Extracellular Nitric Oxide Levels Research Articles

Extracellular nitric oxide sustains root surface redox activity and growth under sudden flooding-induced hypoxic conditions in barley root tips

Main conclusionNitric oxide sustains root tip surface redox activity and restricts lipid peroxidation-triggered cell death in the root tips.In order to gain more insight into the involvement of nitric oxide (NO) in plant response to sudden flooding-induced hypoxic stress, we studied the effect of two NO donors, sodium nitroprusside and S-nitroso-L-glutathione, on short-term partial submergence-induced root growth inhibition, alteration in root surface redox activity, lipid peroxidation and cell death in two barley cultivars (cv.) at their early seedling stage. The short-term hypoxic stress induces root growth arrest in cv. Karmel, accompanied by increased lipid peroxidation and cell death. By contrast, in cv. Slaven, short-term hypoxic conditions cause only reduced root growth rate, associated with elevated extracellular NO level in the root tips. The root tip surface redox activity decreases with the increasing timespan of hypoxic conditions in both cultivars; however, this decrease in redox activity started earlier and was greater in the cv. Karmel in comparison with cv. Slaven. Application of NO donors during hypoxic stress sustains the root redox activity and eliminates the hypoxia-induced lipid peroxidation, accompanied by a partial restoration of root growth after short-term hypoxic stress. These results suggest that extracellular NO plays a key role in maintaining the root tip surface redox activity and in the restriction of lipid peroxidation and cell death under short-term hypoxic stress in the root tips of barley seedlings.

Simultaneous Quantitation of Intra- and Extracellular Nitric Oxide in Single Macrophage RAW 264.7 Cells by Capillary Electrophoresis with Laser-Induced Fluorescence Detection.

Single-cell analysis contributes to the understanding of cellular heterogeneity and behaviors. Nitric oxide (NO) is an important intracellular and intercellular signaling molecule, and the functions of NO are closely related to the balance between intra- and extracellular NO levels. In this manuscript, a convenient and reliable method based on a dual-labeling strategy using capillary electrophoresis (CE) separation with laser-induced fluorescence (LIF) detection has been presented for quantifying intra- and extracellular NO simultaneously in single cells. Followed by single-cell injection, a plug of HEPES buffer containing 1,3,5,7-tetramethyl-8-(3',4'-diaminophenyl)-difluoroboradiaza-s-indacene and disodium 2,6-disulfonate-1,3-dimethyl-5-hexadecyl-8-(3,4-diaminophenyl)-4,4'-difluoro-4-bora-3a,4a-diaza-s-indacene as the labeling reagents for intra- and extracellular NO, respectively, was aspirated from the inlet of the capillary. The on-line derivatization was carried out on the tip of the capillary at room temperature for 20 min. Then, the cell was lysed and NO derivatives were well separated within 14 min, producing mass detection limits (S/N = 3) of 2.4 and 8.1 amol for intra- and extracellular NO, respectively. The proposed method was validated by simultaneous analysis of intra- and extracellular NO in single macrophage cells. The dual labeling-based CE-LIF method holds great promise for research on the functions of NO as well as other bioactive molecules at the single-cell level.

Effects of Ghrelin on iNOS-Derived NO Promoted LPS-Induced Pulmonary Alveolar Epithelial A549 Cells Apoptosis

