Role Of Neuronal Nitric Oxide Research Articles

The role of neuronal nitric oxide and its pathways in the protection and recovery from neurotoxin-induced de novo hypokinetic motor behaviors in the embryonic zebrafish (Danio rerio).

Neuronal nitric oxide (nNO) has been shown to affect motor function in the brain. Specifically, nNO acts in part through regulation of dopamine (DA) release, transporter function, and the elicitation of neuroprotection/neurodegeneration of neurons in conditions such as Parkinson's disease (PD). Recently, the zebrafish has been proposed to be a new model for the study of PD since neurotoxin damage to their nigrostriatal-like neurons exhibit PD-like motor dysfunctions similar to those of mammalian models and human patients. Results from this study demonstrate that treatment of 5 days post fertilization (dpf) fish with a nNO synthase inhibitor as a co-treatment with 6-OHDA facilitates long-term survival and accelerates the recovery from 6-OHDA-induced hypokinesia-like symptoms. These findings are unique in that under conditions of neurotoxin-induced stress, the inhibition of the NO-related S-nitrosylation indirect pathway dramatically facilitates recovery from 6-OHDA treatment but inhibition of the NO-sGC-cGMP direct pathway is essential for survival in 5 dpf treated fish. In conclusion, these results indicate that nNOS and the inhibition of the NO-linked S-nitrosylation pathway plays an important role in antagonizing the protection and recovery of fish from neurotoxin treatment. These data begin to help in the understanding of the role of NO as a neuroprotectant in dopaminergic pathways, particularly those that influence motor dysfunctions.

Role of neuronal nitric oxide in the inhibition of sympathetic vasoconstriction in resting and contracting skeletal muscle of healthy rats

Isoform-specific nitric oxide (NO) synthase (NOS) contributions to NO-mediated inhibition of sympathetic vasoconstriction in resting and contracting skeletal muscle are incompletely understood. The purpose of the present study was to investigate the role of neuronal NOS (nNOS) in the inhibition of sympathetic vasoconstriction in resting and contracting skeletal muscle of healthy rats. We hypothesized that acute pharmacological inhibition of nNOS would augment sympathetic vasoconstriction in resting and contracting skeletal muscle, demonstrating that nNOS is primarily responsible for NO-mediated inhibition of sympathetic vasoconstriction. Sprague-Dawley rats (n = 13) were anesthetized and instrumented with an indwelling brachial artery catheter, femoral artery flow probe, and lumbar sympathetic chain stimulating electrodes. Triceps surae muscles were stimulated to contract rhythmically at 60% of maximal contractile force. In series 1 (n = 9), the percent change in femoral vascular conductance (%FVC) in response to sympathetic stimulations delivered at 2 and 5 Hz was determined at rest and during muscle contraction before and after selective nNOS blockade with S-methyl-l-thiocitrulline (SMTC, 0.6 mg/kg iv) and subsequent nonselective NOS blockade with N(ω)-nitro-l-arginine methyl ester (l-NAME, 5 mg/kg iv). In series 2 (n = 4), l-NAME was injected first, and then SMTC was injected to determine if the effect of l-NAME on constrictor responses was influenced by selective nNOS inhibition. Sympathetic stimulation decreased FVC at rest (-25 ± 7 and -44 ± 8%FVC at 2 and 5 Hz, respectively) and during contraction (-7 ± 3 and -19 ± 5%FVC at 2 and 5 Hz, respectively). The decrease in FVC in response to sympathetic stimulation was greater in the presence of SMTC at rest (-32 ± 6 and -49 ± 8%FVC at 2 and 5 Hz, respectively) and during contraction (-21 ± 4 and -28 ± 4%FVC at 2 and 5 Hz, respectively). l-NAME further increased (P < 0.05) the sympathetic vasoconstrictor response at rest (-47 ± 4 and -60 ± 6%FVC at 2 and 5 Hz, respectively) and during muscle contraction (-33 ± 3 and -40 ± 6%FVC at 2 and 5 Hz, respectively). The effect of l-NAME was not altered by the order of nNOS inhibition. These data demonstrate that NO derived from nNOS and endothelial NOS contribute to the inhibition of sympathetic vasoconstriction in resting and contracting skeletal muscle.

