Reduced neuronal nitric oxide synthase is involved in ischemia-induced hippocampal neurogenesis by up-regulating inducible nitric oxide synthase expression

Nitric oxide (NO), a free radical with signaling functions in the CNS, is implicated in some developmental processes, including neuronal survival, precursor proliferation, and differentiation. However, neuronal nitric oxide synthase (nNOS) -derived NO and inducible nitric oxide synthase (iNOS) -derived NO play opposite role in regulating neurogenesis in the dentate gyrus after cerebral ischemia. In this study, we show that focal cerebral ischemia reduced nNOS expression and enzymatic activity in the hippocampus. Ischemia-induced cell proliferation in the dentate gyrus was augmented in the null mutant mice lacking nNOS gene (nNOS−/−) and in the rats receiving 7-nitroindazole, a selective nNOS inhibitor, after stroke. Inhibition of nNOS ameliorated ischemic injury, up-regulated iNOS expression, and enzymatic activity in the ischemic hippocampus. Inhibition of nNOS increased and iNOS inhibitor decreased cAMP response element-binding protein phosphorylation in the ipsilateral hippocampus in the late stage of stroke. Moreover, the effects of 7-nitroindazole on neurogenesis after ischemia disappeared in the null mutant mice lacking iNOS gene (iNOS−/−). These results suggest that reduced nNOS is involved in ischemia-induced hippocampal neurogenesis by up-regulating iNOS expression and cAMP response element-binding protein phosphorylation.     

Role of neuronal nitric oxide synthase in hypoxia-induced anapyrexia in rats.

Anapyrexia (a regulated decrease in body temperature) is a response to hypoxia that occurs in organisms ranging from protozoans to mammals, but very little is known about the mechanisms involved. Recently, it has been shown that the NO pathway plays a major role in hypoxia-induced anapyrexia. However, very little is known about which of the three different nitric oxide synthase isoforms (neuronal, endothelial, or inducible) is involved. The present study was designed to test the hypothesis that neuronal nitric oxide synthase (nNOS) plays a role in hypoxia-induced anapyrexia. Body core temperature (T(c)) of awake, unrestrained rats was measured continuously using biotelemetry. Rats were submitted to hypoxia, 7-nitroindazole (7-NI; a selective nNOS inhibitor) injection, or both treatments together. Control animals received vehicle injections of the same volume. We observed a significant (P < 0.05) reduction in T(c) of approximately 2.8 degrees C after hypoxia (7% inspired O(2)), whereas intraperitoneal injection of 7-NI at 25 mg/kg caused no significant change in T(c). 7-NI at 30 mg/kg elicited a reduction in T(c) and was abandoned in further experiments. When the two treatments were combined (25 mg/kg of 7-NI and 7% inspired O(2)), we observed a significant attenuation of hypoxia-induced anapyrexia. The data indicate that nNOS plays a role in hypoxia-induced anapyrexia.     

Alpha-Tocopherol Decreases Iron-Induced Hippocampal and Nigral Neuron Loss

There are many studies about iron-induced neuronal hyperactivity and oxidative stress. Some reports also showed that iron levels rise in the brain in some neurodegenerative diseases such as Parkinson’s (PD) and Alzheimer’s disease (AD). It has been suggested that excessive iron level increases oxidative stress and causes neuronal death. Tocopherols act as a free radical scavenger when phenoxylic head group encounters a free radical. We have aimed to identify the effect of α-tocopherol (Vitamin E) on iron-induced neurotoxicity. For this reason, rats were divided into three groups as control, iron, and iron + α-tocopherol groups. Iron chloride (200 mM in 2.5 μl volume) was injected into brain ventricle of iron and iron + α-tocopherol group rats. Same volume of saline (2.5 μl) was given to the rats belonging to control group. Rats of iron + α-tocopherol group received intraperitoneally (i.p.) α-tocopherol (100 mg/kg/day) for 10 days. After 10 days, rats were perfused intracardially under deep urethane anesthesia. Removed brains were processed using standard histological techniques. The numbers of neurons in hippocampus and substantia nigra of all rats were estimated by stereological techniques. Results of present study show that α-tocopherol decreased hippocampal and nigral neuron loss from 51.7 to 12.1% and 41.6 to 17.8%, respectively. Findings of the present study suggest that α-tocopherol may have neuroprotective effects against iron-induced hippocampal and nigral neurotoxicity and it may have a therapeutic significance for neurodegenerative diseases involved iron.     