Background/Aims: In the process of abnormal apoptosis of pulmonary alveolar type II epithelial A549 cells in acute respiratory distress syndrome (ARDS), inducible nitric oxide synthase (iNOS) activity in the lung, nitric oxide (NO) production, and the level of protein S-nitrosylation were increased. However, the role of excessive NO production in sepsis-induced ARDS is controversial. Additionally, ghrelin is a growth hormone that exerts an inhibitory role in cell apoptosis. We examined the effect of NO and S-nitrosylation on apoptosis of A549 cells induced by Lipopolysaccharide (LPS) and molecular mechanism underlying the anti-apoptotic effect of ghrelin in this process. Methods: Flow cytometry and qPCR were used to detect lentiviral infection efficiency and iNOS gene level, respectively. Extracellular and intracellular NO levels were observed by Griess assay kit and DAF-FM DA. Mitochondrial transmembrane potential, apoptosis rate and SNO levels were determined by flow cytometry, Biotin-Switch method and immunofluoresence staining. The expression of iNOS, apoptotic proteins and JNK were assessed by immunoblot analysis. Results: The results showed about two times increase in iNOS expression and intracellular NO levels response to LPS exposure at 24 hours (P< 0.05), while not in extracellular NO levels. NO donors, S-nitroso-N-acetylpenicillamine (SNAP) significantly raised (36.7%, P< 0.05; 38.4%, P< 0.05; 41.8%, P< 0.05) extracellular NO levels without influencing the intracellular NO levels. LPS increased the apoptosis rate (42.4%±2.6% vs 2.8%±1%, P< 0.05) of A549 accompanied by increased Bax levels and decreased Bcl-2 levels through activating JNK signaling, which was reversed when we diminished the iNOS expression in A549 cells using lentiviral vectors encoding iNOS shRNA in the presence of LPS (24.8%±3.8% vs 42.4%±2.6%, P< 0.05). However, the apoptosis rate was increased when SNAP was added (38.8%±1.3% vs 24.8%±3.8%, P< 0.05). Furthermore, we investigated whether ghrelin exert a protective role against LPS-induced apoptosis and the potential mechanism involved in. Ghrelin alone appeared to decrease iNOS expression (32.3%, P< 0.05; 42.3%, P< 0.05), which showed no signifiant difference between LPS+ghrelin group and LPS group. However, this study showed that ghrelin decreased the intracellular NO production (38.9%, P< 0.05), protein S-nitrosylation levels (33.5%, P< 0.05), Bax protein expression (70.2%, P< 0.05), whereas increasing Bcl-2 protein expression (14.1%, P< 0.05) and mitochondrial transmembrane potential (∆ΨM) (20.7%, P< 0.05) in the presence of LPS. Conclusion: The data suggested that NO derived from iNOS induced by LPS stimulation exerts an important role in promoting apoptosis of A549 cells, and ghrelin abolished intracellular NO production and protein S-nitrosylation levels, abrogating the apoptosis of A549 cells partly through inhibiting mitochondrial-dependent pathways.

Tart Cherry Extracts Reduce Inflammatory and Oxidative Stress Signaling in Microglial Cells.

Tart cherries contain an array of polyphenols that can decrease inflammation and oxidative stress (OS), which contribute to cognitive declines seen in aging populations. Previous studies have shown that polyphenols from dark-colored fruits can reduce stress-mediated signaling in BV-2 mouse microglial cells, leading to decreases in nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression. Thus, the present study sought to determine if tart cherries—which improved cognitive behavior in aged rats—would be efficacious in reducing inflammatory and OS signaling in HAPI rat microglial cells. Cells were pretreated with different concentrations (0–1.0 mg/mL) of Montmorency tart cherry powder for 1–4 h, then treated with 0 or 100 ng/mL lipopolysaccharide (LPS) overnight. LPS application increased extracellular levels of NO and tumor necrosis factor-alpha (TNF-α), and intracellular levels of iNOS and cyclooxygenase-2 (COX-2). Pretreatment with tart cherry decreased levels of NO, TNF-α, and COX-2 in a dose- and time-dependent manner versus those without pretreatment; the optimal combination was between 0.125 and 0.25 mg/mL tart cherry for 2 h. Higher concentrations of tart cherry powder and longer exposure times negatively affected cell viability. Therefore, tart cherries (like other dark-colored fruits), may be effective in reducing inflammatory and OS-mediated signals.

Effects of coarse chalk dust particles (2.5-10 μm) on respiratory burst and oxidative stress in alveolar macrophages.