Breast Feeding Increases Vasoconstriction Induced by Electrical Field Stimulation in Rat Mesenteric Artery. Role of Neuronal Nitric Oxide and ATP

ObjectivesThe aim of this study was to investigate in rat mesenteric artery whether breast feeding (BF) affects the vasomotor response induced by electrical field stimulation (EFS), participation by different innervations in the EFS-induced response and the mechanism/s underlying these possible modifications.MethodsExperiments were performed in female Sprague-Dawley rats (3 months old), divided into three groups: Control (in oestrous phase), mothers after 21 days of BF, and mothers that had recovered their oestral cycle (After BF, in oestrous phase). Vasomotor response to EFS, noradrenaline (NA) and nitric oxide (NO) donor DEA-NO were studied. Neuronal NO synthase (nNOS) and phosphorylated nNOS (P-nNOS) protein expression were analysed and NO, superoxide anion (O2 .–), NA and ATP releases were also determined.ResultsEFS-induced contraction was higher in the BF group, and was recovered after BF. 1 µmol/L phentolamine decreased the response to EFS similarly in control and BF rats. NA vasoconstriction and release were similar in both experimental groups. ATP release was higher in segments from BF rats. 0.1 mmol/L L-NAME increased the response to EFS in both control and BF rats, but more so in control animals. BF decreased NO release and did not modify O2 .– production. Vasodilator response to DEA-NO was similar in both groups, while nNOS and P-nNOS expressions were decreased in segments from BF animals.ConclusionBreast feeding increases EFS-induced contraction in mesenteric arteries, mainly through the decrease of neuronal NO release mediated by decreased nNOS and P-nNOS expression. Sympathetic function is increased through the increased ATP release in BF rats.

In VivoEvidence of Increased nNOS Activity in Acute MPTP Neurotoxicity: A Functional Pharmacological MRI Study

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a neurotoxin commonly used to produce an animal model of Parkinson's disease. Previous studies have suggested a critical role for neuronal nitric oxide (NO) synthase- (nNOS-) derived NO in the pathogenesis of MPTP. However, NO activity is difficult to assess in vivo due to its extremely short biological half-life, and so in vivo evidence of NO involvement in MPTP neurotoxicity remains scarce. In the present study, we utilized flow-sensitive alternating inversion recovery sequences, in vivo localized proton magnetic resonance spectroscopy, and diffusion-weighted imaging to, respectively, assess the hemodynamics, metabolism, and cytotoxicity induced by MPTP. The role of NO in MPTP toxicity was clarified further by administering a selective nNOS inhibitor, 7-nitroindazole (7-NI), intraperitoneally to some of the experimental animals prior to MPTP challenge. The transient increase in cerebral blood flow (CBF) in the cortex and striatum induced by systemic injection of MPTP was completely prevented by pretreatment with 7-NI. We provide the first in vivo evidence of increased nNOS activity in acute MPTP-induced neurotoxicity. Although the observed CBF change may be independent of the toxicogenesis of MPTP, this transient hyperperfusion state may serve as an early indicator of neuroinflammation.

The complex contribution of NOS interneurons in the physiology of cerebrovascular regulation

Following the discovery of the vasorelaxant properties of nitric oxide (NO) by Furchgott and Ignarro, the finding by Bredt and coll. of a constitutively expressed NO synthase in neurons (nNOS) led to the presumption that neuronal NO may control cerebrovascular functions. Consequently, numerous studies have sought to determine whether neuraly-derived NO is involved in the regulation of cerebral blood flow (CBF). Anatomically, axons, dendrites, or somata of NO neurons have been found to contact the basement membrane of blood vessels or perivascular astrocytes in all segments of the cortical microcirculation. Functionally, various experimental approaches support a role of neuronal NO in the maintenance of resting CBF as well as in the vascular response to neuronal activity. Since decades, it has been assumed that neuronal NO simply diffuses to the local blood vessels and produce vasodilation through a cGMP-PKG dependent mechanism. However, NO is not the sole mediator of vasodilation in the cerebral microcirculation and is known to interact with a myriad of signaling pathways also involved in vascular control. In addition, cerebrovascular regulation is the result of a complex orchestration between all components of the neurovascular unit (i.e., neuronal, glial, and vascular cells) also known to produce NO. In this review article, the role of NO interneuron in the regulation of cortical microcirculation will be discussed in the context of the neurovascular unit.