Involvement of Nitric Oxide in Spatial Memory Deficits in Status Epilepticus Rats

Status epilepticus (SE) is associated with a significant risk of cognitive impairment, and the increase of nitric oxide (NO) releasing has been reported during SE. We investigated the effects of neuronal nitric oxide synthase (nNOS) inhibitor, 7-nitroindazole (7-NI) and inducible nitric oxide synthase (iNOS) inhibitor, aminoguanidine (AG), on spatial performance of rats in the Morris water maze. Treatment with 7-NI, but not with AG, improved the performance of rats after SE not only in acquisition of the task but also in probe test. Furthermore, the level of SE-induced malondialdehyde (MDA), end product of lipid peroxidation, was significantly decreased only in animals receiving 7-NI injection. Taken together, the results of the present study provided evidence that the NO pathway contributed to oxidative stress after SE, and nNOS/NO pathway may underlie one of the potential mechanisms contributing to SE-induced spatial memory deficits.     

Nitric Oxide Participates in the Brain Ischemic Tolerance Induced by Intermittent Hypobaric Hypoxia in the Hippocampal CA1 Subfield in Rats

Previous studies have shown that intermittent hypobaric hypoxia (IH) preconditioning protected neurons survival from brain ischemia. However, the mechanism remains to be elucidated. The present study explored the role of nitric oxide (NO) in the process by measuring the expression of NO synthase (NOS) and NO levels. Male Wistar rats (100) were randomly assigned into four groups: sham group, IH?+?sham group, ischemia group and IH?+?ischemia group. Rats for IH preconditioning were exposed to hypobaric hypoxia mimicking 5000 m high-altitude (P_B?=?404 mmHg, PO₂?=?84 mmHg) 6 h/day, once daily for 28 days. Global brain ischemia was established by four-vessel occlusion that has been created by Pulsinelli. Rats were sacrificed at 7th day after the ischemia for neuropathological evaluation by thionin stain. In addition, the expression of neuronal NOS (nNOS), inducible NOS (iNOS), and NO content in the hippocampal CA1 subfield were measured at 2nd day and 7th day after the ischemia. Results revealed that global brain ischemia engendered delayed neuronal death (DND), both nNOS and iNOS expression up-regulated, and NO content increased in the hippocampal CA1 subfield. IH preconditioning reduced neuronal injury induced by the ischemia, and prevented the up-regulation of NOS expression and NO production. In addition, l-NAME?+?ischemia group was designed to detect whether depressing NO production could alleviate the DND. Pre-administration of l-NAME alleviated DND induced by the ischemia. These results suggest that IH preconditioning plays a protective role by inhibiting the over expression of NOS and NO content after brain ischemia.     

Nitric oxide produced via neuronal NOS may impair vasodilatation in septic rat skeletal muscle

