Tetrahydrobiopterin and Biogenic Amine Metabolism in the hph-1 Mouse

Abstract: hph-1 mice, which have defective tetrahydrobiopterin biosynthesis due to decreased GTP cyclohydrolase I activity, have been used to investigate the effects of tetrahydrobiopterin deficiency on aromatic l -amino acid monooxygenases and brain monoamine metabolism. Liver tetrahydrobiopterin levels were decreased, and tetrahydrobiopterin deficiency and reduced levels of dopamine, norepinephrine, serotonin, and their metabolites in the brain occurred both pre- and postnatally. Chronic subcutaneous tetrahydrobiopterin elevated brain levels to values higher than those seen in controls but had no effect on monoamine metabolism. In vivo activities of tyrosine hydroxylase and tryptophan hydroxylase were significantly decreased. There was a 30% decrease in the in vitro activity of striatal tyrosine hydroxylase and 50% decrease in liver phenylalanine hydroxylase. Western blotting demonstrated that the lower monooxygenase activities resulted from a reduced absolute amount of tyrosine hydroxylase and phenylalanine hydroxylase protein. The findings suggest involvement of tetrahydrobiopterin in the control of the steady-state concentration of the aromatic l -amino acid monooxygenases. In addition, demonstration of central monoamine changes in the hph-1 mouse make it a possible model system for the investigation of the neuropathological mechanisms in Dopa-responsive dystonia, which has recently been linked with mutations in the gene for GTP cyclohydrolase I.     

Attenuated endothelium-mediated relaxation by acteoside in rat aorta: Role of endothelial [Ca2+]i and nitric oxide/cyclic GMP pathway

Acteoside and other phenylethanoid glycoside are contained in many plants that are widely used in traditional Chinese herbal medicine. Acteoside possesses multiple biological actions. Its effect on the vascular system is, however, incompletely understood. This study was aimed to investigate the role of endothelial [Ca²⁺]_i, nitric oxide (NO), and cyclic GMP in acteoside-induced inhibition of endothelial NO-mediated relaxation in rat aorta. Acteoside reduced endothelial NO-dependent relaxation induced by acetylcholine (Ach) or A23187. Acteoside inhibited Ach-stimulated increase in tissue content of cyclic GMP in endothelium-intact rings. L-NNA abolished the stimulatory effect of Ach. Treatment with acteoside significantly suppressed bradykinin-induced increase in [Ca²⁺]_i of cultured rat aortic endothelial cells. Acute exposure to acteoside (30 μM) did not affect the expression of eNOS mRNA in endothelium-intact rings. In summary, acteoside impairs endothelial NO-mediated aortic relaxation partially through inhibition of agonist-induced endothelial Ca²⁺ mobilization and Ca²⁺-dependent NO production and subsequent suppression of cyclic GMP formation. This novel pharmacological action if occurring in small vessels in vivo, may contribute to the reported anti-inflammatory effect of acteoside against NO-mediated vascular permeability-related acute edema.     

Homocysteine induces oxidative stress by uncoupling of NO synthase activity through reduction of tetrahydrobiopterin

Hyperhomocysteinemia is a risk factor for cardiovascular diseases that induces endothelial dysfunction. Here, we examine the participation of endothelial NO synthase (eNOS) in the homocysteine-induced alterations of NO/O(2)(-) balance in endothelial cells from human umbilical cord vein. When cells were treated for 24 h, homocysteine dose-dependently inhibited thrombin-activated NO release without altering eNOS phosphorylation and independently of the endogenous NOS inhibitor, asymmetric dimethylarginine. The inhibitory effect of homocysteine on NO release was associated with increased production of reactive nitrogen and oxygen species (RNS/ROS) independent of extracellular superoxide anion (O(2)(-)) and was suppressed by the NOS inhibitor L-NAME. In unstimulated cells, L-NAME markedly decreased RNS/ROS formation and the ethidium red fluorescence induced by homocysteine. This eNOS-dependent O(2)(-) synthesis was associated with reduced intracellular levels of both total biopterins (-45%) and tetrahydrobiopterin (-80%) and increased release of 7,8-dihydrobiopterin and biopterin in the extracellular medium (+40%). In addition, homocysteine suppressed the activating effect of sepiapterin on NO release, but not that of ascorbate. The results show that the oxidative stress and inhibition of NO release induced by homocysteine depend on eNOS uncoupling due to reduction of intracellular tetrahydrobiopterin availability.     