The main aim of the present study was to examine in vitro responses of rat alveolar macrophages (AMs) exposed to coarse chalk dust particles (particulate matter in the size range 2.5-10 μm, PM(coarse)) by respiratory burst and oxidative stress. Chalk PM(coarse)-induced respiratory burst in AMs was measured by using a luminol-dependent chemiluminescence (CL) method. Also, the cell viability; lactate dehydrogenase (LDH) release; levels of cellular superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), malondialdehyde (MDA), and acid phosphatase (ACP); plasma membrane ATPase; and extracellular nitric oxide (NO) level were determined 4 h following the treatment with the different dosages of chalk PM(coarse). The results showed that chalk PM(coarse) initiated the respiratory burst of AMs as indicated by strong CL, which was inhibited by diphenyleneiodonium chloride and L-N-nitro-L-arginine methyl ester hydrochloride. It suggested that chalk PM(coarse) induced the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in AMs. This hypothesis was confirmed by the fact that chalk PM(coarse) resulted in a significant decrease of intracellular SOD, GSH, ACP, and ATPase levels and a notable increase of intracellular CAT, MDA content, and extracellular NO level, consequently leading to a decrease of the cell viability and a increase of LDH release. It was concluded that AMs exposed to chalk PM(coarse) can suffer from cytotoxicity which may be mediated by generation of excessive ROS/RNS. Graphical Abstract The possible mechanism of coarse chalk particles-induced adverse effects in AMs.

Inhibition of proteasome-mediated glucocorticoid receptor degradation restores nitric oxide bioavailability in myocardial endothelial cells in vitro.

Glucocorticoids (GCs), including the synthetic GC derivate dexamethasone, are widely used as immunomodulators. One of the numerous side effects of dexamethasone therapy is hypertension arising from reduced release of the endothelium-derived vasodilator nitric oxide (NO). Herein, we described the role of dexamethasone and its glucocorticoid receptor (GR) in the regulation of NO synthesis in vitro using the mouse myocardial microvascular endothelial cell line, MyEND. GC treatment caused a firm decrease of extracellular NO levels, whereas the expression of endothelial NO synthase (eNOS) was not affected. However, GC application induced an impairment of tetrahydrobiopterin (BH4 ) concentrations as well as GTP cyclohydrolase-1 (GTPCH-1) expression, both essential factors for NO production upstream of eNOS. Moreover, dexamethasone stimulation resulted in a substantially decreased GR gene and protein expression in MyEND cells. Importantly, inhibition of proteasome-mediated proteolysis of the GR or overexpression of an ubiquitination-defective GR construct improved the bioavailability of BH4 and strengthened GTPCH-1 expression and eNOS activity. Summarising our results, we propose a new mechanism involved in the regulation of NO signalling by GCs in myocardial endothelial cells. We suggest that a sufficient GR protein expression plays a crucial role for the management of GC-induced harmful adverse effects, including deregulations of vasorelaxation arising from disturbed NO biosynthesis.

Circadian entrainment to light–dark cycles involves extracellular nitric oxide communication within the suprachiasmatic nuclei

The ability to synchronize to light-dark (LD) cycles is an essential property of the circadian clock, located in mammals within the hypothalamic suprachiasmatic nuclei (SCN). Single light pulses activate nitric oxide (NO) intracellular signaling, leading to circadian phase-shifts required for synchronization. In addition, extracellular NO has a role in the SCN paracrine communication of photic phase advances. In this work, the extracellular nitrergic transmission was assessed in steady-state synchronization to LD cycles of locomotor rhythms in the golden hamster (Mesocricetus auratus). Extracellular NO levels were pharmacologically decreased in vivo with the specific scavenger, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO). Hamsters were subjected to LD cycles different from normal 24 h (LD 14 : 10) cycles (i.e. T-cycles), with a single 30-min light pulse presented either every 23 h (T23 cycles), or every 25 h (T25 cycles), thus allowing synchronization by advances or delays, respectively. Acute PTIO intracerebroventricular microinjections, delivered 30 min previous to the light pulse, inhibited synchronization by phase advances to T23 cycles, but did not alter phase delays under T25 cycles. In addition, NO scavenging inhibited light-induced expression of PERIOD1 protein at circadian time 18 (i.e. the time for light-induced phase advances). These findings demonstrate the role of extracellular NO communication within the SCN in the steady-state synchronization to LD cycles.