Impaired vascular responsiveness in sepsis may lead to maldistribution of blood flow in organs. We hypothesized that increased production of nitric oxide (NO) via inducible nitric oxide synthase (iNOS) mediates the impaired dilation to ACh in sepsis. Using a 24-h cecal ligation and perforation (CLP) model of sepsis, we measured changes in arteriolar diameter and in red blood cell velocity (V(RBC)) in a capillary fed by the arteriole, following application of ACh to terminal arterioles of rat hindlimb muscle. Sepsis attenuated both ACh-stimulated dilation and V(RBC) increase. In control rats, arteriolar pretreatment with the NO donors S-nitroso-N-acetylpenicillamine or sodium nitroprusside reduced diameter and V(RBC) responses to a level that mimicked sepsis. In septic rats, arteriolar pretreatment with the "selective" iNOS blockers aminoguanidine (AG) or S-methylisothiourea sulfate (SMT) restored the responses to the control level. The putative neuronal NOS (nNOS) inhibitor 7-nitroindazole also restored the response toward control. At 24-h post-CLP, muscles showed no reduction of endothelial NOS (eNOS), elevation of nNOS, and, surprisingly, no induction of iNOS protein; calcium-dependent constitutive NOS (eNOS+nNOS) enzyme activity was increased whereas calcium-independent iNOS activity was negligible. We conclude that 1) AG and SMT inhibit nNOS activity in septic skeletal muscle, 2) NO could impair vasodilative responses in control and septic rats, and 3) the source of increased endogenous NO in septic muscle is likely upregulated nNOS rather than iNOS. Thus agents released from the blood vessel milieu (e.g., NO produced by skeletal muscle nNOS) could affect vascular responsiveness.     

Interaction between endogenous nitric oxide and carbon monoxide in the pathogenesis of recurrent febrile seizures

The aim of the study was to investigate the interaction between nitric oxygenase (NOS)/nitric oxide (NO) and heme oxygenase (HO)/carbon monoxide (CO) system in the pathogenesis of recurrent febrile seizures (FS). On a rat model of recurrent FS, the ultrastructure of hippocampal neurons was observed under electron microscopy, and expression of neuronal NOS (nNOS) in hippocampus and NO formation in plasma were examined after treatment with ZnPP-IX, an HO-1 inhibitor. In the ultrastructure of hippocampal neurons, the expression of HO-1 in hippocampus and CO formation in plasma were examined after treatment with L-NAME, a NOS inhibitor. We found that hippocampal neurons were injured after recurrent FS. The gene and protein expression of nNOS and HO-1 increased markedly in hippocampus in FS rats, while CO formation in plasma increased markedly and the concentration of NO in plasma increased slightly. ZnPP-IX could worsen the neuronal damage of recurrent FS rats. However, it further increased the expression of nNOS and endogenous production of NO obviously. L-NAME alleviated the neuronal damage of recurrent FS rats, but decreased the expression of HO-1 and CO formation. The results of this study suggested that endogenous NOS/NO and HO/CO systems might interact with each other and therefore play an important regulating role in recurrent FS brain damage.     

Possible involvement of neuronal nitric oxide synthase enzyme in early-phase isoflurane-induced hypotension in rats

This study was conducted to demonstrate the involvement of nitric oxide synthase (NOS) in the early-phase isoflurane-induced hypotension and to ascertain whether this NOS is neuronal NOS (nNOS) or endothelial NOS (eNOS). Mean arterial pressures (MAPs) were directly measured from the femoral arteries of urethane-anesthetized rats. Isoflurane-induced changes in MAP were monitored in rats following pretreatment with vehicle or one of the following NOS inhibitors: L-N^G-monomethyl-L-arginine (L-NMMA), which is non-selective; L-N^G-nitro arginine (L-NOARG), which is more selective for nNOS and eNOS; and 7-nitroindazole (7-NI), which is selective for nNOS. Exposure to 2% isoflurane in oxygen produced a triphasic reduction in MAP, including an early phase in which mean arterial pressure (MAP) fell by 25-30% during the initial 2½ min. This early hypotensive response, but not subsequent phases, was abolished by i.v. pretreatment with either L-NMMA or L-NOARG. The early-phase hypotension was also significantly attenuated by i.p. pretreatment with 7-NI; however, the blockade was not as complete as with L-NMMA or L-NOARG. Cerebella and aorta were removed from vehicle- and 7-NI pretreated rats and assayed for NOS activity by determining the conversion of [¹⁴C]L-arginine to [¹⁴C]L-citrulline. The 7-NI pretreatment significantly reduced NOS activity in the cerebellum but not the aorta. These findings indicate that the early-phase isoflurane-induced hypotension may involve nNOS as well as eNOS. The nNOS may participate in regulation of isoflurane-induced neuronal release of endogenous opioid peptide, which produces a vasodilation that is dependent on NO derived from an action of eNOS.     

