Nitric oxide-induced calcium release via ryanodine receptors regulates neuronal function

Mobilization of intracellular Ca(2+) stores regulates a multitude of cellular functions, but the role of intracellular Ca(2+) release via the ryanodine receptor (RyR) in the brain remains incompletely understood. We found that nitric oxide (NO) directly activates RyRs, which induce Ca(2+) release from intracellular stores of central neurons, and thereby promote prolonged Ca(2+) signalling in the brain. Reversible S-nitrosylation of type 1 RyR (RyR1) triggers this Ca(2+) release. NO-induced Ca(2+) release (NICR) is evoked by type 1 NO synthase-dependent NO production during neural firing, and is essential for cerebellar synaptic plasticity. NO production has also been implicated in pathological conditions including ischaemic brain injury, and our results suggest that NICR is involved in NO-induced neuronal cell death. These findings suggest that NICR via RyR1 plays a regulatory role in the physiological and pathophysiological functions of the brain.     

Nitric oxide-induced damage to mtDNA and its subsequent repair.

Mutations in mitochondrial DNA (mtDNA) have recently been associated with a variety of human diseases. One potential DNA-damaging agent to which cells are continually exposed that could be responsible for some of these mutations is nitric oxide (NO). To date, little information has been forthcoming concerning the damage caused by this gas to mtDNA. Therefore, this study was designed to investigate damage to mtDNA induced by NO and to evaluate its subsequent repair. Normal human fibroblasts were exposed to NO produced by the rapid decomposition of 1-propanamine, 3-(2-hydroxy-2-nitroso-1-propylhydrazino) (PAPA NONOate) and the resultant damage to mtDNA was determined by quantitative Southern blot analysis. This gas was found to cause damage to mtDNA that was alkali-sensitive. Treatment of the DNA with uracil-DNA glycosylase or 3-methyladenine DNA glycosylase failed to reveal additional damage, indicating that most of the lesions produced were caused by the deamination of guanine to xanthine. Studies using ligation-mediated PCR supported this finding. When a 200 bp sequence of mtDNA from cells exposed to NO was analyzed, guanine was found to be the predominantly damaged base. However, there also was damage to specific adenines. No lesions were observed at pyrimidine sites. The nucleotide pattern of damage induced by NO was different from that produced by either a reactive oxygen species generator or the methylating chemical, methylnitrosourea. Most of the lesions produced by NO were repaired rapidly. However, there appeared to be a subset of lesions which were repaired either slowly or not at all by the mitochondria.     

Nitric oxide-induced vasodilation of organic nitrate-tolerant rabbit aorta

It is postulated that the organic nitrate vasodilator agents, including glyceryl trinitrate (GTN) and isosorbide dinitrate (ISDN), are prodrugs, such that biotransformation to the active inorganic metabolite, nitric oxide (NO), occurs prior to the onset of vasodilation. Furthermore, it is proposed that organic nitrate tolerance in vascular tissue involves decreased formation of NO. To test this latter hypothesis, we examined vasodilation induced by NO, GTN, and ISDN in non-tolerant, GTN-tolerant, and ISDN-tolerant rabbit aortic rings (RARs). Isolated RARs were contracted submaximally with phenylephrine; the time of onset of relaxation and percent relaxation of tissue were determined in response to NO (0.3 microM), GTN (0.03 microM), and ISDN (0.12 microM) before and after a 1-h treatment with 500 microM GTN, 500 microM ISDN, or buffer only. The data demonstrated that the response to NO was not changed in GTN-tolerant and ISDN-tolerant tissues, in which there was virtually no GTN-induced or ISDN-induced relaxation. These results are consistent with the postulate that organic nitrate vasodilator drugs must undergo biotransformation to NO before vasodilation can occur and that the mechanism of organic nitrate tolerance involves decreased formation of NO.     

Nitric oxide-induced suspended animation promotes survival during hypoxia

Oxygen plays a key role in energy metabolism. However, there are organisms that survive severe shortfalls in oxygen. Drosophila embryos rapidly arrest development upon severe hypoxia and recover upon restoration of oxygen, even days later. Stabilization of the normally unstable engrailed RNA and protein preserved the localized striped pattern of this embryonic patterning gene during 3 days in hypoxia. Severe hypoxia blocked expression of a heat-shock-inducible lacZ transgene. Cyanide, a metabolic poison, did not immediately block gene expression or turnover, arguing against a passive response to energy limitation. In contrast, nitric oxide, a putative hypoxia signal, induced a reversible arrest of development, gene expression and turnover. Reciprocally, a nitric oxide scavenger allowed continued gene expression and turnover during hypoxia, but it reduced hypoxia tolerance. We suggest that hypoxia-induced stasis preserves the status quo of embryonic processes and promotes survival. Our data implicate nitric oxide as a mediator of this response and provide a system in which to investigate its action.     