Possible involvement of NO/NOS signaling in hippocampal amyloid-β production induced by transient focal cerebral ischemia in aged rats

In the present study, to define the roles of nitric oxide (NO) signaling in amyloid-β (Aβ) production after transient cerebral ischemia, extracellular levels of NO and Aβ were monitored by intracerebral microdialysis in the hippocampus of aged rats exposed to transient middle cerebral artery occlusion and reperfusion (MCAO/R). The results indicated that 1-h MCAO significantly upregulated hippocampal NO and Aβ levels. In addition, the NO elevation preceded the Aβ changes. The Western blotting suggested that acute hypoperfusion could increase the expression of β-secretase 1 (BACE1) but not BACE2. The enhanced NO concentration in acute stage of MCAO/R was coincident with increased eNOS expression, while in subacute stage was coincident with increased iNOS and nNOS. Our results also indicated that pretreatment of L-NAME, one non-selective NOS inhibitor could decrease the BACE1 expression, reverse both NO and Aβ changes and rescue the delayed neuronal death. These preliminary findings indicated that activation of NOS/NO signaling system could trigger Aβ production through BACE1 pathway during acute ischemic episode. The present data may be important in understanding, at least in part, the pathological role of NO/NOS system involved in hippocampal Aβ production and neuronal damage induced by transient cerebral ischemia.

Current Literature Review

To assess whether intracavernosal injections of prostaglandin E1 (PGE1) can influence nitric oxide (NO) release in the corpora in a rat model of penile erection.The extracellular levels of NO were monitored at 100 seconds intervals in the corpus cavernosum of anesthetized rats by using differential normal pulse voltammetry with porphyrin-Nafion coated carbon fiber microelectrodes. The intracavernosal pressure (ICP) was simultaneously recorded. PGE1 was given either as a single dose (ranging from 0.2 to 15 μg.) or as repeated 2 μg. injections in alternate days for two weeks. The NO and ICP responses to electrostimulation of the cavernosal nerve (SCN) was studied in the animals in the repeated treatment schedule at 1, 7, 15 and 30 days after its termination. The levels of the three NO synthase (NOS) isoforms in the cavernous tissue were measured by immunoblotting.Acute PGE1 treatment dose-relatedly increased NO levels in the corpora, with a concomitant ICP increase with the highest dose. Repeated 2 μg. PGE1 injections increased the NO and ICP responses to SCN as compared with intact or vehicle-injected animals. This treatment also increased the penile content of the neuronal and endothelial NOS proteins. The inducible NOS isoform remained unchanged after either vehicle or PGE1 injections. The effects of the repeated PGE1 treatment were greater in the group studied 24 hours after the last injection and decreased progressively thereafter.Stimulation of NO release can contribute to the erectogenic effect of intracavernous PGE1 injections. The increased levels of constitutive NOS isoforms in the corpora could contribute to the improvement of the erectile function reported by some patients following repeated treatment with vasorelaxant agents.