Advances in the study of the peripheral nervous system for erection in animals and humans.

Since Walsh first emphasized the importance of preserving the neurovascular bundle n to protect the cavernous nerve during pelvic surgery, patients' sexual life quality has dramatically improved. Today, nerve-sparing radical prostatectomy is the established gold standard for organ-confined prostate cancer patients. Recent technical advances in functional assessment such as intraoperative electrical stimulation have unveiled new anatomical features and physiological roles. Basic research has advanced understanding of cavernous nerve function, while molecular biology has uncovered the crucial role of neuronal nitric oxide in mediating erection, and has led to new treatments such as phosphodiesterase type-5 inhibitors. A recent focus in cavernous nerve research concerns the nerve distribution external to the neurovascular bundle. The cavernous nerves in humans appear to be distributed more widely beneath the lateral pelvic fascia than in other animals, and electrical stimulation studies suggest possible involvement of these nerves in erection. These findings have prompted new surgical techniques such as the "veil of Aphrodite", or "intrafascial nerve-sparing" procedures. These recent anatomical and physiological studies in humans and animals and their impact are reviewed in this article. Further investigation should stimulate future advances in strategies to preserve erectile function in RP patients.

Lactate‐induced retinal vasodilation implicates neuronal nitric oxide synthesis in minipigs

Abstract Purpose To investigate the role of neuronal nitric oxide (NO) synthesis in the retinal vasodilatory response to lactate in minipigs Methods Ten eyes of 10 minipigs were evaluated.After 1 hour of intravenous infusion of Nω‐nitro‐L‐arginine methylester (L‐NAME),an intravitreal juxta‐arteriolar microinjection of 30µl of L‐lactate 0.5 mol/l (pH= 7.4) was performed through a micropipette.Ten minutes later, an intravitreal juxta‐arteriolar microinjection of 30µl of L‐NAME 0.01 mol/l (pH= 7.4) was performed in all eyes but one which received balanced saline solution (BSS).Retinal arteriolar diameter changes were measured in vivo using a Retinal Vessel Analyzer.The animals’ vital signs were monitored and systemic arterial pressure was maintained stable Results Retinal arteriolar diameter decreased by 4.1% 1 hour after intravenous L‐NAME infusion compared to baseline but the difference did not reach significance.Juxta‐arteriolar injection of L‐lactate induced a significant increase in retinal arteriolar diameter of 22.7% and 28.7% at 5 and 10 minutes respectively(p&lt;0.01). This was followed by a significant decrease of 8.6% 10 min after juxta‐arteriolar injection of L‐NAME(p&lt;0.01).Injection of BBS had no effect in the control eye and retinal arterioles remained dilated under the effect of L‐lactate Conclusion Juxta‐arteriolar administration of L‐lactate induced vasodilation despite inhibition of endothelial‐derived NO by the continuous intravenous infusion of L‐NAME. Moreover,juxta‐arteriolar L‐NAME microinjection significantly suppressed the vasodilatory effect of L‐lactate.These data suggest that neuronal‐derived NO is an important mediator of the lactate‐induced vasodilation in minipigs

Lactate-Induced Retinal Arteriolar Vasodilation Implicates Neuronal Nitric Oxide Synthesis in Minipigs