Effects of chronic treatment with 7-nitroindazole in hyperthyroid rats

This study analyzed the contribution of neuronal nitric oxide synthase (nNOS) to the hemodynamic manifestations of hyperthyroidism. The effects on hyperthyroid rats of the chronic administration of 7-nitroindazole (7-NI), an inhibitor of nNOS, were studied. Six groups of male Wistar rats were used: control, 7-NI (30 mg.kg-1.day-1 by gavage), T(4)50, T(4)75 (50 or 75 microg thyroxine.rat-1.day-1, respectively), T(4)50+7-NI, and T(4)75+7-NI. All treatments were maintained for 4 wk. Body weight, tail systolic blood pressure (SBP), and heart rate (HR) were recorded weekly. Finally, SBP, pulse pressure (PP), and HR were measured in conscious rats, and morphological, metabolic, plasma, and renal variables were determined. Expression of nNOS in the hypothalamus of T(4)75 and control rats was analyzed by Western blot analysis. The response of mean arterial pressure (MAP) to pentolinium (10 mg/kg iv) was used to evaluate the sympathetic contribution to BP in T(4)75 and T(4)75+7-NI rats. T(4) produced an increased hypothalamic nNOS expression and dose-related increases in blood pressure (BP), HR, and PP vs. control rats. 7-NI did not modify BP or any other hemodynamic variable in normal rats. However, 7-NI produced a marked reduction in BP, HR, PP, and food and water intake in both hyperthyroid groups and improved creatinine clearance in the T(4)75 group. Pentolinium produced a greater MAP decrease in the T(4)75+7-NI than in the T(4)75 group. In conclusion, administration of 7-NI attenuates the hemodynamic and metabolic manifestations of hyperthyroidism, suggesting that nNOS contributes to the hyperdynamic circulation of this endocrine disease by modulating sympathetic activity.     

Effect of 7-Nitroindazole Sodium on the Cellular Distribution of Neuronal Nitric Oxide Synthase in the Cerebral Cortex of Hypoxic Newborn Piglets

Cerebral hypoxia results in generation of nitric oxide (NO) free radicals by Ca⁺⁺-dependent activation of neuronal nitric oxide synthase (nNOS). The present study tests the hypothesis that the hypoxia-induced increased expression of nNOS in cortical neurons is mediated by NO. To test this hypothesis the cellular distribution of nNOS was determined immunohistochemically in the cerebral cortex of hypoxic newborn piglets with and without prior exposure to the selective nNOS inhibitor 7-nitroindazole sodium (7-NINA). Studies were conducted in newborn piglets, divided into normoxic (n = 6), normoxic treated with 7-NINA (n = 6), hypoxic (n = 6) and hypoxic pretreated with 7-NINA (n = 6). Hypoxia was induced by lowering the FiO₂ to 0.05–0.07 for 1 h. Cerebral tissue hypoxia was documented by decrease of ATP and phosphocreatine levels in both the hypoxic and 7-NINA pretreated hypoxic groups (P < 0.01). An increase in the number of nNOS immunoreactive neurons was observed in the frontal and parietal cortex of the hypoxic as compared to the normoxic groups (P < 0.05) which was attenuated by pretreatment with 7-NINA (P < 0.05 versus hypoxic). 7-NINA affected neither the cerebral energy metabolism nor the cellular distribution of nNOS in the cerebral cortex of normoxic animals. We conclude that nNOS expression in cortical neurons of hypoxic newborn piglets is NO-mediated. We speculate that nNOS inhibition by 7-NINA will protect against hypoxia-induced NO-mediated neuronal death.     