Unexpected Effects of Nitric Oxide Synthase Inhibitors on Extracellular Nitrite Levels in the Hippocampus in vivo

The aim of this study was to determine whether extracellular nitric oxide levels in the hippocampus of freely moving animals were reduced by the administration of nitric oxide synthase (NOS) inhibitors via a microdialysis probe. Our results show that extracellular nitrite levels were increased following the infusion of N-nitro-L-arginine methyl ester (L-NAME) and 7-nitroindazole (7-NI), in the case of the latter, the response was biphasic. In contrast, infusion of both inhibitors together resulted in a substantial reduction in nitrite when compared to control. More predictably, the infusion of NMDA elicited an increase in extracellular nitrite levels. This effect was biphasic, the second phase occurring some 3 h after the drug infusion period had ended. When NMDA was infused in the presence of L-NAME, no agonist-induced increase in nitrite production was recorded, in fact nitrite levels were found to decline to below control values. There was no immediate increase in nitrite levels when NMDA was infused in the presence of 7-NI, although this may have been partially obscured by the biphasic effect of the inhibitor. It did appear, however, that the second phase of the NMDA-induced response was attenuated by 7-NI. No NMDA-evoked increase in nitrite was evident when the agonist was infused in the presence of a combination of both inhibitors. We have no clear explanation for the data presented here but suggest that up-regulated activity of particular NOS isoforms might compensate for the inhibition of the other by a mechanism yet to be elucidated. In addition, we propose that caution be exercised when interpreting results from in vivo microdialysis studies where NOS inhibitors are administered directly into the brain via a probe.

Neuronal death in primary retinal cultures is related to nitric oxide production, and is inhibited by erythropoietin in a glucose‐sensitive manner

The aim of this work was to investigate the interrelated effects of glucose, nitric oxide (NO) and erythropoietin on neuronal survival in retinal cultures, thereby exploring the mechanism of neuronal death in the diabetic retina. Rat retinal cells were cultured in low (5 mM) or high (15 mM) glucose concentrations. After 9 days, cell viability was assessed by (3,4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and NO production was determined by the Griess reaction. Immunohistochemistry was used to quantify GABA-labelled neurones and cells staining for DNA breakdown. High or low glucose concentrations had no effect on basal NO production or the survival of neurones in culture, but treatment with N-nitro-L-arginine methyl ester reduced extracellular levels of NO and increased neuronal survival at both concentrations of glucose. Erythropoietin decreased cell death and NO levels, but only in cultures grown in low concentrations of glucose. It is concluded that erythropoietin's neurotrophic function in the retina is attenuated at glucose concentrations similar to those which occur in diabetes.

The effects of high- and low-intensity percutaneous stimulation on nitric oxide levels and spike activity in the superficial laminae of the spinal cord

Nitric oxide (NO) was measured using a new electrochemical method with a carbon fibre microelectrode at depths of up to 400μm in the lumbar dorsal horn of the anaesthetised rat. The method allowed extracellular spike recording from single units together with the electrochemical recording at the same electrode. Thirty-six cells with low threshold cutaneous (brush/touch) or wide dynamic range receptive fields (brush/touch plus pinch) were studied. Adequate stimulation of the receptive fields did not alter the extracellular NO level for any cells. Percutaneous needle electrodes inserted into the receptive fields were used to stimulate the cells electrically. Twenty-one cells were stimulated using 10mA current with 0.05ms duration (low intensity) pulses to stimulate predominantly A-fibre afferents. Single shock stimuli gave short latency spike responses but no change in nitric oxide level. Tetanic bursts of stimuli (400 stimuli at 50Hz) generated a burst of spikes (spike count 548±42) and a transient increase in NO (2.61±0.11μM NO). Nitric oxide synthesis inhibition with NG-nitro-l-arginine methyl ester (l-NAME) nearly abolished the stimulus-evoked increase in nitric oxide and increased the response of the cells (spike count 694±34). However, the inhibition of nitric oxide synthesis had no effect on the receptive fields. Fifteen cells were stimulated with shocks using 5ms pulses (high intensity), to recruit C-fibre afferents into the input volley. This more intense stimulation increased the evoked NO release to 3.63±0.15μM and the spike response to 647±54 in control conditions. Following l-NAME, the evoked NO release was reduced and the evoked spike response was significantly decreased. These results show that tetanic activity in afferent fibres increases NO synthesis in the dorsal horn and that inhibition of nitric oxide synthesis may be associated with a selective attenuation of the spike responses to C-fibre inputs. NO may be necessary to maintain proper function of C-fibre afferent synapses when they are subjected to sustained or tetanic inputs.