Adrenergic Stimulation of Cyclic GMP Formation Requires NO-Dependent Activation of Cytosolic Guanylate Cyclase in Rat Pinealocytes

Abstract: Cyclic GMP (cGMP) formation in rat pinealocytes is regulated through a synergistic dual receptor mechanism involving β-and α₁-adrenergic receptors. The effects of N -monomethyl- l -arginine (NMMA), which inhibits nitric oxide (NO) synthase and NO-mediated activation of cytosolic guanylate cyclase, and methylene blue (MB), which inhibits cytosolic guanylate cyclase, were investigated in an attempt to understand the role of NO in adrenergic cGMP formation. Both NMMA and MB inhibited β-adrenergic stimulation of cGMP formation as well as α₁-adrenergic potentiation of β-adrenergic stimulation of cGMP formation, whereas they had no effect in unstimulated pinealocytes. The inhibitory action of NMMA was antagonized by addition of l -arginine. On the basis of these findings it can be concluded that the adrenergic stimulation of cGMP formation involves NO synthesis followed by activation of cytosolic guanylate cyclase.     

Stimulation of Cyclic GMP Accumulation by Sodium Nitroprusside Is Potentiated via a Gs Mechanism in Intact Pinealocytes

Abstract: Cyclic GMP accumulation in pinealocytes is elevated>100-fold by norepinephrine (NE) through a mechanism involving conjoint activation of α₁- and β₁-adrenergic receptors. Little or no stimulation occurs if either α₁- or β₁-adrenergic receptors alone are activated. It appears that α₁-adrenergic effects are mediated by Ca²⁺ acting in part through nitric oxide (NO), and β₁-adrenergic effects are mediated by G_s. In the study presented here we investigated effects of adrenergic agonists or related postreceptor-active agents on stimulation of pineal cyclic GMP accumulation by the NO generator sodium nitroprusside (NP). The cyclic GMP response to NP (1 m M ) was potentiated by NE and isoproterenol (ISO) but not by phenylephrine, indicating that activation of β₁-adrenergic receptors potentiates the effects of NP. Similarly, vasoactive intestinal peptide (VIP), cholera toxin (CTX), and forskolin, all of which are known to mimic the effects of ISO in this system, also potentiated the effects of NP. In contrast, neither dibutyryl cyclic AMP nor agents that elevate intracellular Ca²⁺ levels caused marked potentiation of the effects of NP on pineal cyclic GMP. Depletion (90%) of G_sα by 21-h treatment with CTX reduced β-adrenergic potentiation of NP. These findings indicate that β-adrenergic agonists and VIP potentiate the effects of NP through a mechanism involving G_s. The molecular basis of this action may be an increase in guanylyl cyclase responsiveness to NO.     

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.     

Altered nitric oxide production in mouse brain after administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridin or methamphetamine

Several studies have demonstrated the involvement of reactive nitrogen and oxygen species (RNOS) in the neurotoxic effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridin (MPTP) and methamphetamine (METH), so the contribution of altered nitric oxide synthase (NOS) enzyme function can be suspected. In this study, about 50% increase in nitric oxide (NO) production in the mouse striatum was found between 4 and 12 h after a single MPTP injection, allowing an increased peroxynitrite (ONOO⁻) formation in the target brain region. However, METH injection induced a rapid decrease of NO formation both in mouse striatum and hippocampus, reaching its minimum level at 2 h, and restored to the control value after 6 h in the striatum and 12 h in the hippocampus. The uncoupled function of NOS with increased superoxide (O₂⁻) production after METH injection is suggested.     