Nitric oxide-induced cardioprotection in cultured rat ventricular myocytes

The aim of this study was to investigate the role of nitric oxide (NO) in a cellular model of early preconditioning (PC) in cultured neonatal rat ventricular myocytes. Cardiomyocytes "preconditioned" with 90 min of stimulated ischemia (SI) followed by 30 min reoxygenation in normal culture conditions were protected against subsequent 6 h of SI. PC was blocked by N(G)-monomethyl-L-arginine monoacetate but not by dexamethasone pretreatment. Inducible nitric oxide synthase (NOS) protein expression was not detected during PC ischemia. Pretreatment (90 min) with the NO donor S-nitroso-N-acetyl-L,L-penicillamine (SNAP) mimicked PC, resulting in significant protection. SNAP-triggered protection was completely abolished by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) but was unaffected by chelerythrine or the presence of glibenclamide and 5-hydroxydecanoate. With the use of RIA, SNAP treatment increased cGMP levels, which were blocked by ODQ. Hence, NO is implicated as a trigger in this model of early PC via activation of a constitutive NOS isoform. After exposure to SNAP, the mechanism of cardioprotection is cGMP dependent but independent of protein kinase C or ATP-sensitive K(+) channels. This differs from the proposed mechanism of NO-induced cardioprotection in late PC.     

Nitric oxide-induced mitochondrial dysfunction: implications for neurodegeneration

Excessive generation of nitric oxide (NO) has been implicated in the pathogenesis of several neurodegenerative disorders. Damage to the mitochondrial electron transport chain has also been implicated in these disorders. NO and its toxic metabolite peroxynitrite (ONOO(-)) can inhibit the mitochondrial respiratory chain, leading to energy failure and ultimately cell death. There appears to be a differential susceptibility of brain cell types to NO/ONOO(-), which may be influenced by factors including cellular antioxidant status and the ability to maintain energy requirements in the face of marked respiratory chain damage. Although formation of NO/ONOO(-) following cytokine exposure does not affect astrocyte survival, these molecules may diffuse out and cause mitochondrial damage to neighboring NO/ONOO(-)-sensitive cells such as neurons. Evidence suggests that NO/ONOO(-) causes release of neuronal glutamate, leading to glutamate-induced activation of neuronal NO synthase and generation of further damaging species. While neurons appear able to recover from short-term exposure to NO/ONOO(-), extending the period of exposure results in persistent damage to the respiratory chain and cell death ensues. These findings have important implications for acute infection vs. chronic neuroinflammatory disease states. The evidence for NO/ONOO(-)-mediated mitochondrial damage in neurodegenerative disorders is reviewed and potential therapeutic strategies are discussed.     

Nitric oxide-induced anti-mitogenic effects in high and low responder rat strains.

Nitric oxide (NO) has multiple biologic functions: in the brain it acts as a neuronal messenger; elsewhere, it causes smooth muscle relaxation, inhibition of platelet aggregation, inhibition of leukocyte adhesion, inhibition of tumor growth, and microbiostasis. Our studies show that production of NO is responsible for the unusual unresponsiveness of BN rat spleen cells to mitogens. NG-monomethyl-L-arginine (NGMMA), a potent competitive inhibitor for NO synthase, reverses this defect. Lysed RBC or NGMMA were shown to enhance mitogen-induced spleen cell proliferation only one- to twofold in Lewis rats (that have normal mitogen responsiveness) but act to stimulate BN rat T cells by 10- to 100-fold. NGMMA-enhanced proliferation was significantly diminished by prior depletion of macrophages. Surprisingly, NO did not inhibit IL-2 production in 48-h cultures of BN rat spleen cells, and exogenous IL-2 was ineffective in releasing NO-mediated suppression. These studies indicate that NO produced by macrophages can completely and reversibly inhibit T cell proliferation. The BN rat appears to be unique in its production of very high levels of NO, making it an especially useful animal model for studying the biologic control and functional consequences of NO generation.     

Nitric oxide-induced epidermal growth factor-dependent phosphorylations in A431 tumour cells.