Nitric oxide reduces blood pressure in the nucleus tractus solitarius: a real time electrochemical study

Increasing evidence has demonstrated that nitric oxide (NO) is involved in central cardiovascular regulation. In this study, we directly measured extracellular NO levels, in real-time, in the nucleus tractus solitarius (NTS) of anesthetized cats using Nafion/Porphyrine/o-Phenylenediamine-coated NO sensors. We found that local application of L-arginine (L-Arg) induced NO overflow in NTS and hypotension. These responses were potentiated in the vagotomized animals. Pretreatment with NO synthase (NOS)/guanylate cyclase inhibitor methylene blue, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one or NO scavenger hemoglobin attenuated L-Arg-induced hypotension, suggesting that exogenous supplement of NO suppressed cardiac functions through the NOS/cyclic guanosine monophosphate mechanism. The role of endogenous NO was examined after local application of N G-nitro-L-arginine methyl ester (L-NAME). We found that L-NAME suppressed endogenous NO levels in NTS and elicited hypertension and tachycardia. Taken together, our data suggest that NO is tonically released in the NTS to inhibit blood pressure.

The nNOS/cGMP signal transducing system is involved in the cardiovascular responses induced by activation of NMDA receptors in the rostral ventrolateral medulla of cats

Nitric oxide (NO) is synthesized from l-arginine by NO synthase (NOS). NO stimulates the soluble form of guanylyl cyclase (sGC) and induces accumulation of cyclic guanosine monophosphate (cGMP). The purpose of this study was to examine whether the cardiovascular responses induced by N-methyl- d-aspartate (NMDA) in the rostral ventrolateral medulla (RVLM) depend on the actions of NOS and sGC. In anesthetized cats, the extracellular NO level was measured by in vivo voltammetry using a nafion/porphyrine/o-phenylenediamine-coated carbon-fiber electrode. Microinjection of NMDA into the RVLM produced hypertension and bradycardia associated with NO formation. These NMDA-induced responses were attenuated by prior injections of 7-nitroindazole, a neuronal NO synthase (nNOS) inhibitor, and 1H- [1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a sGC inhibitor. These findings suggest that NO is involved in the NMDA-induced cardiovascular responses in the RVLM.

Reciprocal regulation of nitric oxide and glutamate in the nucleus tractus solitarii of rats

Nitric oxide (NO) and glutamate are both important mediators of the central cardiovascular regulation in the nucleus tractus solitarii. Our previous studies revealed that the central cardiovascular effects of NO in the nucleus tractus solitarii could be inhibited by glutamate receptor blockade. On the other hand, nitric oxide synthase (NOS) inhibitor attenuated the cardiovascular effects of glutamate. Thus, NO and glutamatergic systems appear to interact in central cardiovascular regulation. The present study examined whether NO and glutamate may affect each other's release/production in the nucleus tractus solitarii. A microdialysis probe was implanted into the nucleus tractus solitarii of male Sprague–Dawley rats, and the changes in the extracellular levels of glutamate and NO were determined by high performance liquid chromatography coupled with electrochemical detection and an NO analyzer, respectively. The results showed that NO solution elicited >10 fold increases in the extracellular level of glutamate, which returned to normal 60 min after the end of NO perfusion. The NO donor N-acetyl-penicillamine (SNAP) had an effect similar to NO solution. Furthermore, the glutamate level was reduced to 61% of basal value by perfusion with the NOS inhibitor, N G-monomethyl- l-arginine ( l-NMMA). When glutamate receptor agonist N-methyl- d-aspartic acid (NMDA) or α-amino-3-hydroxy-5-methylixoxazole-4-propionic acid (AMPA) was administered into the nucleus tractus solitarii, the extracellular NO level was increased by 70–100%, whereas glutamate receptor antagonists (MK-801 hydrogen maleate and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)) did not alter the basal levels of NO. These results suggest that NO and glutamate may enhance each other's release/production in the nucleus tractus solitarii. This reciprocal regulation of NO and glutamate may be important in central cardiovascular control in the nucleus tractus solitarii.

