Enhanced expression of neuronal nitric oxide synthase in islets of exercise-trained rats

It is well known that glucose-stimulated insulin secretion (GSIS) decreases after exercise training. In the present study, we investigated the effects of exercise training (9 weeks of running) on the activity of glucokinase (GK), the production of nitric oxide (NO), and the protein expressions of both glucose transporter-2 (GLUT-2) and NO synthase (NOS) in rat pancreatic islets. Exercise training significantly reduced GSIS, with decreases in GK activity and GLUT-2 protein expression. The NO releases and cGMP contents were higher in the islets of trained rats than in those of control rats. Exercise training enhanced cNOS activity, the protein expression of both neuronal nitric oxide synthase (nNOS) and calmodulin, and NADPH-cytochrome c reductase activity in the homogenates of islets. Thus, exercise training-induced reduction of GSIS would result from, at least in part, decreases in both glucose entry and the first step in glycolytic utilization of glucose. Moreover, exercise training could enhance the protein expression of nNOS, which in turn enhances two catalytic activities of nNOS, an NO production and a cytochrome c reductase activity.     

In search of potent and selective inhibitors of neuronal nitric oxide synthase with more simple structures

In certain neurodegenerative diseases damaging levels of nitric oxide (NO) are produced by neuronal nitric oxide synthase (nNOS). It, therefore, is important to develop inhibitors selective for nNOS that do not interfere with other NOS isoforms, especially endothelial NOS (eNOS), which is critical for proper functioning of the cardiovascular system. While we have been successful in developing potent and isoform-selective inhibitors, such as lead compounds 1 and 2, the ease of synthesis and bioavailability have been problematic. Here we describe a new series of compounds including crystal structures of NOS-inhibitor complexes that integrate the advantages of easy synthesis and good biological properties compared to the lead compounds. These results provide the basis for additional structure–activity relationship (SAR) studies to guide further improvement of isozyme selective inhibitors.     

Plasma membrane calcium ATPase 4b inhibits nitric oxide generation through calcium-induced dynamic interaction with neuronal nitric oxide synthase

The activation and deactivation of Ca²⁺- and calmodulindependent neuronal nitric oxide synthase (nNOS) in the central nervous system must be tightly controlled to prevent excessive nitric oxide (NO) generation. Considering plasma membrane calcium ATPase (PMCA) is a key deactivator of nNOS, the present investigation aims to determine the key events involved in nNOS deactivation of by PMCA in living cells to maintain its cellular context. Using time-resolved F?rster resonance energy transfer (FRET), we determined the occurrence of Ca²⁺-induced protein-protein interactions between plasma membrane calcium ATPase 4b (PMCA4b) and nNOS in living cells. PMCA activation significantly decreased the intracellular Ca²⁺ concentrations ([Ca²⁺]_i), which deactivates nNOS and slowdowns NO synthesis. Under the basal [Ca²⁺]_i caused by PMCA activation, no protein-protein interactions were observed between PMCA4b and nNOS. Furthermore, both the PDZ domain of nNOS and the PDZ-binding motif of PMCA4b were essential for the protein-protein interaction. The involvement of lipid raft microdomains on the activity of PMCA4b and nNOS was also investigated. Unlike other PMCA isoforms, PMCA4 was relatively more concentrated in the raft fractions. Disruption of lipid rafts altered the intracellular localization of PMCA4b and affected the interaction between PMCA4b and nNOS, which suggest that the unique lipid raft distribution of PMCA4 may be responsible for its regulation of nNOS activity. In summary, lipid rafts may act as platforms for the PMCA4b regulation of nNOS activity and the transient tethering of nNOS to PMCA4b is responsible for rapid nNOS deactivation.     

