Brain Neuronal Nitric Oxide Synthase Research Articles

Effects of Aerobic Physical Training on Thermoregulatory Adjustments During Physical Exercise in Spontaneously Hypertensive Rats

This study aimed to investigate the effects of physical training on thermoregulatory adjustments during running exercise and its effects on brain neuronal nitric oxide synthase (n‐NOS) expression. Male spontaneously hypertensive (SHR) and normotensive Wistar rats (NWR) at 8‐weeks age old were divided into four groups: untrained NWR (NWR‐U), trained NWR (NWR‐T), untrained SHR (SHR‐U) and trained SHR (SHR‐T). Experimental procedures were approved by the CEUA‐UFMG (#21/2015). Aerobic exercise training was performed on a treadmill running for 8 weeks. On the day of the experiments, the rats were subjected to a constant‐speed (60% of Vmáx) protocol until fatigue at 25°C. Tail skin temperature (Ts) and abdominal temperature (Ta) were measured every minute throughout the exercise trials. The preoptic area (POA) and paraventricular nuclei (PVN) of hypothalamus was collected to quantification of n‐NOS(ser852) by western blot. During exercise, the SHR‐U group showed greater increase in Ta compared to with NWR‐U group from the 16th min until the 23th min (min 23: 1.62 ± 0.15 °C vs. 1.21 ± 0.12 °C, p<0.05) and lower Ts from the 1th until the 16th min of exercise (min 16: 0.09 ± 0.95 °C vs. 1.39 ± 0.41 °C, p<0.05). Physical training in SHRs group did not change Ta response during exercise (fatigue: SHR‐T: 1.54 ± 0.29 °C vs. SHR‐U: 1.70 ± 0.18 °C, p<0.05) but caused a greater increase in Ts in SHR‐T group (min 12: −0.54 ± 0.93 °C vs. −2.64 ± 0.80 °C, p<0.05). Physical training in NWR group did not change Ta response during exercise (fatigue: NWR‐T: 1.62 ± 0.26 °C vs. NWR‐U: 1.71 ± 0.26 °C, p<0.05) but caused a greater increase in Ts in NWR‐T group (fatigue: 3.65 ± 0.38 °C vs. 1.60 ± 0.47 °C, p<0.05). The p‐nNOS expression in APO was lower in SHR‐U group when compared to the NWR‐U (0.06 ± 0.01 u.a. vs. 0.09 ± 0.01 u.a., p<0.05). Physical training increased the p‐nNOS expression in the APO in the SHR group (0.10 ± 0.01 u.a. vs. 0.06 ± 0.01 u.a., p<0.05). The p‐nNOS expression in PVN was similar between SHR‐U and NWR‐U groups (SHR‐U: 1.10 ± 0.19 u.a vs. NWR NT: 0.94 ± 0.13 u.a., p>0.05). Physical training increased p‐NOS expression in the APO in both SHR group (1.58 ± 0.17 u.a. vs. 1.10 ± 0.19 u.a., p<0.05) and NWR group (1.56 ± 0.25 u.a vs. 0.94 ± 0.13 u.a., p<0.05). We conclude that physical training increases the efficiency of thermoregulatory control during exercise in hypertensive animals, which can be explained, at least in part, to an increase in the activity of neuronal nitric oxide synthase in trained animals.Support or Funding InformationFapemig, Capes, and CNPq.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Chronic cocaine induces HIF-VEGF pathway activation along with angiogenesis in the brain.