To investigate the role of neuronal nitric oxide (NO) synthesis in the retinal vasodilatory response to lactate in minipigs. Thirteen eyes of 13 minipigs were evaluated. Ten eyes received an intravenous infusion of N(omega)-nitro-L-arginine methyl ester (L-NAME). After 1 hour, the same eyes received an intravitreous juxta-arteriolar microinjection of 30 microL of L-lactate 0.5 M (pH 7.4) through a micropipette. Ten minutes later, 9 of 10 eyes received an intravitreous juxta-arteriolar microinjection of 30 microL of L-NAME 0.01 M (pH 7.4), and 1 received physiologic saline solution (PSS). The remaining three eyes received a microinjection of 30 microL of L-lactate 0.5 M (pH 7.4), without intravenous or intravitreous L-NAME. The three eyes that received juxta-arteriolar injection of L-lactate only showed a reproducible increase in retinal arteriolar diameter that persisted during the entire study period (maximum effect at 20 minutes, 40.9% +/- 3.2%). Retinal arteriolar diameter decreased by 4.1% 1 hour after intravenous L-NAME when compared with baseline but the difference did not reach significance. The juxta-arteriolar injection of L-lactate induced a significant increase in retinal arteriolar diameter (22.7% and 28.7% at 5 and 10 minutes, respectively; P < 0.01), followed by a significant decrease (8.6%; P < 0.01) 10 minutes after juxta-arteriolar injection of L-NAME. Injection of PSS had no effect on retinal arteriolar diameter. Juxta-arteriolar administration of L-lactate induced vasodilation, which was also observed with continuous intravenous infusion of L-NAME. Moreover, juxta-arteriolar L-NAME microinjection significantly suppressed the vasodilatory effect of L-lactate. These data suggest that neuronal-derived NO is an important mediator of lactate-induced vasodilation in minipigs.

Role of neuronal nitric oxide in the regulation of vasopressin expression and release in response to inhibition of catecholamine synthesis and dehydration

We used neuronal nitric oxide synthase (nNOS) gene knockout mice to study the effects of catecholamines and neuronal nitric oxide on vasopressin expression in the hypothalamic neurosecretory centers. nNOS gene deletion did not change the level of vasopressin mRNA in the supraoptic or paraventricular nuclei. In contrast, vasopressin immunoreactivity was lower in nNOS deficient mice than in wild-type animals. Dehydration increased vasopressin mRNA levels and decreased vasopressin immunoreactivity in both wild-type and nNOS knockout mice, but these responses were more marked in the nNOS knockout mice. Treatment with alpha-mpt, a pharmacologic inhibitor of catecholamine synthesis, resulted in increased vasopressin mRNA levels in wild-type mice and in reduced vasopressin immunoreactivity in both wild-type and nNOS knockout mice. From these results, we conclude: (1) neuronal nitric oxide suppresses vasopressin expression under basal conditions and during activation of the vasopressinergic system by dehydration; (2) catecholamines limit vasopressin expression; (3) nNOS is required for the effects of catecholamines on vasopressin expression.

Role of nitric oxide in cerebral blood flow changes during kainate seizures in mice: genetic and pharmacological approaches

The role of neuronal nitric oxide (NO) in the cerebrovascular response to partial seizures induced by intrahippocampal injection of kainate (KA) was investigated in mice deleted for the neuronal NO synthase gene (nNOS-/-) and in wild-type controls (WT). A second group of WT mice received the nNOS inhibitor, 7-nitroindazole (WT-7NI). Local cerebral blood flow (LCBF) was measured using the quantitative (14)C-iodoantipyrine method. Within the epileptic focus, all three groups of seizing mice (WT, WT-7NI, and nNOS-/-) showed significant 26-88% LCBF increases in ipsilateral hippocampus, compared to saline-injected mice. Contralaterally to the epileptic focus, KA seizures induced a 21-47% LCBF decreases in hippocampus and limbic cortex of WT mice and in most contralateral brain structures of nNOS-/- mice, while WT-7NI mice showed no contralateral CBF change. Neuronal NO appears to be not involved in the cerebrovascular response within the epileptic focus, but may rather have a role in the maintenance of distant LCBF regulation during seizures.