Electron transfer is activated by calmodulin in the flavin domain of human neuronal nitric oxide synthase

The objective of this study was to clarify the mechanism of electron transfer in the human neuronal nitric oxide synthase (nNOS) flavin domain using the recombinant human nNOS flavin domains, the FAD/NADPH domain (contains FAD- and NADPH-binding sites), and the FAD/FMN domain (the flavin domain including a calmodulin-binding site). The reduction by NADPH of the two domains was studied by rapid-mixing, stopped-flow spectroscopy. For the FAD/NADPH domain, the results indicate that FAD is reduced by NADPH to generate the two-electron-reduced form (FADH(2)) and the reoxidation of the reduced FAD proceeds via a neutral (blue) semiquinone with molecular oxygen or ferricyanide, indicating that the reduced FAD is oxidized in two successive one-electron steps. The neutral (blue) semiquinone form, as an intermediate in the air-oxidation, was unstable in the presence of O(2). The purified FAD/NADPH domain prepared under our experimental conditions was activated by NADP(+) but not NAD(+). These results indicate that this domain exists in two states; an active state and a resting state, and the enzyme in the resting state can be activated by NADP(+). For the FAD/FMN domain, the reduction of the FAD-FMN pair of the oxidized enzyme with NADPH proceeded by both one-electron equivalent and two-electron equivalent mechanisms. The formation of semiquinones from the FAD-FMN pair was greatly increased in the presence of Ca(2+)/CaM. The air-stable semiquinone form, FAD-FMNH(.), was further rapidly reduced by NADPH with an increase at 520 nm, which is a characteristic peak of the FAD semiquinone. Results presented here indicate that intramolecular one-electron transfer from FAD to FMN is activated by the binding of Ca(2+)/CaM.     

Chiral linkers to improve selectivity of double-headed neuronal nitric oxide synthase inhibitors

To develop potent and selective nNOS inhibitors, new double-headed molecules with chiral linkers that derive from natural amino acids or their derivatives have been designed. The new structures contain two ether bonds, which greatly simplifies the synthesis and accelerates structure optimization. Inhibitor (R)-6b exhibits a potency of 32 nM against nNOS and is 475 and 244 more selective for nNOS over eNOS and iNOS, respectively. Crystal structures show that the additional binding between the aminomethyl moiety of 6b and the two heme propionates in nNOS, but not eNOS, is the structural basis for its high selectivity. This work demonstrates the importance of stereochemistry in this class of molecules, which significantly influences the potency and selectivity of the inhibitors. The structure–activity information gathered here provides a guide for future structure optimization.     

The antithyroid agent 6-n-propyl-2-thiouracil is a mechanism-based inactivator of the neuronal nitric oxide synthase isoform

6-n-Propyl-2-thiouracil (6-PTU), the antithyroid agent, produces a time-, concentration-, and turnover-dependent inactivation of the NO synthetic capability of the neuronal nitric oxide synthase isoform irreversible by either arginine or (6R)-5,6,7,8-tetrahydro-L-biopterin. By contrast 6-PTU produces an inhibition of the cytokine-inducible and endothelial nitric oxide synthases fully reversible by arginine. The inactivation of neuronal nitric oxide synthase by 6-PTU follows first order kinetics, and is inhibited competitively by both arginine and (6R)-5,6,7,8-tetrahydro-L-biopterin, but is not accompanied by either a loss of heme-CO binding, heme fluorescence, or disassembly of dimeric structure. 2-Thiouracil behaves qualitatively identically to 6-PTU. Turnover-dependent inactivation of neuronal nitric oxide synthase by [2-14C]-2-thiouracil is accompanied by incorporation of radioactivity into the polypeptide chain. Ca2+-dependent NO formation by GH3 pituitary cells is inhibited by 6-PTU in a manner enhanced by depletion of either extracellular arginine or intracellular (6R)-5,6,7,8-tetrahydro-L-biopterin. These observations establish that 6-PTU is an alternate substrate, mechanism-based inactivator of the neuronal nitric oxide synthase isoform with the ability to suppress cellular NO formation.     

Phosphorylation of neuronal nitric oxide synthase at Ser in the dentate gyrus of rat brain after transient forebrain ischemia

