Group I mGluR regulates the polarity of spike-timing dependent plasticity in substantia gelatinosa neurons

The spinal synaptic plasticity is associated with a central sensitization of nociceptive input, which accounts for the generation of hyperalgesia in chronic pain. However, how group I metabotropic glutamate receptors (mGluRs) may operate spinal plasticity remains essentially unexplored. Here, we have identified spike-timing dependent synaptic plasticity in substantia gelatinosa (SG) neurons, using perforated patch-clamp recordings of SG neuron in a spinal cord slice preparation. In the presence of bicuculline and strychnine, long-term potentiation (LTP) was blocked by AP-5 and Ca2+ chelator BAPTA-AM. The group I mGluR antagonist AIDA, PLC inhibitor U-73122, and IP3 receptor blocker 2-APB shifted LTP to long-term depression (LTD) without affecting acute synaptic transmission. These findings provide a link between postsynaptic group I mGluR/PLC/IP3-gated Ca2+ store regulating the polarity of synaptic plasticity and spinal central sensitization.     

Angiotensin-(1-7) increases neuronal potassium current via a nitric oxide-dependent mechanism

Actions of angiotensin-(1-7) [Ang-(1-7)], a heptapeptide of the renin-angiotensin system, in the periphery are mediated, at least in part, by activation of nitric oxide (NO) synthase (NOS) and generation NO(·). Studies of the central nervous system have shown that NO(·) acts as a sympathoinhibitory molecule and thus may play a protective role in neurocardiovascular diseases associated with sympathoexcitation, such as hypertension and heart failure. However, the contribution of NO in the intraneuronal signaling pathway of Ang-(1-7) and the subsequent modulation of neuronal activity remains unclear. Here, we tested the hypothesis that neuronal NOS (nNOS)-derived NO(·) mediates changes in neuronal activity following Ang-(1-7) stimulation. For these studies, we used differentiated catecholaminergic (CATH.a) neurons, which we show express the Ang-(1-7) receptor (Mas R) and nNOS. Stimulation of CATH.a neurons with Ang-(1-7) (100 nM) increased intracellular NO levels, as measured by 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM) fluorescence and confocal microscopy. This response was significantly attenuated in neurons pretreated with the Mas R antagonist (A-779), a nonspecific NOS inhibitor (nitro-L-arginine methyl ester), or an nNOS inhibitor (S-methyl-L-thiocitrulline, SMTC), but not by endothelial NOS (eNOS) or inhibitory NOS (iNOS) inhibition {L-N-5-(1-iminoethyl)ornithine (L-NIO) and 1400W, respectively}. To examine the effect of Ang-(1-7)-NO(·) signaling on neuronal activity, we recorded voltage-gated outward K(+) current (I(Kv)) in CATH.a neurons using the whole cell configuration of the patch-clamp technique. Ang-(1-7) significantly increased I(Kv), and this response was inhibited by A-779 or S-methyl-L-thiocitrulline, but not L-NIO or 1400W. These findings indicate that Ang-(1-7) is capable of increasing nNOS-derived NO(·) levels, which in turn, activates hyperpolarizing I(Kv) in catecholaminergic neurons.     

Spinal astrocytic activation is involved in a virally-induced rat model of neuropathic pain

Postherpetic neuralgia (PHN), the most common complication of herpes zoster (HZ), plays a major role in decreased life quality of HZ patients. However, the neural mechanisms underlying PHN remain unclear. Here, using a PHN rat model at 2 weeks after varicella zoster virus infection, we found that spinal astrocytes were dramatically activated. The mechanical allodynia and spinal central sensitization were significantly attenuated by intrathecally injected L-α-aminoadipate (astrocytic specific inhibitor) whereas minocycline (microglial specific inhibitor) had no effect, which indicated that spinal astrocyte but not microglia contributed to the chronic pain in PHN rat. Further study was taken to investigate the molecular mechanism of astrocyte-incudced allodynia in PHN rat at post-infection 2 weeks. Results showed that nitric oxide (NO) produced by inducible nitric oxide synthase mediated the development of spinal astrocytic activation, and activated astrocytes dramatically increased interleukin-1β expression which induced N-methyl-D-aspartic acid receptor (NMDAR) phosphorylation in spinal dorsal horn neurons to strengthen pain transmission. Taken together, these results suggest that spinal activated astrocytes may be one of the most important factors in the pathophysiology of PHN and "NO-Astrocyte-Cytokine-NMDAR-Neuron" pathway may be the detailed neural mechanisms underlying PHN. Thus, inhibiting spinal astrocytic activation may represent a novel therapeutic strategy for clinical management of PHN.     