Cocaine induces vasoconstriction in cerebral vessels, which with repeated use can result in transient ischemic attacks and cerebral strokes. However, the neuroadaptations that follow cocaine’s vasoconstricting effects are not well understood. Here, we investigated the effects of chronic cocaine exposure (2 and 4 weeks) on markers of vascular function and morphology in the rat brain. For this purpose we measured nitric oxide (NO) concentration in plasma, brain neuronal nitric oxide synthase (nNOS or NOS1), HIF-1α, and VEGF expression in different brain regions, i.e., middle prefrontal cortex, somatosensory cortex, nucleus accumbens, and dorsal striatum, using ELISA or Western blot. Additionally, microvascular density in these brain regions was measured using immunofluorescence microscopy. We showed that chronic cocaine significantly affected NOS1, HIF-1α and VEGF expression, in a region- and cocaine treatment-time- dependent manner. Cerebral microvascular density increased significantly in parallel to these neurochemical changes. Furthermore, significant correlations were detected between VEGF expression and microvascular density in cortical regions (middle prefrontal cortex and somatosensory cortex), but not in striatal regions (nucleus accumbens and dorsal striatum). These results suggest that following chronic cocaine use, as cerebral ischemia developed, NOS1, the regulatory protein to counteract blood vessel constriction, was upregulated; meanwhile, the HIF-VEGF pathway was activated to increase microvascular density (i.e., angiogenesis) and thus restore local blood flow and oxygen supply. These physiological responses were triggered presumably as an adaptation to minimize ischemic injury caused by cocaine. Therefore, effectively promoting such physiological responses may provide novel and effective therapeutic solutions to treat cocaine-induced cerebral ischemia and stroke.

Maternal magnesium sulfate fetal neuroprotective effects to the fetus: inhibition of neuronal nitric oxide synthase and nuclear factor kappa-light-chain-enhancer of activated B cells activation in a rodent model

Maternal magnesium administration has been shown to protect the preterm fetus from white- and gray-matter injury, although the mechanism is unknown. The purpose of the study is to test the following hypotheses: (1) maternal infections/inflammation activate fetal neuronal N-methyl-D-aspartate receptors that up-regulate neuronal nitric oxide synthase and nuclear factor kappa-light-chain-enhancer of activated Bcells pathways; and (2) maternal magnesium sulfate attenuates fetal brain neuronal nitric oxide synthase and nuclear factor kappa-light-chain-enhancer of activated B cells activation through N-methyl-D-aspartate receptors. Pregnant rats at embryonic day 16 and embryonic day 18 (n= 6, 48 total) received injections of intraperitoneal lipopolysaccharide 500 μg/kg or saline at time 0. Dams were randomized for treatment with subcutaneous magnesium sulfate (270 mg/kg) or saline for 2 hours prior to and following lipopolysaccharide/saline injections. At 4 hours after lipopolysaccharide administration, fetal brains were collected from the 4treatment groups (lipopolysaccharide/saline, lipopolysaccharide/magnesium sulfate, saline/magnesium sulfate, saline/saline), and phosphoneuronal nitric oxide synthase, nuclear factor kappa-light-chain-enhancer of activated B cells p65, and chemokine (C-C motif) ligand 2 protein levels were determined by Western blot. An additional group of pregnant rats (n= 5) received N-methyl-D-aspartate-receptor antagonist following the lipopolysaccharide injection to study magnesium sulfate protective mechanism. Lipopolysaccharide (lipopolysaccharide/saline) significantly increased fetal brain phosphoneuronal nitric oxide synthase, nuclear factor kappa-light-chain-enhancer of activated B cells p65, and chemokine (C-C motif) ligand 2 protein levels compared to the saline/saline group at both embryonic day 16 (phosphoneuronal nitric oxide synthase 0.23 ± 0.01 vs 0.11 ± 0.01 U; nuclear factor kappa-light-chain-enhancer of activated B cells 0.24 ± 0.01 vs 0.14 ± 0.01 U; chemokine (C-C motif) ligand 2 0.28 ± 0.01 vs .01 ± 0.01 U) and embryonic day 18 (phosphoneuronal nitric oxide synthase 0.28 ± 0.01 vs 0.12 ± 0.01 U; nuclear factor kappa-light-chain-enhancer of activated B cells 0.12 ± 0.01 vs 0.1 ± 0.01 U; chemokine (C-C motif) ligand 2 0.27 ± 0.01 vs 0.11 ± 0.01 U). Magnesium sulfate treatment to lipopolysaccharide dams (lipopolysaccharide/magnesium sulfate) significantly decreased fetal brain phosphoneuronal nitric oxide synthase, nuclear factor kappa-light-chain-enhancer of activated B cells, and chemokine (C-C motif) ligand 2 protein levels compared to lipopolysaccharide/saline dams at both embryonic day 16 (neuronal nitric oxide synthase 0.17 ± 0.02 U; nuclear factor kappa-light-chain-enhancer of activated B cells 0.17 ± 0.03 U; chemokine (C-C motif) ligand 2 0.18 ± 0.01 U) and embryonic day 18 (phosphoneuronal nitric oxide synthase 0.1 ± 0.01 U; nuclear factor kappa-light-chain-enhancer of activated B cells 0.09 ± 0.01 U; chemokine (C-C motif) ligand 2 0.21 ± 0.01 U). Notably, maternal lipopolysaccharide at embryonic day 16 activated nuclear factor kappa-light-chain-enhancer of activated B cells twice as often compared to dams induced at embryonic day 18. N-methyl-D-aspartate-receptor antagonist decreased fetal brain phosphoneuronal nitric oxide synthase and nuclear factor kappa-light-chain-enhancer of activated B cells levels comparable to magnesium sulfate. Lipopolysaccharide-simulated inflammation during pregnancy may cause brain injury through activation of neuronal nitric oxide synthase and nuclear factor kappa-light-chain-enhancer of activated B cells pathways and, potentially, production of excitotoxic nitric oxide and inflammatory mediators. The increased susceptibility to brain injury in preterm fetuses may be due to enhanced nuclear factor kappa-light-chain-enhancer of activated B cells activation. Magnesium sulfate protective effects may be secondary, in part, to inhibition of neuronal nitric oxide synthase and nuclear factor kappa-light-chain-enhancer of activated B cells activation and decrease proinflammatory cytokine production through blocking nuclear factor kappa-light-chain-enhancer of activated B cells receptors.