We previously demonstrated that calmodulin-dependent protein kinase IIα (CaM-KIIα) phosphorylates nNOS at Ser⁸⁴⁷ in the hippocampus after forebrain ischemia; this phosphorylation attenuates NOS activity and might contribute to resistance to post-ischemic damage. We also revealed that cyclic AMP-dependent protein kinase (PKA) could phosphorylate nNOS at Ser¹⁴¹²in vitro. In this study, we focused on chronological and topographical changes in the phosphorylation of nNOS at Ser¹⁴¹² after rat forebrain ischemia. The hippocampus and adjacent cortex were collected at different times, up to 24 h, after 15 min of forebrain ischemia. NOS was partially purified from crude samples using ADP agarose gel. Neuronal NOS, phosphorylated (p)-nNOS at Ser¹⁴¹², PKA, and p-PKA at Thr¹⁹⁷ were studied in the rat hippocampus and cortex using Western blot analysis and immunohistochemistry. Western blot analysis revealed that p-nNOS at Ser¹⁴¹² significantly increased between 1 and 6 h after reperfusion in the hippocampus, but not in the cortex. PKA was cosedimented with nNOS by ADP agarose gel. Immunohistochemistry revealed that phosphorylation of nNOS at Ser¹⁴¹² and PKA at Thr¹⁹⁷ occurred in the subgranular layer of the dentate gyrus. Forebrain ischemia might thereby induce temporary activation of PKA at Thr¹⁹⁷, which then phosphorylates nNOS at Ser¹⁴¹² in the subgranular layer of the dentate gyrus.     

Induction of nitric oxide synthase expression by Vibrio vulnificus cytolysin.

The pore-forming cytolysin of Vibrio vulnificus (VVC) causes severe hypotension and vasodilatation in vivo. Under the condition of bacterial sepsis, large amounts of nitric oxide (NO) produced by inducible NO synthase (iNOS) can contribute to host-induced tissue damage causing hypotension and septic shock. In this study, we investigated the effect of purified VVC on NO production in mouse peritoneal macrophages. VVC induced NO production in the presence of interferon-gamma. Increased NO production was not affected by polymyxin B, and heat inactivation of cytolysin abolished the NO-inducing capability. NO production was induced at the same concentration range of cytolysin for pore formation, as evidenced by the release of preloaded 2-deoxy-d-[(3)H]glucose. At the higher concentrations of cytolysin causing the depletion of cellular ATP, no NO production was observed. Increased expression of iNOS and activation of NFkappaB by VVC were confirmed by Western blotting and gel shift assay, respectively. These results suggest the role of cytolysin as an inducer of iNOS and NO production in macrophage and as a possible virulence determinant in V. vulnificus infection.     

Spectrofluorimetric analysis of the interaction of amyloid peptides with neuronal nitric oxide synthase: Implications in Alzheimer's disease

Background

The deposition of aggregated β-amyloid peptide senile plaques and the accumulation of arginine within the astrocytes in the brain of an Alzheimer's patient are classic observations in the neuropathology of the disease. It would be logical, in the aetiology and pathogenesis, to investigate arginine-metabolising enzymes and their intimate association with amyloid peptides.

Methods

Neuronal nitric oxide synthase (nNOS) was isolated, purified and shown, through fluorescence quenching spectroscopy and fluorescence resonance energy transfer (FRET), to interact with structural fragments of Aβ_1–40 and be catalytic towards amyloid fibril formation.

Results

Only one binding site on the enzyme was available for binding. Two amyloid peptide fragments of Aβ_1–40 (Aβ_17–28 and Aβ_25–35) had Stern–Volmer values (K_SV) of 0.111 μM⁻¹ and 0.135 μM⁻¹ indicating tight binding affinity to nNOS and easier accessibility to fluor molecules during binding. The polarity of this active site precludes binding of the predominantly hydrophobic amyloid peptide fragments contained within Aβ_17–28 and within two glycine zipper motifs [G-X-X-X-G-X-X-X-G] [Aβ_29–37] and bind to the enzyme at a site remote to the active region.

Conclusions

The interaction and binding of Aβ_17–28 and Aβ_25–35 to nNOS causes the movement of two critical tryptophan residues of 0.77 nm and 0.57 nm respectively towards the surface of the enzyme.

General significance

The binding of Aβ-peptide fragments with nNOS has been studied by spectrofluorimetry. The information and data presented should contribute towards understanding the mechanism for deposition of aggregated Aβ-peptides and fibrillogenesis in senile plaques in an AD brain.     