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.     

Stimulation of the Cyclic GMP Pathway by NO Induces Expression of the Immediate Early Genes c-fos and junB in PC12 Cells

Abstract: Stimulation of several second messenger pathways induces the expression of immediate early genes such as c- fos , c- jun , junB , and junD , but little is known about their induction via the stimulation of the cyclic GMP pathway. Here we looked at the expression of early genes in pheochromocytoma PC12 cells after activation of cytosolic guanylate cyclase by sodium nitroprusside. This compound spontaneously releases NO, a molecule known to be involved in cell communication. We found that expression of c- fos and junB but not of c- jun or junD is increased upon activation of cyclic GMP pathway. c- fos mRNA expression was the most activated (fourfold at 30 min), whereas junB response was more modest (2.2-fold activation at 60 min). Nuclear extracts of stimulated cells show increased binding capacity to the AP1 binding site consistent with the dose-response curve. The activating effect of nitroprusside could be reproduced by dipyridamole, a selective cyclic GMP phosphodiesterase inhibitor and by 8- p -chlorophenylthio-cyclic GMP, a permeant selective cyclic GMP-dependent protein kinase activator, and abolished by KT5823, an inhibitor of that kinase. The results show that NO promotes early gene activation and AP1 binding enhancement through the stimulation of the cyclic GMP pathway.     

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.     

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.     

Angiotensin-Induced Cyclic GMP Production Is Mediated by Multiple Receptor Subtypes and Nitric Oxide in N1E-115 Neuroblastoma Cells

Angiotensin II (AngII) elicited a rapid and dose-related production of intracellular cyclic GMP (cGMP) in murine neuroblastoma N1E-115 cells. The agonist-induced rise in cGMP levels was blocked in a monophasic fashion by the AT1-selective antagonist DuP 753 or the nonselective antagonist [Sarc1,Ile8]-AngII, and both antagonists produced complete inhibition of the cGMP response elicited by submaximal concentrations of AngII. In contrast, the AT2-selective antagonist CGP 42112A inhibited the cGMP response biphasically. At lower antagonist concentrations, agonist-induced cGMP production was only partially inhibited, whereas complete inhibition was observed only when the concentration of CGP 42112A was increased sufficiently to interact with both AT1 and AT2 receptor subtypes. AngII also increased inositol trisphosphate (InsP3) levels in N1E-115 cells. However, the InsP3 response was mediated exclusively by the AT1 receptor subtype because it was inhibited by lower, AT1-selective concentrations of DuP 753, whereas only higher, nonselective concentrations of CGP 42112A were effective. Finally, the stimulatory effects of AngII on cGMP production appeared to be mediated by the intracellular formation of nitric oxide in that they were attenuated by the nitric oxide synthase inhibitor, N-monomethyl-L-arginine. Collectively, these results suggest that the AngII-elicited rise in cGMP levels may require an interaction between AT1-mediated mobilization of intracellular Ca2+, as well as some partial role of AT2 receptors.     

Ionic Mechanisms Implicated in the Stimulation of Cerebellar Cyclic GMP Levels by N-Methyl-D-Aspartate