Free radical production from the interaction of 2-chloroethyl vesicants (mustard gas) with pyridine nucleotide-driven flavoprotein electron transport systems

The biochemical sequelae to chloroethyl mustard exposure correspond very well to toxic processes initiated by free radicals. Additionally, mustard solutions contain spontaneously formed cyclic onium ions which produce carbon free radicals when reduced electrochemically. Therefore, we hypothesized that the onium ions of sulfur or nitrogen mustards might produce carbon free radicals upon being reduced enzymatically, and that these radicals might constitute a metabolic activation. We set out to document radical production using an in vitro metabolic system and electron paramagnetic resonance (EPR). Our system consisted of NADPH, one of several pyridine nucleotide-driven flavoprotein reductases, cytochrome c as a terminal electron acceptor, various sulfur or nitrogen mustards and the spin trap α-[4-pyridyl-1-oxide]- N- tert-butylnitrone in buffer. Reactions were started by adding the reductase to the other materials, vortexing and immediately transferring the mixture to a 10 mm EPR flat cell. Repeated scans on a Bruker ESP 300E EPR spectrometer produced a triplet of doublets with hyperfine splitting constants of a N = 15.483 G and a H = 2.512 G. The outcome supported our hypothesis that carbon-centered free radicals are produced when mustard-related onium ions are enzymatically reduced. The EPR results varied little with the chloroethyl compound used or with porcine or human cytochrome P450 reductase, the reductase domain of rat brain neuronal nitric oxide synthase or rat liver thioredoxin reductase. Our results offer new insight into the basis for mustard-induced vesication and the outcome of exposure to different mustards. The free radical model provides an explanation for similarities in the lesions arising from mustard exposure and energy-based lesions such as those from heat, ultraviolet and nuclear radiation as well as damage across tissue types such as skin, eyes or airway epithelium.

Brain neuronal nitric oxide synthase neuron-mediated sympathoinhibition is enhanced in hypertensive Dahl rats

To elucidate the role of central neurons containing neuronal nitric oxide synthase (nNOS neurons) in the sympathetic nervous system in hypertensive Dahl salt-sensitive (DS) rats. Dahl rats were fed either a regular-salt (0.4% NaCl) or high-salt (8% NaCl) diet for 4 weeks. The effect of intracerebroventricular administration of S-methyl-L-thiocitrulline, a selective nNOS inhibitor, on renal sympathetic nerve activity was examined in chronically instrumented conscious DS rats. The activity and protein amount of brain nNOS was evaluated by enzyme assay and western blot analysis. The distribution and number of nNOS neurons in the brainstem were examined immunohistochemically in hypertensive and normotensive DS rats. S-methyl-L-thiocitrulline induced a larger increase in tonic renal sympathetic nerve activity generated before baroreflex-mediated inhibition in hypertensive DS rats than normotensive DS rats. Hypertensive DS rats showed increased nNOS activity in the brainstem, but not in the diencephalon or cerebellum. High nNOS activity was confirmed by an increase in the amount of nNOS protein. nNOS Neurons were localized in several nuclei throughout the brainstem; the dorsolateral periaqueductal gray, pedunculopontine tegmental nucleus, dorsal raphe nucleus, laterodorsal tegmental nucleus, lateral parabrachial nucleus, rostral ventrolateral medulla, nucleus tractus solitarius and raphe magnus. The number of nNOS neurons in these nuclei, except for the two raphes, was significantly greater in hypertensive than in normotensive DS rats. These findings suggest that central nNOS-mediated sympathoinhibition may be enhanced in salt-sensitive hypertensive Dahl rats. The upregulated nNOS-mediated inhibition may occur in the central sympathetic control system generated before baroreflex-mediated inhibition.