Inhibition of nNOS reduces ischemic cell death through down-regulating calpain and caspase-3 after experimental stroke

In vitro nitric oxide (NO) regulates calpain and caspase-3 activation, and in vivo neuronal nitric oxide synthase (nNOS), calpain and caspase-3 participate in the ischemic brain injury. Our objective was to investigate whether nNOS was involved in the ischemic brain injury through activating calpain and caspase-3 during experimental stroke. Rats received 1-h ischemia by intraluminant filament, and then reperfused for 23 h (R 23 h). nNOS inhibitor 7-nitroindozale (7-NI, 50 mg/kg) was administrated intraperitoneally 5 min before ischemia. Our data showed that treatment with 7-NI markedly reduced neurological deficits, the brain swelling, and the infarct volume at R 23 h. Enzyme studies revealed significant suppression of the activities of m-calpain and caspase-3 in penumbra and core, and the activities of μ-calpain in penumbra, but not in core, in 7-NI-treated rats versus vehicle-treated rats. Western blot analysis demonstrated that 7-NI markedly increased the levels of MAP-2 and spectrin in penumbra and core compared with vehicle-treated rats. Histopathological studies displayed that 7-NI significantly reduced the necrotic cell death in penumbra and core, and apoptotic cell death in penumbra, but not in core. These data demonstrate the involvement of NO produced by nNOS in the ischemic neuronal injury through affecting the activation of calpain and caspase-3 in penumbra and core after experimental stroke, which provides a new perspective on possible mechanisms of action of nNOS inhibition in cerebral ischemia.     

A calcium channel blocker flunarizine attenuates the neurotoxic effects of iron

Iron is a metal highly concentrated in liver and brain tissue, and known to induce neuronal hyperactivity and oxidative stress. It has been established that iron levels rise in the brain in some neurodegenerative diseases such as Parkinson's and Alzheimer's diseases (AD). A body of evidence indicates a link between neuronal death and intracellular excessive calcium accumulation. The aim of the present study was to investigate the effects of a calcium antagonist, flunarizine, on neurotoxicity induced by intracerebroventricular (i.c.v.) iron injection. For this reason rats were divided into three groups as control, iron and iron+flunarizine groups. Animals in iron and iron+flunarizine groups received i.c.v. FeCl₃ injection (200 mM, 2.5 μl), while control rats received the same amount of saline into the cerebral ventricles. Rats in iron+flunarizine group also received i.c.v. flunarizine (1 μM, 2 μl) following FeCl₃ injection. All animals were kept alive for ten days following the operation and animals in iron+flunarizine group received intraperitoneal (i.p.) flunarizine injections once a day (10 mg/kg/day) during this period. After ten days, rats were sacrificed. The total numbers of neurons in hippocampus of all rats were estimated with the latest, unbiased stereological techniques. Findings of the present study suggest that flunarizine may attenuate the neurotoxic effects of iron injection by inhibiting the cellular influx of excessive calcium ions.     

beta-Adrenoceptor and nNOS-derived NO interactions modulate hypoglycemic pial arteriolar dilation in rats

We examined the relative contributions from nitric oxide (NO) and catecholaminergic pathways in promoting cerebral arteriolar dilation during hypoglycemia (plasma glucose congruent with 1.4 mM). To that end, we monitored the effects of beta-adrenoceptor (beta-AR) blockade with propranolol (Pro, 1.5 mg/kg iv), neuronal nitric oxide synthase (nNOS) inhibition with 7-nitroindazole (7-NI, 40 mg/kg ip) or ARR-17477 (300 microM, via topical application), or combined intravenous Pro + 7-NI or ARR-17477 on pial arteriolar diameter changes in anesthetized rats subjected to insulin-induced hypoglycemia. Additional experiments, employing topically applied TTX (1 microM), addressed the possibility that the pial arteriolar response to hypoglycemia required neuronal transmission. Separately, Pro and 7-NI elicited modest but statistically insignificant 10-20% reductions in the normal ~40% increase in arteriolar diameter accompanying hypoglycemia. However, combined Pro-7-NI was accompanied by a >80% reduction in the hypoglycemia-induced dilation. On the other hand, the combination of intravenous Pro and topical ARR-17477 did not affect the hypoglycemia response. In the presence of TTX, the pial arteriolar response to hypoglycemia was lost completely. These results suggest that 1) beta-ARs and nNOS-derived NO interact in contributing to hypoglycemia-induced pial arteriolar dilation; 2) the interaction does not occur in the vicinity of the arteriole; and 3) the vasodilating signal is transmitted via a neuronal pathway.     