N-Methyl-D-aspartate (NMDA) increases cyclic GMP levels in immature rat cerebellar slices incubated in magnesium-containing Krebs buffer in vitro. This effect is blocked by 2-amino-5-phosphonovalerate and by D-alpha-aminoadipate, but not by glutamic acid diethyl ester or gamma-D-glutamylaminomethylsulfonic acid, indicating specific involvement of the NMDA receptor. The response produced by NMDA is abolished by removal of calcium from the medium, proportional to the concentration of extracellular calcium, and blocked by a number of inorganic (Ni2+, Co2+, Cd2+, La3+, Mn2+) calcium antagonists. The responses to NMDA are not blocked by barium or strontium and persist when these ions are substituted for calcium in the incubation medium. The effects of NMDA are blocked by, but are not particularly sensitive to, the organic voltage-dependent calcium channel antagonists. Nifedipine (10 microM) produces partial inhibition of the effects of NMDA, which are also antagonized by high (greater than 200 microM) concentrations of diltiazem and verapamil. The effects of NMDA are tetrodotoxin insensitive but are abolished by omission of sodium from the medium and inhibited by a tetrodotoxin-insensitive sodium channel blocker, Zn2+. The results suggest that calcium channel opening is a consequence of NMDA receptor activation in this model. However, the sodium dependence of the response argues against the use of receptor-operated calcium channels, whereas the weak activity of the organic voltage-sensitive calcium channel antagonists argues either against the use of voltage-dependent calcium channels, or that those implicated in the effects of NMDA are insensitive to these agents.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Eclosion Hormone Stimulates Cyclic GMP Levels in Manduca sexta Nervous Tissue via Arachidonic Acid Metabolism with Little or No Contribution from the Production of Nitric Oxide

The neuropeptide eclosion hormone acts directly on the nervous system of the tobacco hornworm, Manduca sexta, to trigger ecdysis behavior at the end of each molt. Previous studies have shown that the action of eclosion hormone is mediated via the intracellular messenger cyclic GMP. In the present study we have investigated the mechanisms involved in the eclosion hormone-stimulated increases in cyclic GMP. No stimulation of guanylate cyclase was seen in homogenized nervous tissue, suggesting that eclosion hormone does not directly stimulate a membrane-bound form of guanylate cyclase. Nitric oxide synthase inhibitors, N-methylarginine and nitroarginine, had no effect on eclosion hormone-stimulated cyclic GMP levels. By contrast, 4-bromophenacyl bromide, an inhibitor of arachidonic acid release, and nordihydroguaiaretic acid, an inhibitor of arachidonic acid metabolism, almost completely abolished the eclosion hormone-stimulated cyclic GMP increase. We hypothesize that eclosion hormone receptors are coupled to a lipase, activation of which causes the release of arachidonic acid. Either the arachidonic acid directly stimulates the soluble guanylate cyclase or further metabolism of arachidonic acid yields compounds that activate guanylate cyclase.     

Benzodiazepine-sensitive GABA(A) receptors limit the activity of the NMDA/NO/cyclic GMP pathway: a microdialysis study in the cerebellum of freely moving rats

In the cerebellum, infusion of NMDA (200 microM) for 20 min evoked a marked (200%) increase of extracellular cyclic GMP (cGMP) levels. The selective GABA(A) receptor agonist muscimol (0.01-100 microM) was able to counteract the NMDA effect with an EC(50) of 0.65 microM; the inhibitory effect of muscimol (10 microM) was prevented by bicuculline (50 microM). Diazepam (10 microM) significantly potentiated the muscimol (1 microM) inhibition; furthermore, when coinfused with 0.1 microM muscimol (a concentration not affecting, on its own, the cGMP response to NMDA), diazepam (10 microM) reduced the NMDA effect. Similar results were obtained with zolpidem (0.1-1 microM). Finally, local infusion of the benzodiazepine site antagonist flumazenil (10 microM), together with muscimol and diazepam, almost completely restored the effect of NMDA on extracellular cGMP levels. It is concluded that GABA(A) receptors potently control the NMDA/nitric oxide/cGMP pathway in the cerebellum in vivo. In terms of the alpha subunit composition, we can deduce that the cerebellar GABA(A) receptor does not contain alpha(6) or beta(4) subunits because it is diazepam-sensitive. Moreover, the observation that zolpidem is active at a rather low concentration, in combination with localization studies present in the literature, tend to exclude the presence of alpha(5) subunits in the receptor composition and suggest the involvement of an alpha(1) subunit.     