Nitric oxide regulates AKT phosphorylation and nuclear translocation in cultured retinal cells

Previous studies have shown that nitric oxide (NO) inhibits apoptosis of retinal neurons in culture through the canonical cyclic GMP/protein kinase G (PKG)-dependent pathway, but also involving multiple kinase pathways, such as phosphatidylinositol 3′ kinase (PI3k) and AKT. NO and AKT exhibit survival-promoting properties and display important roles in both CNS development and plasticity. The purpose of this study was to evaluate the effects of exogenous NO, derived from the NO donor S-nitroso-N-acetylpenicillamin (SNAP), or endogenous NO, produced from l-arginine, on AKT phosphorylation in cultured chick retinal neurons. Our results demonstrate that SNAP or l-arginine enhances AKT phosphorylation on both serine-473 and threonine-308 residues in a concentration and time-dependent manner. This effect was mediated by the activation of soluble guanylyl cyclase and PKG, since it was blocked by the respective enzyme inhibitors ODQ or LY83583 and KT5823, as well as by transduction with shRNA lentiviruses coding PKGII shRNA, and mimicked by the respective enzyme activators YC-1 and 8-Bromo cyclic GMP, and also by the cyclic GMP phosphodiesterase inhibitor zaprinast. In addition, LY294002 or wortmannin suppressed the SNAP effect, indicating the involvement of phosphoinositide 3′ kinase. Moreover, the mTOR inhibitor KU0063794 blocked SNAP-induced AKT phosphorylation at both residues, suggesting the participation of the mTORC2 complex in the process. Glutamate and NMDA also promoted AKT phosphorylation and a nitric oxide synthase inhibitor abrogated these effects, revealing a mechanism involving the activation of NMDA receptors and NO production. We have also found that SNAP and l-arginine induced AKT translocation into the nucleus of retinal neurons as well as other neuronal cell lines. SNAP also protects retinal cells from death induced by hydrogen peroxide and this effect was blocked by the phosphoinositide 3′ kinase inhibitor LY294002. We therefore conclude that NO produced from endogenous or exogenous sources promotes AKT activation and its shuttling to the nucleus, probably participating in neuronal survival pathways important during CNS development.     

eNOS, nNOS, cGMP and protein kinase G mediate the inhibitory effect of pancreastatin, a chromogranin A-derived peptide, on growth and proliferation of hepatoma cells

Pancreastatin (PST), a chromogranin A-derived peptide, has an anti-insulin metabolic effect and inhibits growth and proliferation by producing nitric oxide (NO) in HTC rat hepatoma cells. When NO production is blocked, a proliferative effect prevails due to the activation a Galphaq/11-phospholipase C-beta (PLC-beta) pathway, which leads to an increase in [Ca2+]i, protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) activation. The aim of the present study was to investigate the NO synthase (NOS) isoform that mediates these effects of PST on HTC hepatoma cells and the possible roles of cyclic GMP (cGMP) and cGMP-dependent protein kinase. DNA and protein synthesis in response to PST were measured as [3H]-thymidine and [3H]-leucine incorporation in the presence of various pharmacological inhibitors: N-monomethyl-L-arginine (NMLA, nonspecific NOS inhibitor), L-NIO (endothelial nitric oxide synthase (eNOS) inhibitor), espermidine (neuronal nitric oxide synthase (nNOS) inhibitor), LY83583 (guanylyl cyclase inhibitor), and KT5823 (protein kinase G inhibitor, (PKG)). L-NIO, similarly to NMLA, reverted the inhibitory effect of PST on hepatoma cell into a stimulatory effect on growth and proliferation. Nevertheless, espermidine also prevented the inhibitory effect of PST, but there was no stimulation of growth and proliferation. When guanylyl cyclase activity was blocked, there was again a reversion of the inhibitory effect into a stimulatory action, suggesting that the effect of NO was mediated by the production of cGMP. PKG inhibition prevented the inhibitory effect of PST, but there was no stimulatory effect. Therefore, the inhibitory effect of PST on growth and proliferation of hepatoma cells may be mainly mediated by eNOS activation. In turn, the effect of NO may be mediated by cGMP, whereas other pathways in addition to PKG activation seem to mediate the inhibition of DNA and protein synthesis by PST in HTC hepatoma cells.     