Binding of L-arginine and imidazole suggests heterogeneity of rat brain neuronal nitric oxide synthase.

Nitric oxide synthase (NOS) is inhibited by imidazole, which binds to the heme in a low-spin complex absorbing at 428 nm. Conversion by L-arginine of this complex into a high-spin species absorbing at 395 nm is a common method to determine the binding parameters of Arg. However, both Arg-competitive and noncompetitive inhibition of NOS by imidazole has been reported, and optical studies with neuronal NOS provided no evidence for imidazole affecting Arg binding. We investigated the cause for these paradoxical observations with recombinant rat brain neuronal NOS. Imidazole bound to nNOS with a K(d)(app) of 50 microM; tetrahydrobiopterin (BH4) lowered the affinity of nNOS for imidazole 4-fold. The enzyme behaved heterogeneously with respect to Arg binding. Most of nNOS (65-80%) showed competition between Arg and imidazole. In the presence of BH4, a K(d)(Arg) of 1 microM could be estimated for this fraction, as well as apparent association and dissociation rate constants of 2.5 x 10(6) M(-1) x s(-1) and 2.5 s(-1). A second fraction of nNOS (20-30%) exhibited little or no competition. Consequently, Arg binding did not cause dissociation of the imidazole complex for this fraction, and complete generation of the high-spin state by Arg could not be achieved in the presence of imidazole. A third fraction (< or =10%) bound Arg with low affinity (K(d) 1-2 mM). Because of this heterogeneity, titration curves with Arg became almost uninterpretable. We propose that this heterogeneous response of nNOS toward Arg and imidazole is underlying the apparently conflicting results reported in the literature.

Effects of pH on the structure and function of neuronal nitric oxide synthase.

We investigated how pH affects rat brain neuronal nitric oxide synthase (nNOS) with regard to spin-state equilibrium and the thiolate ligand bond of the haem group, catalytic activity, and monomerleft and right arrow dimer equilibrium. At neutral pH, nNOS containing 1 equiv. of (6R)-5,6,7,8-tetrahydro-l-biopterin (BH4) per dimer was mostly high-spin (lambdamax at 398 nm), whereas the BH4-free enzyme consisted of a mixture of the high-spin and two low-spin forms (lambdamax at 418 nm, and at 376 and 456 nm respectively). With BH4-free nNOS, an appreciable high-spin fraction was only observed between pH 7 and 8; at pH 6 and 9, the 418 and 376/456 nm low-spin forms predominated respectively. With nNOS containing 1 equiv. of BH4 per dimer, similar observations were made, but these involved only half of the enzyme; the other half, presumably the BH4-containing subunits, remained high-spin. Since the spin state in the BH4-free subunit appeared little affected by the state of the other subunit, we conclude that, in dimeric nNOS, the two haem groups function independently. Low pH destabilized thiolate binding and the interaction between NOS subunits, as indicated by CO-binding studies and gel electrophoresis respectively. Formation of l-citrulline was optimal between pH 7.0 and 7.5; the decrease in NOS activity at lower pH proved to be due to uncoupling of NADPH oxidation, resulting in increased formation of H2O2. At high pH strict coupling of l-arginine and NADPH oxidation was maintained, even in the absence of exogenous BH4. The possible pathophysiological implications of the uncoupling at low pH are discussed.

Influence of different classes of NO synthase inhibitors in the pig gastric fundus.