Akt-mediated signaling is induced by cytokines and cyclic adenosine monophosphate and suppresses hepatocyte inducible nitric oxide synthase expression independent of MAPK P44/42

Cyclic AMP inhibits the expression of nitric oxide synthase (Harbrecht et al., 1995 [1]) in hepatocytes but the mechanism for this effect is incompletely understood. Cyclic AMP can activate several intracellular signaling pathways in hepatocytes including Protein Kinase A (PKA), cAMP regulated guanine nucleotide exchange factors (cAMP-GEFs), and calcium-mediated Protein Kinases. There is considerable overlap and cross-talk between many of these signaling pathways, however, and how these cascades regulate hepatocyte iNOS is not known. We hypothesized that Akt mediates the effect of cAMP on hepatocyte iNOS expression. Hepatocytes cultured with cytokines and dbcAMP increased Akt phosphorylation up to 2 h of culture. Akt phosphorylation was inhibited by the PI3K inhibitor LY294002 (10 μM), farnyltranferase inhibitor FTI-276, or transfection with a dominant negative Akt. The cyclic AMP-induced suppression of cytokine-stimulated iNOS was partially reversed by LY294002 and FTI-276. LY294002 also increased NFκB nucleus translocation by Western blot analysis in nuclear extracts. Cyclic AMP increased phosphorylation of Raf1 at serine 259 which was blocked by LY294002 and associated with decreased MAPK P44/42 phosphorylation. However, inhibition of MAPK P44/42 signaling with PD98059 failed to suppress cytokine-induced hepatocyte iNOS expression and did not enhance the inhibitory effect of dbcAMP on iNOS production. A constitutively active MAPK P44/42 plasmid had no effect on cytokine-stimulated NO production. These data demonstrate that dbcAMP regulates hepatocyte iNOS expression through an Akt-mediated signaling mechanism that is independent of MAPK P44/42.     

Regulation of iNOS function and cellular redox state by macrophage Gch1 reveals specific requirements for tetrahydrobiopterin in NRF2 activation

Inducible nitric oxide synthase (iNOS) is a key enzyme in the macrophage inflammatory response, which is the source of nitric oxide (NO) that is potently induced in response to proinflammatory stimuli. However, the specific role of NO production, as distinct from iNOS induction, in macrophage inflammatory responses remains unproven. We have generated a novel mouse model with conditional deletion of Gch1, encoding GTP cyclohydrolase 1 (GTPCH), an essential enzyme in the biosynthesis of tetrahydrobiopterin (BH4) that is a required cofactor for iNOS NO production. Mice with a floxed Gch1 allele (Gch1^fl/fl) were crossed with Tie2cre transgenic mice, causing Gch1 deletion in leukocytes (Gch1^fl/flTie2cre). Macrophages from Gch1^fl/flTie2cre mice lacked GTPCH protein and de novo biopterin biosynthesis. When activated with LPS and IFNγ, macrophages from Gch1^fl/flTie2cre mice induced iNOS protein in a manner indistinguishable from wild-type controls, but produced no detectable NO, as judged by L-citrulline production, EPR spin trapping of NO, and by nitrite accumulation. Incubation of Gch1^fl/flTie2cre macrophages with dihydroethidium revealed significantly increased production of superoxide in the presence of iNOS expression, and an iNOS-independent, BH4-dependent increase in other ROS species. Normal BH4 levels, nitric oxide production, and cellular redox state were restored by sepiapterin, a precursor of BH4 production by the salvage pathway, demonstrating that the effects of BH4 deficiency were reversible. Gch1^fl/flTie2cre macrophages showed only minor alterations in cytokine production and normal cell migration, and minimal changes in basal gene expression. However, gene expression analysis after iNOS induction identified 78 genes that were altered between wild-type and Gch1^fl/flTie2cre macrophages. Pathway analysis identified decreased NRF2 activation, with reduced induction of archetypal NRF2 genes (gclm, prdx1, gsta3, nqo1, and catalase) in BH4-deficient Gch1^fl/flTie2cre macrophages. These findings identify BH4-dependent iNOS regulation and NO generation as specific requirements for NRF2-dependent responses in macrophage inflammatory activation.