Neurogenic nitric oxide release increases in mesenteric arteries from ouabain hypertensive rats

We investigated whether chronic ouabain treatment changes the vasoconstrictor responses induced by electrical field stimulation (EFS) in endothelium-denuded rat superior mesenteric arteries and a possible role of neuronal nitric oxide (NO). Mesenteric arteries from untreated and ouabain-treated rats (approximately equal to 8.0 microg/kg per day, for 5 weeks) were used in this study. Vascular reactivity was analyzed by isometric tension recording. Expression of the neuronal NO synthase isoform was analyzed by Western blot. Noradrenaline release was evaluated in segments incubated with [H]noradrenaline. Systolic (SBP) and diastolic (DBP) blood pressure were higher in ouabain-treated rats than in untreated rats (SBP, untreated: 120 +/- 3.5 mmHg versus ouabain-treated: 150 +/- 4.7 mmHg, P < 0.01; DBP, untreated: 87 +/- 3.0 mmHg versus ouabain-treated: 114 +/- 2.6 mmHg, P < 0.001). EFS-induced vasoconstrictions were smaller in arteries from ouabain-treated rats than in those from untreated animals, while the EFS-induced [H]noradrenaline release and the vasoconstriction induced by exogenous noradrenaline (1 nmol/l-10 micromol/l) remained unmodified. The non-selective NO synthase (NOS) inhibitor, N-nitro-L-arginine methyl ester (100 micromol/l), increased the EFS-induced vasoconstriction in mesenteric arteries from both groups, although the effect was more pronounced in segments from ouabain-treated rats. The selective neuronal NOS inhibitor, 7-nitroindazole (7-NI; 100 micromol/l) increased EFS-induced contraction only in segments from ouabain-treated rats. Neuronal NOS expression was greater in the mesenteric arteries from ouabain-treated rats than in those from untreated animals. Sodium nitroprusside (0.1 nmol/l-10 micromol/l) induced a similar vasodilatation in segments from both groups. These results suggest that chronic ouabain treatment is accompanied by an increase in neuronal NO release that reduces EFS-induced vasoconstriction.

The role of neuronal nitric oxide in the vagal control of cardiac interval of the rat heart in vitro

The aim of this study was to examine the role of neuronal nitric oxide (NO) on vagal regulation of the rat heart in vitro using the neuronal nitric oxide synthase (nNOS) inhibitor 1-(2-trifluoromethylphenyl) imidazole (TRIM). All experiments were carried out in the presence of the β-adrenoreceptor antagonist atenolol (4 μM). Right thoracic vagus, or its cardiac branch, was stimulated at frequencies of 2, 4, 8, 16 and 32 Hz (pulse duration 1 ms, 20 V, for 20 s) before and after addition of TRIM (0.14 mM) and cardiac interval (ms) assessed. There was a significant positive linear correlation between cardiac interval and vagal frequency giving a slope of 2.76±0.8 ms/Hz (slope±S.E. slope; data pooled from eight rats) which was significantly attenuated following TRIM to 0.4±0.6 ms/Hz ( P<0.05 ANOVA; n=8 rats). Nicotine applied in cumulative concentrations (0.03, 0.1, 0.3, 0.5, 1 mM) caused a linear concentration-dependent increase in cardiac interval, with a slope of 403±72 ms/mM ( n=10 rats) which was significantly attenuated after treatment with hexamethonium (28 μM), to 190±36 ms/mM ( n=10 rats, P<0.05 ANOVA), and atropine (3 μM) 100±31 ms/mM ( n=9 rats, P<0.05 ANOVA) but not following TRIM (0.14 mM) 262±48 ms/mM ( n=9 rats, P<0.05 ANOVA). These results suggest that NO facilitates vagal effects on the rat heart in vitro by an action at the pre-ganglionic/post-ganglionic synapse.

The role of neuronal nitric oxide in the regional neurovascular coupling: Voxel-based comparison between perfusion and metabolic PET images

In order to clarify the role of neuronal nitric oxide in cerebral neurovascular coupling, we measured the CBF and CMRglc with PET in α-chloralose anesthetized cats during electrical somatosensory stimulation, and under the treatment of a neuronal nitric oxide inhibitor, 7-nitroindazole (7-NI). The voxel-based analysis was performed with the use of globally normalized CBF and CMRglc values to identify the increased areas of CBF and CMRglc produced by somatosensory stimulation, and the uncoupled areas between their changes induced by the 7-NI. Somatosensory stimulation in the left forepaw elicited an increase in both the CBF and CMRglc in the left cerebellum, the right thalamus and the right somatosensory cortex. Injection of 7-NI induced global uncoupling (20% decrease in the global CBF, but not in the global CMRglc), whereas the regional uncoupling during activation was observed in the ipsilateral cerebellum. Thus, neuronal nitric oxide may play a predominant role in the coupling of the regional perfusion and metabolism in the cerebellum, but less so in the neocortex.