The nNos/cGMP mediation of the depressor response to NMDA receptor stimulation in the caudal ventrolateral medulla

Using in vivo voltammetry to directly measure extracellular nitric oxide (NO) levels, our previous studies suggested that the neuronal NO synthase (nNOS) and cyclic guanosine monophosphate (cGMP) signal transducing systems are involved in the cardiovascular responses elicited by activation of N-methyl-D-aspartate (NMDA) receptors in the rostral ventrolateral medulla. In this study, we examined if the depressor responses elicited by activation of NMDA receptors in the caudal ventrolateral medulla (CVLM) also depend on the actions of nNOS and soluble guanylyl cyclase. In anesthetized cats, microinjection of NMDA into the CVLM produced hypotension and bradycardia associated with NO formation. These NMDA-induced responses were attenuated by prior injections of 2-amino-5-phosphonopentanoate (a NMDA receptor competitive antagonist), 7-nitroindazole (a nNOS inhibitor) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (an inhibitor of soluble guanylyl cyclase). These findings suggest that NO is also involved in the NMDA-induced depressor responses of the CVLM.     

Nitric oxide regulation of lingual blood flow in the rat.

The purpose of this study was to determine the role of nitric oxide in the maintenance of basal lingual blood flow in the anesthetized rat. By using laser-Doppler flowmetry, blood flow was measured from the tongue before and after treatment with the nonselective inhibitor of nitric oxide synthase, L-NAME (0.2, 2.0, and 20 mg/kg), or the selective neuronal nitric oxide synthase inhibitor, 7-nitroindazole (40 mg/kg). Other groups of rats were treated with saline, D-NAME (2.0 mg/kg), L-arginine (200 mg/kg), L-arginine + L-NAME (200 + 2.0 mg/kg), or the 7-nitroindazole vehicle. L-NAME produced a dose-related depression in blood flow in the tongue (concurrent with increased arterial blood pressure), which was attenuated by prior administration of L-arginine. Lingual blood flow depression was not seen after administration of the inactive stereoisomer, D-NAME. In addition, the neuronally specific nitric oxide synthase inhibitor, 7-nitroindazole, failed to produce a significant depression of lingual blood flow. These results suggest that the tonic release of nitric oxide from the vascular endothelium plays an important role in maintaining basal blood flow in the tongue and that neuronally released nitric oxide is not involved in maintaining basal circulation in this vascular bed.     

Fenfluramine-induced serotonergic neurotoxicity in mice: lack of neuroprotection by inhibition/ablation of nNOS

Previous studies have implicated a role for nitric oxide (NO) and peroxynitrite in methamphetamine-induced dopaminergic neurotoxicity. The present study was undertaken to investigate whether NO is involved in serotonergic neurotoxicity caused by fenfluramine. In the first experiment, the effect of the neuronal nitric oxide synthase (nNOS) inhibitor 7-nitroindazole (7-NI; 25 mg/kg x 4) on fenfluramine (25 mg/kg x 4)-induced serotonergic neurotoxicity in Swiss Webster mice was investigated. In the second experiment, the effect of fenfluramine (25 mg/kg x 4) on nNOS (-/-) and wild-type (WT) mice was investigated. Fenfluramine induced hypothermia in all three mouse strains, and 7-NI had no thermoregulatory effect. Selective depletion of 5-HT and 5-HT transporter binding sites in the striatum, frontal cortex and hippocampus in all three mouse strains was observed, with no evidence of dopaminergic neurotoxicity. In the first experiment, 7-NI did not attenuate serotonergic neurotoxicity in Swiss Webster mice. In the second experiment, nNOS(-/-) and WT mice were equally sensitive to serotonergic neurotoxicity. These findings suggest that NO and peroxynitrite do not mediate fenfluramine-induced serotonergic neurotoxicity, and that NO is a selective mediator of amphetamines-induced dopaminergic neurotoxicity.     

