caused no significant changes in O(2) consumption (SNAP 7.1+/-0.8 vs. control 6.4+/-0.7) or cyclic GMP (SNAP 14.2+/-1.8 vs. control 12.1+/-1.3). In controls, guanylyl cyclase activity was significantly stimulated by SNAP (216.7+/-20.0 SNAP vs. 34.4+/-2.5 pmol/mg/min base), while this increase was blunted after L-NAME (115.9+/-24.5 SNAP vs. 24.9+/-4.7 base). These results demonstrated that chronic NOS blockade followed by washout blunts the response to exogenous NO, with little effect on cyclic GMP or myocardial O(2) consumption. This was related to reduced guanylyl cyclase activity after chronic L-NAME. These results suggest that, unlike many receptor systems, the NO-cyclic GMP signal transduction system becomes downregulated upon chronic inhibition.