Treatment with 7-nitroindazole enhances kainic acid induced cholinergic neurotoxicity in the rat striatum: a neuroprotective role for neuronal nitric oxide.

1. In this study we investigated the effect of 7-nitroindazole (7-NI), a preferential inhibitor of neuronal nitric oxide synthase (nNOS), on kainic acid (KA) induced neurotoxicity in rats. Choline acetyltransferase activity (CAT), a cholinergic marker, and histological changes were employed to assess neurotoxicity. 2. In control rats, the local intrastriatal injection of 0.5 microg of KA reduced CAT from 22.9 +/- 2.2 to 14.7 +/- 2.0 nmol/h/mg tissue ((38 +/- 6)% reduction) (P < 0.001). Greater reductions in CAT were observed with 1 and 2 microg of KA ((70 +/- 6)% and (80 +/- 3)%, respectively). 7-NI aggravated KA-induced cholinergic and histological damage. KA reduced CAT by (68.2 +/- 4)% in 7-NI-treated rats, by (38 +/- 6)% in saline-treated controls, and by (41 +/- 4)% in peanut-oil- (7-NI-vehicle-) treated rats (P = 0.0047). 3. After KA, CAT activity averaged 14.3 +/- 2.0 in peanut-oil-treated rats and 7.9 +/- 1.0 nmol/h/mg tissue in 7-NI- (peanut-oil-) treated rats (P = 0.015). Similarly to changes in CAT, 7-NI treatment aggravated KA-induced histological changes indicative of neuronal damage (acute ischemic neuronal changes, disorganization of myelinated fibers bundle, and vacuolation changes of the neuropil). Treatment with 7-NI was not associated with increased mortality. 4. Our findings suggest that neuronal NO plays a neuroprotective action on excitotoxicity.

A role for free radicals and nitric oxide in delayed recovery in aged rats with chronic constriction nerve injury

Using a reversible chronic constriction injury (CCI) model of neuropathic pain, we previously demonstrated that changes in thermal hyperalgesia correlate with the changes in peripheral microvascular blood flow in the affected paw, and that recovery can be assessed by normalization of both behavioral and vascular responses. Using the same model, this study examined age-related changes in recovery after nerve injury and the involvement of free radicals and nitric oxide (NO) in these changes. Four loose, nonconstrictive ligatures were applied to the sciatic nerve in the right, mid-thigh region of young and old (3 and 24 months) Sprague Dawley rats. All rats were monitored weekly (for 8–10 weeks) for their thermal threshold using a 46°C water bath and some groups were used to examine endothelial and smooth muscle-dependent microvascular responses to substance P (SP) and sodium nitroprusside (SNP), respectively. These substances were perfused over the base of blisters raised on the footpad innervated by the injured nerve. Free radical activity in the sciatic nerve was assessed by measuring the activity of xanthine oxidase (XO) and lipid hydroperoxides (LPO). Young rats showed signs of recovery (reduction in thermal hyperalgesia and improvement of peripheral microvascular blood flow) from the fifth week. No signs of recovery were observed in old rats for 8 weeks, with some reduction in thermal hyperalgesia observed by weeks 9 and 10. XO activity was significantly higher in young injured nerves compared to sham (400%) and was even significantly greater in old injured nerves (680%). Similarly, old injured nerves showed 300% increase in LPO levels compared to sham. The role of reactive oxygen species (ROS) in delayed recovery in old rats was examined using the antioxidant tirilazad mesylate. Tirilazad (20 mg/kg) was injected intramuscularly (im) in the mid-thigh region starting on day 1 post CCI, (early treatment) or day 7 (late treatment). Levels of LPO in the injured sciatic nerves were significantly reduced using either early or late treatment, however tirilazad had opposing effects on recovery, prolonging or alleviating thermal hyperalgesia, respectively. The role of neuronal nitric oxide (nNO) was then examined using the specific neuronal nitric oxide synthase (nNOS) inhibitor, 3-bromo-7-nitroindazole (3Br-7NI) (10 mg/kg). 3Br-7NI resulted in a significant alleviation of thermal hyperalgesia with improvement in the vascular responses from weeks 5 and 6 onwards. A combination of 3Br-7NI and tirilazad treatment was also used but did not show an additive effect. The results suggest that ROS and nNO contribute to delayed recovery of injured nerves in old rats and to the maintenance of thermal hyperalgesia and the reduction in microvascular blood flow in the area innervated by the injured nerve. The results also raise the notion that possible interaction of free radicals with NO to form peroxynitrite might be responsible for such delayed recovery. Ironically, this study also reveals a positive role for free radicals in tissue repair and raises the notion that early intervention with antioxidants could exert a negative effect on repair of injured nerves.