Characterization of Drosophila nitric oxide synthase: a biochemical study

The heme and flavin-binding domains of Drosophila nitric oxide synthase (DNOS) were expressed in Escherichia coli using the expression vector pCW. The denatured molecular mass of the expressed protein was 152kDa along with a proteolytically cleaved product of 121kDa. The DNOS heme protein exhibited very low Ca(2+)/calmodulin-dependent NO synthase activity. The trypsin digestion patterns were different from nNOS. The full-length DNOS protein had high degree of stability against trypsin. The activity assay of trypsin-digested protein confirmed the same result. Urea dissociation profile of DNOS full-length protein showed that the reductase domain activity was much more susceptible towards urea than the oxygenase domain activity. Urea gradient gel of DNOS full-length protein established distinct transition of dissociation and unfolding in the range 3-4M urea. Reductase domain activity of full-length DNOS protein against external electron acceptors like cytochrome c indicated slow electron transfer from FMN. The bacterial expression of DNOS full-length protein represents an important development in structure-function studies of this enzyme and comparison with other mammalian NOS enzymes which is evolutionary significant.     

Monitoring the activation of neuronal nitric oxide synthase in brain tissue and cells with a potentiometric immunosensor

An all solid state potentiometric immunosensor (ASPI) has been developed to study the activation process of neuronal nitric oxide synthase (nNOS), the enzyme involved in the synthesis of nitric oxide generated under physiological conditions. At first, an all solid state H⁺-selective ISE was fabricated with the carboxylated poly(vinyl chloride) (PVC-COOH) film containing H⁺ ionophore, antibody was then immobilized on the polymer layer. The immunocomplex formation was detected by monitoring pH change due to interaction between urease labeled secondary antibody and antigen. Experimental parameters such as the amount of phosphorylated nNOS immobilized on the electrode surface and pH responses due to the antibody–antigen reaction were studied in detail. The calibration plot of the potentiometric potential vs. phosphorylated nNOS concentration exhibited a linear relationship in the range of 3.4–340.0 μg/ml. The calibration sensitivity of the phosphorylated nNOS immunosensor was −0.073 ± 0.002 mV/μg ml⁻¹. The detection limit of nNOS was determined to be 0.2 μg/ml based on five-time measurements (95% confidence level, k = 3, n = 5). The reliability of the immunosensor was examined with rat brain tissues as well as neuronal cells, and the results shown were good, implying a promising approach for a novel electrochemical immunosensor platform with potential applications to clinical diagnosis.     

电针对去卵巢大鼠学习记忆能力及海马神经元型一氧化氮合酶mRNA表达的影响

目的研究电针对去卵巢大鼠学习记忆能力及海马神经元型一氧化氮合酶（nNOS）mRNA表达的影响。方法采用卵巢切除大鼠模型,造成低雌激素记忆障碍,去势2周后进行电针刺激,连续治疗3个月。Morris水迷宫测试空间学习记忆能力,酶联免疫吸附分析（ELISA）检测血清雌二醇（E2）浓度,实时荧光定量PCR检测检测nNOSmRNA的相对表达量。结果与假手术组比较,模型组大鼠逃避潜伏期时间明显延长,跨越平台次数明显减少,血清E2浓度和海马nNOSmR—NA表达显著降低（P〈O．01）;与模型组比较,电针组和假电针组治疗后逃避潜伏期缩短,跨越平台次数增加,血清E2浓度和海马nNOSmRNA表达均显著升高,电针组升高更明显（P〈O．01）。结论电针能够提高去卵巢大鼠学习记忆能力,其机制可能与升高体内雌激素浓度上调海马nNOSmRNA的表达有关。     

The localization of neuronal nitric oxide synthase may influence its role in neuronal precursor proliferation and synaptic maintenance

Neuronal nitric oxide synthase (nNOS) is implicated in some developmental processes, including neuronal survival, differentiation, and precursor proliferation. To define the roles of nNOS in neuronal development, we utilized the olfactory system as a model. We hypothesized that the role of nNOS may be influenced by its localization. nNOS expression was developmentally regulated in the olfactory system. During early postnatal development, nNOS was expressed in developing neurons in the olfactory epithelium (OE), while in the adult its expression was restricted to periglomerular (PG) cells in the olfactory bulb (OB). At postnatal week 1 (P1W), loss of nNOS due to targeted gene deletion resulted in a decrease in immature neurons in the OE due to decreased proliferation of neuronal precursors. While the pool of neuronal precursors and neurogenesis normalized in the nNOS null mouse by P6W, there was an overgrowth of mitral or tufted cells dendrites and a decreased number of active synapses in the OB. Cyclic GMP (cGMP) immunostaining was reduced in the OE and in the glomeruli of the OB at early postnatal and adult ages, respectively. Our results suggest that nNOS appears necessary for neurogenesis in the OE during early postnatal development and for glomerular organization in the OB in the adult. Thus, the location of nNOS, either within cell bodies or perisynaptically, may influence its developmental role.     