Nitric oxide (NO*) strongly inhibits the proliferation of human A431 tumour cells. It also inhibits tyrosine phosphorylation of a 170-kDa band corresponding to the epidermal growth factor receptor (EGFR) and induces the phosphorylation at tyrosine residue(s) of a 58-kDa protein which we have denoted NOIPP-58 (nitric oxide-induced 58-kDa phosphoprotein). The NO*-induced phosphorylation of NOIPP-58 is strictly dependent on the presence of EGF. Phosphorylation of NOIPP-58 and inhibition of the phosphorylation of the band corresponding to EGFR are both cGMP-independent processes. We also demonstrate that the p38 mitogen-activated protein kinase (p38MAPK) pathway is activated by NO* in the absence and presence of EGF, whereas the activity of the extracellular signal-regulated protein kinase 1/2 (ERK1/2) and the c-Jun N-terminal kinase 1/2 (JNK1/2) pathways are not significantly affected or are slightly decreased, respectively, on addition of this agent. Moreover, we show that the p38MAPK inhibitor, SB202190, induces rapid vanadate/peroxovanadate-sensitive dephosphorylation of prephosphorylated EGFR and NOIPP-58. We propose that the dephosphorylation of both NOIPP-58 and EGFR are mediated by a p38MAPK-controlled phosphotyrosine-protein phosphatase (PYPP). Activation of the p38MAPK pathway during nitrosative stress probably prevents the operation of this PYPP, allowing NOIPP-58, and in part EGFR, to remain phosphorylated and therefore capable of generating signalling events.     

Nitric oxide-induced S-glutathionylation and inactivation of glyceraldehyde-3-phosphate dehydrogenase

S-Nitrosylation of protein thiol groups by nitric oxide (NO) is a widely recognized protein modification. In this study we show that nitrosonium tetrafluoroborate (BF4NO), a NO+ donor, modified the thiol groups of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by S-nitrosylation and caused enzyme inhibition. The resultant protein-S-nitrosothiol was found to be unstable and to decompose spontaneously, thereby restoring enzyme activity. In contrast, the NO-releasing compound S-nitrosoglutathione (GSNO) promoted S-glutathionylation of a thiol group of GAPDH both in vitro and under cellular conditions. The GSH-mixed protein disulfide formed led to a permanent enzyme inhibition, but upon dithiothreitol addition a functional active GAPDH was recovered. This S-glutathionylation is specific for GSNO because GSH itself was unable to produce protein-mixed disulfides. During cellular nitrosative stress, the production of intracellular GSNO might channel signaling responses to form protein-mixed disulfide that can regulate intracellular function.     

Nitric oxide-induced downregulation of leptin production by 3T3-L1 adipocytes.

Leptin secreted mainly by adipocytes plays an important role in insulin sensitivity in metabolic syndrome. Inducible nitric oxide synthase (iNOS) in 3T3-L1 adipocytes is induced by lipopolysaccharide (LPS) and several proinflammatory cytokines such as tumor necrosis factor-alpha and interferon-gamma (IFN-gamma). Because the role of iNOS-derived nitric oxide (NO) in adipocyte function has not been fully clarified, the question that we addressed in the present study was whether iNOS-derived NO is involved in regulation of leptin secretion by adipocytes. Incubation of 3T3-L1 adipocytes for 12h with a mixture of IFN-gamma and LPS caused not only a 55% reduction in leptin secretion and a 52% reduction in leptin mRNA, but also significant induction of iNOS at both protein and mRNA levels. Inhibition of leptin secretion that had been induced by the IFN-gamma-LPS mixture was completely nullified by NOS inhibitors such as Nomega-monomethyl-L-arginine and aminoguanidine. Treatment of adipocytes with NO donors such as an NONOate and S-nitrosoglutathione produced an effect on leptin secretion similar to that of the IFN-gamma-LPS mixture. It is likely therefore that NO mediates downregulation of leptin caused by the IFN-gamma-LPS mixture in 3T3-L1 adipocytes, which suggests an important role for NO in adipocyte functions.     

NMDA receptors in sensory information processing.

During the past year electrophysiological studies, particularly in the visual and somatosensory systems, have begun to uncover the specific roles played by NMDA receptors in the processing of sensory information. Many of the features of NMDA-receptor-mediated sensory responses reflect known properties of the receptor.     