Extracellular accumulation of nitric oxide, hydrogen peroxide, and glutamate in astrocytic cultures following glutathione depletion, complex I inhibition, and/or lipopolysaccharide-induced activation

Dopaminergic neuronal death in substantia nigra in Parkinson’s disease is accompanied by depletion of reduced glutathione levels and inhibition of complex I activity which occur partially in normal or activated cells. The relationship between neuronal death and altered glial function is not known, but this may involve the release of toxic mediators from astrocytes and microglia, which in turn cause neuronal injury. We have examined the effects of l-buthionine-[ S, R]-sulfoximine ( l-BSO)-induced glutathione depletion, inhibition of complex I activity by 1-methyl-4-phenylpyridinium (MPR +), and/or lipopolysaccharide (LPS)-induced activation on the extracellular accumulation of nitric oxide (NO), hydrogen peroxide (H 2O 2), and glutamate in primary cultures of rat forebrain astrocytes. Depletion of glutathione levels by up to 90% did not alter NO, H 2O 2, or glutamate levels in cultured astrocytes. Inhibition of complex I activity by up to 43% had no effect on extracellular NO accumulation, but increased H 2O 2 and glutamate levels. LPS-induced activation of cultured astrocytes increased extracellular levels of NO, H 2O 2, and glutamate. Extracellular accumulation of NO and H 2O 2 caused by LPS was markedly less in glutathione-depleted or complex I-inhibited astrocytic cultures compared to normal astrocytic cultures. In conclusion, complex I inhibition or activation of glial cells, alone or in combination with glutathione depletion, results in the extracellular accumulation of glutamate and the formation of NO and H 2O 2, which in turn may form highly toxic peroxynitrite and hydroxyl radicals. Thus, altered glial function leading to oxidative stress and excitotoxicity may contribute to the initiation or progression of neuronal death in substantia nigra in Parkinson’s disease.

GABA A and GABA B receptors modulating basal and footshock-induced nitric oxide releases in rat prefrontal cortex

Using an in vivo brain microdialysis technique, we measured extracellular levels of nitric oxide (NO) metabolites (NO x −) in the medial prefrontal cortex (mPFC) upon perfusion of γ-aminobutyric acid (GABA) receptor antagonists as well as agonists, and also examined the effects of GABA receptor agonists on mild intermittent footshock-induced NO releases in the mPFC in conscious rats. Perfusion of either bicuculline methiodide, a GABA A receptor antagonist, or saclofen, a GABA B receptor antagonist, through a microdialysis probe resulted in dose-dependent increases in NO x − levels. Higher-dose perfusion of either muscimol (50 μM), a GABA A receptor agonist, or baclofen (250 μM), a GABA B receptor agonist resulted in a significant decrease in NO x − levels. The elevated levels of NO x − after mild intermittent footshock were attenuated by perfusion of either muscimol (10 μM) or baclofen (50 μM), either of which alone did not affect basal NO x − levels. These findings are likely to provide helpful clues to our understanding of the inhibitory modulation of basal and footshock-induced NO metabolites releases by GABA A and GABA B receptors in the mPFC.

Effects of nitric oxide stimulation on the brain.