Light-induced Photoreceptor Apoptosis in Vivo Requires Neuronal Nitric-oxide Synthase and Guanylate Cyclase Activity and Is Caspase-3-independent

Apoptosis is the mode of photoreceptor cell death in inherited and induced retinal degeneration. However, the molecular mechanisms of photoreceptor cell death in human cases and animal models of retinal dystrophies remain undefined. Exposure of Balb/c mice to excessive levels of white light results in photoreceptor apoptosis. This study delineates the molecular events occurring during and subsequent to the induction of retinal degeneration by exposure to white light in Balb/c mice. We demonstrate an early increase in intracellular calcium levels during photoreceptor apoptosis, an event that is accompanied by significant superoxide generation and mitochondrial membrane depolarization. Furthermore, we show that inhibition of neuronal nitric-oxide synthase (nNOS) by 7-nitroindazole is sufficient to prevent retinal degeneration implicating a key role for neuronal nitric oxide (NO) in this model. We demonstrate that inhibition of guanylate cyclase, a downstream effector of NO, also prevents photoreceptor apoptosis demonstrating that guanylate cyclase too plays an essential role in this model. Finally, our results demonstrate that caspase-3, frequently considered to be one of the key executioners of apoptosis, is not activated during retinal degeneration. In summary, the data presented here demonstrate that light-induced photoreceptor apoptosis in vivo is mediated by the activation of nNOS and guanylate cyclase and is caspase-3-independent.

Lessons learned from nitric oxide synthase knockout animals

Nitric oxide (NO) is generated by 3 major isoforms of nitric oxide synthase (NOS) with complex and overlapping patterns of expression. This article presents several examples of how gene targeted mice lacking endothelial and neuronal isoforms have showed various roles of NO. Neuronal NOS knockout mice are resistant to global and focal cerebral ischemia, confirming a role for neuronal NO in cellular toxicity after stroke. Endothelial NOS knockout mice have increased susceptibility to stroke consistent with a vascular protective effect of NO. They are hypertensive and lack endothelium dependent relaxing factor activity. Analysis of cardiac function shows roles for NO in suppression of inotropic responses to beta-adrenergic agonists and in mediating basal diastolic relaxation. Endothelial NOS knockout mice respond to vascular injury with increased neointimal proliferation, consistent with a physiological role for NO to suppress smooth muscle cell proliferation.

Role of neuronal nitric oxide in methamphetamine neurotoxicity and protection by nNOS inhibitor

Abstract Methamphetamine (METH) is a potent psychostimulant known to produce neurotoxicity. The dopaminergic pathway is particularly sensitive to METH. Recent studies showed that 7-nitroindazole (7-NI), a selective inhibitor of neuronal nitric oxide synthase (nNOS), provided protection against METH neurotoxicity both in vitro and in vivo. The present studies were conducted to determine the nNOS activity in various regions of the brain of young adult male Sprague-Dawley rats treated with different doses of METH. Rats were injected ip with 5, 10, 20, and 40 mg/kg and 24 h after the rats were sacrificed and the brain regions (hippocampus, frontal cortex, and cerebellum) were quickly dissected. The cytosolic fractions were prepared, and the nNOS activity was determined using the 3H-citrulline assay. The results showed that nNOS activity was significantly increased in all three brain regions of rats treated with METH. The increase was dose dependent reaching a maximum of 40-100% over the control values. Rats treated with 7NI 30 min prior to METH injection provided protection against the toxicity and also showed a reduction of nNOS activity. The activation of nNOS is known to increase the synthesis of NO which is involved in the regulation of several neurotransmitter pathways including catecholaminergic system. Reducing the METH-induced production of NO by pretreatment with selective inhibitor of nNOS, 7-NI, provided protection against METH neurotoxicity.