The inhibitory potency of different classes of nitric oxide synthase (NOS) inhibitors (amino acid-based substances, guanidines, isothioureas, imidazoles and indazoles) versus peripheral neuronal NOS in the pig gastric fundus was investigated by studying their influence on electrically induced relaxations in non-adrenergic noncholinergic conditions. Circular muscle strips were mounted for isotonic registration in the presence of atropine and guanethidine, and tone was raised with 5-hydroxytryptamine. Electrical field stimulation (40 V, 0.1 ms, 4 Hz, 10 s) induced short-lasting relaxations. The inhibitory effect of 1-phenylimidazole could not be evaluated because it nearly abolished the 5-hydroxytryptamine-induced tone of the tissues. 7-Nitroindazole, imidazole, 2-iminobiotin and aminoguanidine did not inhibit the electrically induced relaxations, while the other 9 substances tested were able to do so. The influence of the incubation period was tested by studying the inhibitory effect after incubation for 10 up to 60 min. For N(G)-nitro-L-arginine methyl ester (L-NAME), N(G)-nitro-L-arginine (L-NNA), L-N5-(1-iminoethyl)-ornithine (L-NIO), L-N6-(1-iminoethyl)-lysine (L-NIL), S-methyl-L-thiocitrulline and S-isopropyl isothiourea there was a moderate increase in the inhibitory effect up to 30 min of incubation so that they were incubated for 30 min to study their inhibitory potency. For L-thiocitrulline, S-methyl isothiourea and S-ethyl isothiourea, an incubation period of 60 min was used. The 9 substances concentration-dependently inhibited the electrically induced relaxations with a maximal inhibitory effect of approximately 80% except for S-methyl isothiourea (Emax of 53%). The overall order of potency was: S-isopropyl isothiourea > S-ethyl isothiourea > or = S-methylL-thiocitrulline > or = L-NNA > L-NIO > L-NAME > S-methyl isothiourea > L-thiocitrulline > L-NIL. While the potency for S-isopropyl isothiourea (EC50: 3.1 x 10(-5) M, n = 6) to S-methyl isothiourea (EC50: 11.5 x 10(-5) M, n = 5) was in the same range, the potency of L-thiocitrulline and L-NIL was clearly lower. This study showed several compounds to be potent inhibitors of peripheral neuronal NOS in the pig gastric fundus while some compounds, that were reported to inhibit brain neuronal NOS were not effective. The EC50 values found for the effective substrates in this functional study may be a guideline for the concentrations required to evaluate the role of NO in NANC neurotransmission in gastrointestinal smooth muscle preparations.

Delineation of the arginine- and tetrahydrobiopterin-binding sites of neuronal nitric oxide synthase.

Nitric oxide synthase (EC 1.14.13.39) catalyses the conversion of arginine, NADPH and oxygen to nitric oxide and citrulline, using haem, (6R)-5,6,7,8-tetrahydro-l-biopterin (tetrahydrobiopterin), calmodulin, FAD and FMN as cofactors. The enzyme consists of a central calmodulin-binding sequence flanked on the N-terminal side by a haem-binding region that contains the arginine and tetrahydrobiopterin sites and on the C-terminal side by a region homologous with NADPH:cytochrome P-450 reductase. By using domain boundaries defined by limited proteolysis of full-length enzyme, recombinant haem-binding regions of rat brain neuronal nitric oxide synthase were expressed and purified. Two proteins were made in high yield: one, corresponding to residues 221-724, contained bound haem and tetrahydrobiopterin and was able to bind Nomega-nitro-l-arginine (nitroarginine) or arginine; the other, containing residues 350-724, contained bound haem but was unable to bind tetrahydrobiopterin, nitroarginine or arginine. These results showed that rat brain neuronal nitric oxide synthase contains a critical determinant for arginine/tetrahydrobiopterin binding between residues 221 and 350. Limited proteolysis with chymotrypsin of the former protein resulted in a new species with an N-terminal residue 275 that retained the ability to bind nitroarginine, further defining the critical region for arginine binding as being between 275 and 350. Comparison of the sequences of nitric oxide synthase and the tetrahydrobiopterin-requiring amino acid hydroxylases revealed a similarity in the region between residues 470 and 600, suggesting that this might represent the core region of the pterin-binding site. The stoichiometries of binding of substrate and cofactors to the recombinant domains were not more than 0.5 mol/mol of monomer, suggesting that there might be a single high-affinity site per dimer.