Nitric oxide-induced bacteriostasis and modification of iron-sulphur proteins in Escherichia coli

The nitric oxide (NO) cytotoxicity has been well documented in bacteria and mammalian cells. However, the underlying mechanism is still not fully understood. Here we report that transient NO exposure effectively inhibits cell growth of Escherichia coli in minimal medium under anaerobic growth conditions and that cell growth is restored when the NO-exposed cells are either supplemented with the branched-chain amino acids (BCAA) anaerobically or returned to aerobic growth conditions. The enzyme activity measurements show that dihydroxyacid dehydratase (IlvD), an iron-sulphur enzyme essential for the BCAA biosynthesis, is completely inactivated in cells by NO with the concomitant formation of the IlvD-bound dinitrosyl iron complex (DNIC). Fractionation of the cell extracts prepared from the NO-exposed cells reveals that a large number of different protein-bound DNICs are formed by NO. While the IlvD-bound DNIC and other protein-bound DNICs are stable in cells under anaerobic growth conditions, they are efficiently repaired under aerobic growth conditions even without new protein synthesis. Additional studies indicate that L-cysteine may have an important role in repairing the NO-modified iron-sulphur proteins in aerobically growing E. coli cells. The results suggest that cellular deficiency to repair the NO-modified iron-sulphur proteins may directly contribute to the NO-induced bacteriostasis under anaerobic conditions.     

Nitric oxide-induced mitochondrial fission is regulated by dynamin-related GTPases in neurons

Mitochondria are present as tubular organelles in neuronal projections. Here, we report that mitochondria undergo profound fission in response to nitric oxide (NO) in cortical neurons of primary cultures. Mitochondrial fission by NO occurs long before neurite injury and neuronal cell death. Furthermore, fission is accompanied by ultrastructural damage of mitochondria, autophagy, ATP decline and generation of free radicals. Fission is occasionally asymmetric and can be reversible. Strikingly, mitochondrial fission is also an early event in ischemic stroke in vivo. Mitofusin 1 (Mfn1) or dominant-negative Dynamin related protein 1 (Drp1(K38A)) inhibits mitochondrial fission induced by NO, rotenone and Amyloid-beta peptide. Conversely, overexpression of Drp1 or Fis1 elicits fission and increases neuronal loss. Importantly, NO-induced neuronal cell death was mitigated by Mfn1 and Drp1(K38A). Thus, persistent mitochondrial fission may play a causal role in NO-mediated neurotoxicity.     

Glycine-dependent activation of NMDA receptors

N-methyl-d-aspartate (NMDA) receptors are the only neurotransmitter receptors whose activation requires two distinct agonists. Heterotetramers of two GluN1 and two GluN2 subunits, NMDA receptors are broadly distributed in the central nervous system, where they mediate excitatory currents in response to synaptic glutamate release. Pore opening depends on the concurrent presence of glycine, which modulates the amplitude and time course of the glutamate-elicited response. Gating schemes for fully glutamate- and glycine-bound NMDA receptors have been described in sufficient detail to bridge the gap between microscopic and macroscopic receptor behaviors; for several receptor isoforms, these schemes include glutamate-binding steps. We examined currents recorded from cell-attached patches containing one GluN1/GluN2A receptor in the presence of several glycine-site agonists and used kinetic modeling of these data to develop reaction schemes that include explicit glycine-binding steps. Based on the ability to match a series of experimentally observed macroscopic behaviors, we propose a model for activation of the glutamate-bound NMDA receptor by glycine that predicts apparent negative agonist cooperativity and glycine-dependent desensitization in the absence of changes in microscopic binding or desensitization rate constants. These results complete the basic steps of an NMDA receptor reaction scheme for the GluN1/GluN2A isoform and prompt a reevaluation of how glycine controls NMDA receptor activation. We anticipate that our model will provide a useful quantitative instrument to further probe mechanisms and structure–function relationships of NMDA receptors and to better understand the physiological and pathological implications of endogenous fluctuations in extracellular glycine concentrations.     

Agonist-specific gating of NMDA receptors

The mechanism by which ligand binding at extracellular receptor domains gates a transmembrane ion-conducting pore is insufficiently understood. Examining a channel's activation reaction in the presence of agonists with distinct efficacies may inform this issue and may help identify agonist-dependent transitions. We have recently applied this approach to NMDA receptors composed of GluN1 and GluN2A subunits. Based on our results with several subunit-specific partial agonists we concluded that agonist effects were distributed over several of the multiple transitions that make up NMDA receptor gating and that these changes were subunit independent. Here we examine an additional GluN2A partial agonist, 4-fluoro-D, L-glutamic acid, and we summarize the observed kinetic changes of all nine partial agonists investigated. These results support the premise that regardless of the subunit-type to which they bind, agonists influence multiple equilibria within the NMDA receptor reaction and may stabilize a slightly different family of conformers.     