P2X7 receptors in satellite glial cells mediate high functional expression of P2X3 receptors in immature dorsal root ganglion neurons

Background

In addition to their central effects, opioids cause peripheral analgesia. There is evidence showing that peripheral activation of kappa opioid receptors (KORs) inhibits inflammatory pain. Moreover, peripheral ??-opioid receptor (MOR) activation are able to direct block PGE₂-induced ongoing hyperalgesia However, this effect was not tested for KOR selective activation. In the present study, the effect of the peripheral activation of KORs on PGE₂-induced ongoing hyperalgesia was investigated. The mechanisms involved were also evaluated.

Results

Local (paw) administration of U50488 (a selective KOR agonist) directly blocked, PGE₂-induced mechanical hyperalgesia in both rats and mice. This effect was reversed by treating animals with L-NMMA or N-propyl-L-arginine (a selective inhibitor of neuronal nitric oxide synthase, nNOS), suggesting involvement of the nNOS/NO pathway. U50488 peripheral effect was also dependent on stimulation of PI3K??/AKT because inhibitors of these kinases also reduced peripheral antinociception induced by U50488. Furthermore, U50488 lost its peripheral analgesic effect in PI3K?? null mice. Observations made in vivo were confirmed after incubation of dorsal root ganglion cultured neurons with U50488 produced an increase in the activation of AKT as evaluated by western blot analyses of its phosphorylated form. Finally, immunofluorescence of DRG neurons revealed that KOR-expressing neurons also express PI3K?? (? 43%).

Conclusions

The present study indicates that activation of peripheral KORs directly blocks inflammatory hyperalgesia through stimulation of the nNOS/NO signaling pathway which is probably stimulated by PI3K??/AKT signaling. This study extends a previously study of our group suggesting that PI3K??/AKT/nNOS/NO is an important analgesic pathway in primary nociceptive neurons.     

nNOS mediated mitochondrial injury in LPS stimulated oligodendrocytes

Products of inflammation and the activation of nitric oxide synthase have been proposed as a mechanism of oligodendrocyte injury in CNS inflammation. There are currently three well described and known isoforms of NOS. Of these, neuronal NOS (nNOS) was initially discovered in neurons, endothelial NOS (eNOS) in vascular endothelium, while the inducible form of NOS (iNOS) is known to be activated in oligodendrocytes, astrocytes and microglia. We examined the activation of nNOS and the down stream effects of NO production in oligodendrocyte precursor cells (OPC) and MO3.13 cell line following culture with LPS. Our studies show that both MO3.13 cells and OPC are susceptible to the cellular injury resulting from LPS mediated activation and NO production. Activation of the TLR4 receptor with LPS led to decrease in cell viability that was associated with loss of mitochondrial membrane potential and impaired enzymatic activity of complex I and complex IV protein of the respiratory chain. 7-NI, a known inhibitor of nNOS was able to rescue of cells from LPS mediated mitochondrial damage. Loss of mitochondrial function was associated with translocation of cytochrome C and apoptosis inducing factor to the cytosol, setting the stage for apoptosis. Phosphorylation of PI3K and Akt was required for optimal activation of NOS. These studies provide a biochemical basis for nNOS mediated oligodendrocyte injury and suggest similar mechanisms may play a role in diseases characterized by oligodendrocyte loss and demyelination.     

Loss of nitric oxide-mediated down-regulation of NMDA receptors in neurofilament aggregate-bearing motor neurons in vitro: implications for motor neuron disease

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disorder in which excitotoxicity has been implicated as a cause for cell death. To examine neurofilament (NF) aggregate-mediated sensitization of motor neurons to NMDA excitotoxicity, we examined NMDA receptor expression and the impact of NO donors (NOC12 or NOC5) or sodium cyanide (NaCN) on calcium influx and viability in dissociated motor neurons derived from wt and hNFL+/+ (NF aggregate-forming) mice. Alterations in intracellular calcium were assayed using Oregon Green calcium dye and the extent of apoptosis using active caspase-3 immunoreactivity. Although NF aggregate-bearing neurons demonstrated increased intracellular calcium levels and enhanced cell death in response to NMDA receptor activation, this was not associated with increased NMDA receptor expression. The down-regulation of the NMDA receptor using NO donors decreased calcium influx and caspase-3 activation in aggregate-bearing neurons, but had no effect on wt cultures. The converse was observed with NaCN in which intracellular calcium levels increased significantly in wt cultures in association with increased cell death. No effect was observed in aggregate-bearing neurons. These findings suggest that the presence of NF aggregates renders motor neurons more susceptible to NMDA-mediated excitotoxicity, and that this can be reversed by NO.     