Is neuronal nitric oxide involved in adjuvant-induced joint inflammation?

Several reports have described a role of macrophagic, endothelial and synoviocytal nitric oxide (NO) in inflammation, immunity and sensory processes in joint diseases. In view of the role of the peripheral nervous system in arthritis and owing to the presence of NO-producing neurons in primary sensory neurons, we have investigated the possible role of neuronal NO during adjuvant-induced joint inflammation in rats. Neural nitric oxide synthase production in sensory ganglia and the spinal cord was investigated by in situ hybridization and immunocytochemistry. Neuronal NO synthase mRNA expression and neuronal NO synthase immunoreactivity increased in lumbar dorsal root ganglia in arthritic rats compared to those of normal rats, whereas neuronal NO synthase mRNA expression decreased in lamina X and lamina I–II of the lumbar spinal cord. The administration of the selective neuronal NO synthase inhibitor 7-nitro indazole, reduced the joint inflammation, whereas the administration of the inducible NO synthase selective inhibitor, aminoguanidine, had no effect on inflammation when administered daily from the third day after adjuvant. These findings could indicate a role for neural NO in adjuvant arthritis.

A neuromodulatory role for neuronal nitric oxide in the rabbit renal artery.

1. The effects of the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) on vasoconstrictor responses to transmural electrical nerve stimulation and noradrenaline were examined in the rabbit isolated renal artery with and without an intact endothelium. In addition, the effect of removing the endothelium from the renal artery on vasoconstrictor responses to transmural electrical nerve stimulation and noradrenaline was also investigated. Immunohistofluorescence techniques were carried out to determine if there were any nitrergic nerves supplying the renal artery. 2. The vasoconstriction produced in response to transmural electrical nerve stimulation (2-64 Hz) was significantly enhanced in the presence of L-NAME (3 x 10(-6), 10(-5), 3 x 10(-5) and 10(-4) M). 3. L-NAME (3 x 10(-6), 10(-5), 3 x 10(-5) and 10(-4) M) did not significantly affect the maximum vasoconstriction produced in response to noradrenaline. However, the noradrenaline dose-response curve was significantly shifted to the left by the addition of L-NAME (3 x 10(-6), 10(-5), 3 x 10(-5) and 10(-4) M). 4. The increase in the amplitude of the vasoconstriction, produced in response to transmural electrical nerve stimulation (16 Hz) and noradrenaline (10(-5) M) in the presence of L-NAME (10(-5) M) was not observed when L-arginine (10(-3) M) was added in addition to L-NAME (10(-5) M). 5. Removing the endothelium did not significantly affect the response to transmural electrical nerve stimulation (1-64 Hz). The maximum vasoconstriction in response to noradrenaline was also unaffected by the removal of the endothelium. The pD2 value for noradrenaline obtained from vessels with no endothelium was significantly greater than the pD2 value obtained from vessels with an intact endothelium (5.90 +/- 0.11 and 5.16 +/- 0.03, respectively). 6. On renal artery segments with no endothelium L-NAME (3 x 10(-5) M) significantly enhanced the response to transmural electrical nerve stimulation (2-64 Hz). L-NAME did not affect the maximum response to noradrenaline. However, there was a significant shift to the right of the noradrenaline doseresponse curve in the presence of L-NAME (3 x 10(-5) M). 7. Both nitric oxide synthase-containing and NADPH-diaphorase stained nerves were located on the adventitial-medial border of the rabbit renal artery. 8. The present study has suggested a presynaptic inhibitory action for nitric oxide (probably derived from identified perivascular nitrergic nerves), on perivascular sympathetic vasoconstrictor nerve mediated responses of the rabbit renal artery. In contrast, the enhancement of the response to noradrenaline by L-NAME can be attributed to inhibition of the synthesis of endothelium-derived nitric oxide.