Nitric oxide-induced cell death in the heart: the role of autophagy

There is unequivocal evidence of autophagy in the heart, both in human hearts from patients who experienced heart failure and in experimental models of myocardial ischemia and reperfusion. Whether autophagy is involved in the pathophysiology of these conditions is controversial as studies suggest inhibition of Beclin 1 can increase or decrease cardiomyocyte cell injury. Increased beclin 1 expression, however, has been consistently identified in myocardial ischemia/reperfusion. Because of the role of nitric oxide (NO) in myocardial ischemia/reperfusion as well as in heart failure, we sought to determine whether NO and its byproduct peroxynitrite alter the expression of some genes involved in autophagy in the heart. Neonatal mouse cardiomyocytes were treated with SIN-1 (3-morpholinosydnonimine), which releases NO and accelerates formation of peroxynitrite. Gene expression was evaluated using RNA labeled and hybridized to cDNA microarrays. SIN-1 treatment induced significant changes in five caspases. In contrast, there were no changes in three genes involved in autophagy, namely beclin 1, Atg5l and Atg12l. Several different time periods were examined; a short time period, 2h, to more closely model myocardial ischemia reperfusion and a long time period, 20 h, that more closely represents sustained injury. In summary, evidence to date suggests that NO is not involved in increased beclin 1 expression in ischemia/reperfusion injury in the heart and would be unlikely to account for the signs of autophagy in the hearts of patients with heart failure.     

Nitric oxide-induced conversion of cellular chelatable iron into macromolecule-bound paramagnetic dinitrosyliron complexes

One of the most important biological reactions of nitric oxide (nitrogen monoxide, *NO) is its reaction with transition metals, of which iron is the major target. This is confirmed by the ubiquitous formation of EPR-detectable g=2.04 signals in cells, tissues, and animals upon exposure to both exogenous and endogenous *NO. The source of the iron for these dinitrosyliron complexes (DNIC), and its relationship to cellular iron homeostasis, is not clear. Evidence has shown that the chelatable iron pool (CIP) may be at least partially responsible for this iron, but quantitation and kinetic characterization have not been reported. In the murine cell line RAW 264.7, *NO reacts with the CIP similarly to the strong chelator salicylaldehyde isonicotinoyl hydrazone (SIH) in rapidly releasing iron from the iron-calcein complex. SIH pretreatment prevents DNIC formation from *NO, and SIH added during the *NO treatment "freezes" DNIC levels, showing that the complexes are formed from the CIP, and they are stable (resistant to SIH). DNIC formation requires free *NO, because addition of oxyhemoglobin prevents formation from either *NO donor or S-nitrosocysteine, the latter treatment resulting in 100-fold higher intracellular nitrosothiol levels. EPR measurement of the CIP using desferroxamine shows quantitative conversion of CIP into DNIC by *NO. In conclusion, the CIP is rapidly and quantitatively converted to paramagnetic large molecular mass DNIC from exposure to free *NO but not from cellular nitrosothiol. These results have important implications for the antioxidative actions of *NO and its effects on cellular iron homeostasis.     

Ethanol sensitivity of NMDA receptors

NMDA receptors are ionotropic glutamate receptors assembled of subunits of the NR1 and of the NR2 family (NR2A–NR2D). The subunit diversity largely affects the pharmacological properties of NMDA receptors and, hence, gives rise to receptor heterogeneity. As an overall result of studies on recombinant and native NMDA receptors, ethanol inhibits the function of receptors containing the subunits NR2A and/or NR2B to a greater extent than those containing NR2C or NR2D. For example, in rat cultured mesencephalic neurons, NR2C expression was developmentally increased, whereas expression of NR2A and NR2B was decreased. These changes coincided with a developmental loss of sensitivity of NMDA responses to ethanol and ifenprodil, a non-competitive NMDA receptor antagonist that shows selectivity for NR2B-containing receptors. Also in rat locus coeruleus neurons, the low ethanol sensitivity of somatic NMDA receptors could be explained by a prominent expression of NR2C. The inhibitory site of action for ethanol on the NMDA receptor is not yet known. Patch–clamp studies suggest a target site exposed to or only accessible from the extracellular environment. Apparently, amino acid residue Phe⁶³⁹, located in the TM3 domain of NR1, plays a crucial role in the inhibition of NMDA receptor function by ethanol. Since this phenylalanine site is common to all NMDA and non-NMDA receptor (AMPA/kainate receptor) subunits, this observation is consistent with accumulating evidence for a similar ethanol sensitivity of a variety of NMDA and non-NMDA receptors, but it cannot explain the differences in ethanol sensitivity observed with different NR2 subunits.