Inflammatory neurodegeneration mediated by nitric oxide,glutamate, and mitochondria

In inflammatory, infectious, ischemic, and neurodegenerative pathologies of th central nervous system (CNS) glia become “activated” by inflammatory mediators, and express new proteins such as the inducible isoform of nitric oxide synthase (iNOS). Although these activated glia have beneficial roles, in vitro they potently kill cocultured neurons, and there is increasing evidence that they contribute to pathology in vivo. Nitric oxide (NO) from iNOS appears to be a key mediator of such glial-induced neuronal death. The high sensitivity of neurons to NO is partly due to NO causing inhibition of respiration, rapid glutamate release from both astrocytes and neurons, and subsequent excitotoxic death of the neurons. NO is a potent inhibitor of mitochondrial respiration, due to reversible binding of NO to cytochrome oxidase in competition with oxygen, resulting in inhibition of energy production and sensitization to hypoxia. Activated astrocytes or microglia cause a potent inhibition of respiration in cocultured neurons due to glial NO inhibiting cytochrome oxidase within the neurons, resulting in ATP depletion and glutamate release. In some conditions, glutamate-induced neuronal death can itself be mediated by N-methyl-d-aspartate (NMDA)-receptor activation of the neuronal isoform of NO synthase (nNOS) causing mitochondrial damage. In addition NO can be converted to a number of reactive derivatives such as peroxynitrite, NO₂, N₂O₃, and S-nitrosothiols that can kill cells in part by inhibiting mitochondrial respiration or activation of mitochondrial permeability transition, triggering neuronal apoptosis or necrosis.     

Neuron-glia communication via nitric oxide is essential in establishing antennal-lobe structure in Manduca sexta.

Nitric oxide synthase recently has been shown to be present in olfactory receptor cells throughout development of the adult antennal (olfactory) lobe of the brain of the moth Manduca sexta. Here, we investigate the possible involvement of nitric oxide (NO) in antennal-lobe morphogenesis. Inhibition of NO signaling with a NO synthase inhibitor or a NO scavenger early in development results in abnormal antennal lobes in which neuropil-associated glia fail to migrate. A more subtle effect is seen in the arborization of dendrites of a serotonin-immunoreactive neuron, which grow beyond their normal range. The effects of NO signaling in these types of cells do not appear to be mediated by activation of soluble guanylyl cyclase to produce cGMP, as these cells do not exhibit cGMP immunoreactivity following NO stimulation and are not affected by infusion of a soluble guanylyl cyclase inhibitor. Treatment with Novobiocin, which blocks ADP-ribosylation of proteins, results in a phenotype similar to those seen with blockade of NO signaling. Thus, axons of olfactory receptor cells appear to trigger glial cell migration and limit arborization of serotonin-immunoreactive neurons via NO signaling. The NO effect may be mediated in part by ADP-ribosylation of target cell proteins.     

GroEL1, a heat shock protein 60 of Chlamydia pneumoniae, induces lectin-like oxidized low-density lipoprotein receptor 1 expression in endothelial cells and enhances atherogenesis in hypercholesterolemic rabbits

Lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1) plays a major role in oxidized low-density lipoprotein-induced vascular inflammation. Chlamydia pneumoniae has been found in atherosclerotic lesions and is related to atherosclerotic pathogenesis, although its specific mechanism remains unknown. This study was conducted to investigate the mechanisms of LOX-1 expression in GroEL1 (a heat shock protein from C. pneumoniae)-administered human coronary artery endothelial cells (HCAECs) and atherogenesis in hypercholesterolemic rabbits. We demonstrated that in the hypercholesterolemic rabbit model, GroEL1 administration enhanced fatty streak and macrophage infiltration in atherosclerotic lesions, which may be mediated by elevated LOX-1 expression. In in vitro study using HCAECs, stimulation with GroEL1 increased TLR4 and LOX-1 expression. Increased LOX-1 expression was downregulated by Akt activation and PI3K-mediated endothelial NO synthase activation. PI3K inhibitor and NO synthase inhibitor induced LOX-1 mRNA production, whereas the NO donor ameliorated the increasing effect of LOX-1 mRNA in GroEL1-stimulated HCAECs. LOX-1 expression was regulated by NADPH oxidase, which mediates reactive oxygen species production and intracellular MAPK signaling pathway in GroEL1-stimulated HCAECs. Treatment with polyethylene-glycol-conjugated superoxide dismutase, apocynin, or diphenylene iodonium significantly decreased GroEL1-induced LOX-1 expression, as did the knockdown of Rac1 gene expression by RNA interference. In conclusion, the GroEL1 protein may induce LOX-1 expression in endothelial cells and atherogenesis in hypercholesterolemic rabbits. The elevated level of LOX-1 in vitro may be mediated by the PI3K-Akt signaling pathway, endothelial NO synthase activation, NADPH oxidase-mediated reactive oxygen species production, and MAPK activation in GroEL1-stimulated HCAECs. The GroEL1 protein of C. pneumoniae may contribute to vascular inflammation and cardiovascular disorders.     

Inhibitory effect of nitric oxide on voltage-dependent calcium currents in rat dorsal root ganglion cells

The effect of nitric oxide (NO) on calcium current (I(Ca)) and intracellular calcium concentration ([Ca(2+)](i)) in primarily cultured dorsal root ganglion (DRG) neurons was investigated from neonatal rats. I(Ca) and [Ca(2+)](i) were simultaneously recorded using perforated-patch technique in combination with fluorescence measurement from single DRG neurons. NO donors, sodium nitroprusside (SNP) and S-nitro-N-acetylpenicillamine (SNAP), inhibited I(Ca) in small-diameter neurons without significant change in voltage-dependence of activation and activation time constants. SNP and SNAP also reduced the transient [Ca(2+)](i) peak accompanied by I(Ca). Inhibition by NO was reproducible, but gradually desensitized. In some DRG neurons, SNP and SNAP increased basal [Ca(2+)](i) in concentration of 10 microM with little effect on NO-induced inhibition of I(Ca). 8-Br-cGMP, a permeable cGMP analog, mimicked the effects of SNP and SNAP. These results suggest that, in DRG neurons, NO has inhibitory effect on I(Ca), which is independent of NO-induced increase of basal [Ca(2+)](i), through cGMP-dependent pathway.     

Carnosic acid, a pro-electrophilic compound, inhibits LPS-induced activation of microglia

In the previous studies, we reported that carnosic acid (CA) protects cortical neurons by activating the Keap1/Nrf2 pathway, which activation is initiated by S-alkylation of the critical cysteine thiol of the Keap1 protein by the "electrophilic"quinone-type CA. Here, we found that the pro-electrophilic CA inhibited the in vitro lipopolysaccharide (LPS)-induced activation of cells of the mouse microglial cell line MG6. LPS induced the expression of IL-1β and IL-6, typical inflammatory cytokines released from microglial cells. CA inhibited the NO production associated with a decrease in the level of inducible NO synthase. Neither CA nor LPS affected cell survival at the concentrations used here. These actions of CA seemed to be mediated by induction of phase 2 genes (gclc, gclm, nqo1 and xct). We propose that an inducer of phase 2 genes may be a critical regulator of microglial activation. Thus, CA is a unique pro-electrophilic compound that provides both a protective effect on neurons and an anti-inflammatory one on microglia through induction of phase 2 genes.     

Insulin rescues retinal neurons from apoptosis by a phosphatidylinositol 3-kinase/Akt-mediated mechanism that reduces the activation of caspase-3.