Nitric oxide (NO) is known as the neurotransmitter responsible for penile erection. Recent experimental studies showed that NO is recognized as an important messenger mediating male copulatory behavior and penile erection in the central nervous system. In this article, the roles of central NO on modulation of male copulatory behavior and penile erection in male rats are reviewed mainly based on our previous experimental results. We focused on two brain areas in the hypothalamus for this purpose: the medial preoptic area (MPOA) and paraventricular nucleus (PVN). Increase in extracellular NO level in the MPOA facilitates copulatory behaviors, while decreases in the NO level reduce them. The altered NO level in the PVN increases frequency of reflexive erections, but does not affect copulatory behavior. Taken together, the current results and previous reports suggest that central NO may modulate male sexual functions and NO in two distinct brain areas and may play different roles through activation of different neurotransmitters. Activities of NO in the hypothalamus are affected by the existence of gonadal steroids. Moreover, our experimental results clearly showed that the extracellular NO level in the MPOA and PVN decreased with aging, and testosterone replacement in aged rats restored NO levels in both brain areas. These results postulate that changes of central NO with aging and following hormone replacement might partly contribute to changes in male rats sexual behavior corresponding to aging and replacement. Moreover, our recent report demonstrated that long-term testosterone replacement in aged rats is more effective for restoration of sexual behavior, which may be partly induced by central NO, than high-dose, short-term replacement.

REPEATED PGE1 TREATMENT ENHANCES NITRIC OXIDE AND ERECTION RESPONSES TO NERVE STIMULATION IN THE RAT PENIS BY UPREGULATING CONSTITUTIVE NOS ISOFORMS

To assess whether intracavernosal injections of prostaglandin E1 (PGE1) can influence nitric oxide (NO) release in the corpora in a rat model of penile erection. The extracellular levels of NO were monitored at 100 seconds intervals in the corpus cavernosum of anesthetized rats by using differential normal pulse voltammetry with porphyrin-Nafion coated carbon fiber microelectrodes. The intracavernosal pressure (ICP) was simultaneously recorded. PGE1 was given either as a single dose (ranging from 0.2 to 15 microg.) or as repeated 2 microg. injections in alternate days for two weeks. The NO and ICP responses to electrostimulation of the cavernosal nerve (SCN) was studied in the animals in the repeated treatment schedule at 1, 7, 15 and 30 days after its termination. The levels of the three NO synthase (NOS) isoforms in the cavernous tissue were measured by immunoblotting. Acute PGE1 treatment dose-relatedly increased NO levels in the corpora, with a concomitant ICP increase with the highest dose. Repeated 2 microg. PGE1 injections increased the NO and ICP responses to SCN as compared with intact or vehicle-injected animals. This treatment also increased the penile content of the neuronal and endothelial NOS proteins. The inducible NOS isoform remained unchanged after either vehicle or PGE1 injections. The effects of the repeated PGE1 treatment were greater in the group studied 24 hours after the last injection and decreased progressively thereafter. Stimulation of NO release can contribute to the erectogenic effect of intracavernous PGE1 injections. The increased levels of constitutive NOS isoforms in the corpora could contribute to the improvement of the erectile function reported by some patients following repeated treatment with vasorelaxant agents.

Androgen-dependent nitric oxide release in rat penis correlates with levels of constitutive nitric oxide synthase isoenzymes.

Androgens are known to influence penile erection and nitric oxide synthase (NOS) activity in cavernosal tissue homogenates. The present study was an assessment of the effects of castration and androgen replacement on the in vivo release of nitric oxide (NO), and of the simultaneously recorded intracavernosal pressure (ICP) changes elicited by electrostimulation of the cavernosal nerves (SCN) in the anesthetized rat. The extracellular levels of NO in the corpora were monitored electrochemically using porphyrin microsensors. The content of NOS isoenzymes in corporal homogenates was determined by immunoblotting. The responses of castrated rats with or without testosterone (T) implants were compared to those of intact animals. Castration virtually abolished both the NO and the ICP responses to SCN. There was a concomitant significant decrease in the content of both the neuronal (nNOS) and the endothelial (eNOS) isoenzymes in the cavernosal tissue. All these effects of castration were prevented by T replacement. The NO response to SCN was positively correlated with the levels of nNOS and eNOS, especially when the values of the two isoforms were added (r = 0.71, P < 0.001). These data suggest that the facilitatory action of androgens on penile erection involves the up-regulation of both constitutive NOS isoenzymes in the corpora cavernosa.