Effect of potential amine prodrugs of selective neuronal nitric oxide synthase inhibitors on blood–brain barrier penetration

Several prodrug approaches were taken to mask amino groups in two potent and selective neuronal nitric oxide synthase (nNOS) inhibitors containing either a primary or secondary amino group to lower the charge and improve blood–brain barrier (BBB) penetration. The primary amine was masked as an azide and the secondary amine as an amide or carbamate. The azide was not reduced to the amine under a variety of in vitro and ex vivo conditions. Despite the decrease in charge of the amino group as an amide and as carbamates, BBB penetration did not increase. It appears that the uses of azides as prodrugs for primary amines or amides and carbamates as prodrugs for secondary amines are not universally effective for CNS applications.     

Inhibition of cerebellar nitric oxide synthase and cyclic GMP production by melatonin via complex formation with calmodulin

Constitutive rat cerebellar nitric oxide synthase (NOS) activity is shown to be inhibited by physiological concentrations of the pineal hormone melatonin. The inhibition was dose-dependent and was coupled to an inhibition of the cyclic GMP production activated by L-arginine. Results also show that calmodulin appears to be involved in this process because its presence in the incubation medium was able to prevent the effect of melatonin on both NOS activity and cyclic GMP production. Moreover, polyacrylamide gel electrophoresis studies suggest that melatonin can interact with calmodulin modifying the binding of the peptide to the synthetic NOS peptide encompassing the calmodulin-binding domain of constitutive NOS from rat cerebellum, the natural mechanism by which calmodulin activates cerebellar NOS. J. Cell. Biochem. 65:430–442. © 1997 Wiley-Liss, Inc.     

Differential localization of neuronal nitric oxide synthase immunoreactivity and NADPH-diaphorase activity in the cat spinal cord

The distributions of neuronal nitric oxide synthase immunoreactivity (NOS-IR) and NADPH-diaphorase (NADPH-d) activity were compared in the cat spinal cord. NOS-IR in neurons around the central canal, in superficial laminae (I and II) of the dorsal horn, in the dorsal commissure, and in fibers in the superficial dorsal horn was observed at all levels of the spinal cord. In these regions, NOS-IR paralleled NADPH-d activity. The sympathetic autonomic nucleus in the rostral lumbar and thoracic segments exhibited prominent NOS-IR and NADPH-d activity, whereas the parasympathetic nucleus in the sacral segments did not exhibit NOS-IR or NADPH-d activity. Within the region of the sympathetic autonomic nucleus, fewer NOS-IR cells were identified compared with NADPH-d cells. The most prominent NADPH-d activity in the sacral segments occurred in fibers within and extending from Lissauer's tract in laminae I and V along the lateral edge of the dorsal horn to the region of the sacral parasympathetic nucleus. These afferent projections did not exhibit NOS-IR; however, NOS-IR and NADPH-d activity were demonstrated in dorsal root ganglion cells (L7-S2). The results of this study demonstrate that NADPH-d activity is not always a specific histochemical marker for NO-containing neural structures.     

Down-regulation of adrenal neuronal nitric oxide synthase mRNAs and proteins after deoxycorticosterone acetate-salt treatment in rats