The ability of insulin to protect neurons from apoptosis was examined in differentiated R28 cells, a neural cell line derived from the neonatal rat retina. Apoptosis was induced by serum deprivation, and the number of pyknotic cells was counted. p53 and Akt were examined by immunoblotting after serum deprivation and insulin treatment, and caspase-3 activation was examined by immunocytochemistry. Serum deprivation for 24 h caused approximately 20% of R28 cells to undergo apoptosis, detected by both pyknosis and activation of caspase-3. 10 nm insulin maximally reduced the amount of apoptosis with a similar potency as 1.3 nm (10 ng/ml) insulin-like growth factor 1, which acted as a positive control. Insulin induced serine phosphorylation of Akt, through the phosphatidylinositol (PI) 3-kinase pathway. Inhibition of PI 3-kinase with wortmannin or LY294002 blocked the ability of insulin to rescue the cells from apoptosis. SN50, a peptide inhibitor of NF-kappaB nuclear translocation, blocked the rescue effect of insulin, but neither insulin or serum deprivation induced phosphorylation of IkappaB. These results suggest that insulin is a survival factor for retinal neurons by activating the PI 3-kinase/Akt pathway and by reducing caspase-3 activation. The rescue effect of insulin does not appear to be mediated by NF-kappaB or p53. These data suggest that insulin provides trophic support for retinal neurons through a PI 3-kinase/Akt-dependent pathway.     

Brain-derived neurotrophic factor accelerates nitric oxide donor-induced apoptosis of cultured cortical neurons

Brain-derived neurotrophic factor (BDNF) is known to have important functions in neuronal survival, differentiation, and plasticity. In addition to its role as a survival-promoting factor, BDNF reportedly can enhance neuronal cell death in some cases, for example, the death caused by excitotoxicity or glucose deprivation. The cellular mechanism of the death-enhancing effect of BDNF remains unknown, in contrast to that of its survival-promoting effect. In this work, we found that BDNF markedly accelerated the nitric oxide (NO) donor-induced death of cultured embryonic cortical neurons. BDNF increased the number of cells with nuclear condensation and DNA fragmentation 24 h after treatment with the NO donor, but it did not change the number of those cells 36 h after the treatment. The BDNF-accelerated death of cortical neurons was inhibited by the addition of actinomycin D or cycloheximide. These results suggest that BDNF can accelerate apoptotic cell death elicited by NO donor. TrkB-IgG and K252a blocked the BDNF-induced acceleration of the death, indicating that the death-accelerating effect by BDNF is mediated by TrkB. In addition, the BDNF-accelerated apoptosis was inhibited by the addition of SB202190 and SB203580, specific inhibitors of p38 mitogen-activated protein kinase (MAPK), and U0126, a specific inhibitor of MAPK/ERK kinase 1, indicating that the activation of both p38 MAPK and ERK is involved in the signaling cascade of the BDNF-accelerated, NO donor-induced apoptosis.     

The involvement of tyrosine kinases, cyclic AMP/protein kinase A, and p38 mitogen-activated protein kinase in IL-13-mediated arginase I induction in macrophages: its implications in IL-13-inhibited nitric oxide production

In macrophages, L-arginine can be used by NO synthase and arginase to form NO and urea, respectively. Therefore, activation of arginase may be an effective mechanism for regulating NO production in macrophages through substrate competition. Here, we examined whether IL-13 up-regulates arginase and thus reduces NO production from LPS-activated macrophages. The signaling molecules involved in IL-13-induced arginase activation were also determined. Results showed that IL-13 increased arginase activity through de novo synthesis of the arginase I mRNA and protein. The activation of arginase was preceded by a transient increase in intracellular cAMP, tyrosine kinase phosphorylation, and p38 mitogen-activated protein kinase (MAPK) activation. Exogenous cAMP also increased arginase activity and enhanced the effect of IL-13 on arginase induction. The induction of arginase was abolished by a protein kinase A (PKA) inhibitor, KT5720, and was down-regulated by tyrosine kinase inhibitors and a p38 MAPK inhibitor, SB203580. However, inhibition of p38 MAPK had no effect on either the IL-13-increased intracellular cAMP or the exogenous cAMP-induced arginase activation, suggesting that p38 MAPK signaling is parallel to the cAMP/PKA pathway. Furthermore, the induction of arginase was insensitive to the protein kinase C and p44/p42 MAPK kinase inhibitors. Finally, IL-13 significantly inhibited NO production from LPS-activated macrophages, and this effect was reversed by an arginase inhibitor, L-norvaline. Together, these data demonstrate for the first time that IL-13 down-regulates NO production through arginase induction via cAMP/PKA, tyrosine kinase, and p38 MAPK signalings and underline the importance of arginase in the immunosuppressive activity of IL-13 in activated macrophages.