The aim of this study was to evaluate the possible changes of adrenal neuronal nitrite oxide synthase (nNOS) messenger RNA (mRNA) and protein of rats after deoxycorticosterone acetate (DOCA)-salt treatment. We determined adrenal nNOS expression in 12 vehicle-treated and 13 DOCA-salt-treated rats by in situ hybridization, immunohistochemistry, and multiplex RT-PCR methods. Adrenal nNOS was also detected by Western blot in five vehicle-treated and five DOCA-salt-treated rats. The results showed that adrenal nNOS mRNA and nNOS immunoreactivities were mainly localized in the medulla and some in the regions of zona glomerulosa. DOCA-salt treatment inactivated nNOS mRNA and peptide expression prominent in the adrenal medulla and slight in the zona glomerulosa. The relative quantities of nNOS mRNA in the adrenals of the DOCA-salt-treated group was 8.8-fold decreased. At the same time, the relative quantities of steroid acute regulatory protein mRNA and phenylethanolamine N-methyltransferase mRNA in the adrenals of the DOCA-salt-treated group were significantly decreased. Western blots showed that total adrenal nNOS were 3.7-fold down-regulated after DOCA-salt treatment. Our results indicated that the down-regulation of adrenal nNOS synthesis might be associated with the inactivation of adrenal function in face of volume expansion.     

Peripheral neuronal nitric oxide synthase activity mediates the antinociceptive effect of Crotalus durissus terrificus snake venom, a delta- and kappa-opioid receptor agonist

Previous work has shown that nitric oxide (NO) mediates the antinociceptive effect of Crotalus durissus terrificus venom on carrageenin-induced hyperalgesia. In the present study the role of constitutive neuronal or of inducible form of nitric oxide synthase on venom effect was determined. The rat paw prostaglandin E(2) (PGE(2))-induced mechanical hyperalgesia model was used for nociceptive evaluation. The venom (200 microg/kg) administered per oz immediately before prostaglandin induced antinociception that persisted for 120 h. The characterisation of the antinociceptive effect of the venom in this model of hyperalgesia showed that kappa and delta-opioid receptors are involved in this effect. 7-nitroindazole (7-NI), a neuronal nitric oxide synthase (NOS) inhibitor, but not L-N(6)-(1-iminoethyl)lysine (L-NIL), an inhibitor of the inducible form of NOS, injected by intraplantar (i.pl.) route, antagonized the antinociceptive effect of the venom. The i.pl. administration of 1H-(1,2,4)oxadiazolo[4,3-a]quinoxaline-1-one (ODQ), a selective guanylate cyclase inhibitor, blocked antinociception, whereas Rp-cGMP triethylamine, a cGMP-dependent protein kinase inhibitor, partially reversed this effect. These data indicate that peripheral kappa- and delta-opioid receptors are involved in the antinociceptive effect of Crotalus durissus terrificus on prostaglandin E(2)-induced hyperalgesia. Peripheral nitric oxide, generated by neuronal NO synthase, and cGMP/PKc are responsible, at least partially, for the molecular mechanisms of venom effect.     

Binding kinetics of calmodulin with target peptides of three nitric oxide synthase isozymes

Efficient electron transfer from reductase domain to oxygenase domain in nitric oxide synthase (NOS) is dependent on the binding of calmodulin (CaM). Rate constants for the binding of CaM to NOS target peptides was only determined previously by surface plasmon resonance (SPR) (Biochemistry 35, 8742-8747, 1996) suggesting that the binding of CaM to NOSs is slow and does not support the fast electron transfer in NOSs measured in previous and this studies. To resolve this contradiction, the binding rates of holo Alexa 350 labeled T34C/T110W CaM (Alexa-CaM) to target peptides from three NOS isozymes were determined using fluorescence stopped-flow. All three target peptides exhibited fast k_on constants at 4.5 °C: 6.6 × 10⁸ M^− 1 s^− 1 for nNOS_726-749, 2.9 × 10⁸ M^− 1 s^− 1 for eNOS_492-511 and 6.1 × 10⁸ M^− 1 s^− 1 for iNOS_507-531, 3-4 orders of magnitude faster than those determined previously by SPR. Dissociation rates of NOS target peptides from Alexa-CaM/peptide complexes were measured by Ca²⁺ chelation with ETDA: 3.7 s^− 1 for nNOS_726-749, 4.5 s^− 1 for eNOS_492-511, and 0.063 s^− 1 for iNOS_507-531. Our data suggest that the binding of CaM to NOS is fast and kinetically competent for efficient electron transfer and is unlikely rate-limiting in NOS catalysis. Only iNOS_507-531 was able to bind apo Alexa-CaM, but in a very different conformation from its binding to holo Alexa-CaM.