Nitric oxide synthase and cGMP-mediated stimulation of renin secretion

The interaction between nitric oxide (NO) and renin is controversial. cAMP is a stimulating messenger for renin, which is degraded by phosphodiesterase (PDE)-3. PDE-3 is inhibited by cGMP, whereas PDE-5 degrades cGMP. We hypothesized that if endogenous cGMP was increased by inhibiting PDE-5, it could inhibit PDE-3, increasing endogenous cAMP, and thereby stimulate renin. We used the selective PDE-5 inhibitor zaprinast at 20 mg/kg body wt ip, which we determined would not change blood pressure (BP) or renal blood flow (RBF). In thiobutabarbital (Inactin)-anesthetized rats, renin secretion rate (RSR) was determined before and 75 min after administration of zaprinast or vehicle. Zaprinast increased cGMP excretion from 12.75 +/- 1.57 to 18.67 +/- 1.87 pmol/min (P < 0.003), whereas vehicle had no effect. Zaprinast increased RSR sixfold (from 2.95 +/- 1.74 to 17.62 +/- 5.46 ng ANG I. h(-1) x min(-1), P < 0.024), while vehicle had no effect (from 4.08 +/- 2.02 to 3.87 +/- 1.53 ng ANG I x h(-1) x min(-1)). There were no changes in BP or RBF. We then tested whether the increase in cGMP could be partially due to the activity of the neuronal isoform of NO synthase (nNOS). Pretreatment with the nNOS inhibitor 7-nitroindazole (7-NI; 50 mg/kg body wt) did not change BP or RBF but attenuated the renin-stimulating effect of zaprinast by 40% compared with vehicle. In 7-NI-treated animals, zaprinast-stimulated cGMP excretion was attenuated by 48%, from 9.17 +/- 1.85 to 13.60 +/- 2.15 pmol/min, compared with an increase from 10.94 +/- 1.90 to 26.38 +/- 3.61 pmol/min with zaprinast without 7-NI (P < 0.04). This suggests that changes in endogenous cGMP production at levels not associated with renal hemodynamic changes are involved in a renin-stimulatory pathway. One source of this cGMP may be nNOS generation of NO in the kidney.     

Neuroprotective MK801 is associated with nitric oxide synthase during hypoxia/reoxygenation in rat cortical cell cultures.

The neuroprotective effect of MK801 against hypoxia and/or reoxygenation-induced neuronal cell injury and its relationship to neuronal nitric oxide synthetase (nNOS) expression were examined in cultured rat cortical cells. Treatment of cortical neuronal cells with hypoxia (95% N(2)/5% CO(2)) for 2 h followed by reoxygenation for 24 h induced a release of lactate dehydrogenase (LDH) into the medium, and reduced the protein level of MAP-2 as well. MK801 attenuated the release of LDH and the reduction of the MAP-2 protein by hypoxia, suggesting a neuroprotective role of MK801. MK801 also diminished the number of nuclear condensation by hypoxia/reoxygenation. The NOS inhibitors 7-nitroindazole (7-NI) and N (G)-nitro-L-arginine methyl ester (L-NAME), as well as the Ca(2+) channel blocker nimodipine, reduced hypoxia-induced LDH, suggesting that nitric oxide (NO) and calcium homeostasis contribute to hypoxia and/or the reoxygenation-induced cell injury. The levels of nNOS immunoactivities and mRNA by RT-PCR were enhanced by hypoxia with time and, down regulated following 24 h reoxygenation after hypoxia, and were attenuated by MK801. In addition, the reduction of nNOS mRNA levels by hypoxia/reoxygenation was also diminished by MK801. Further delineation of the mechanisms of NO production and nNOS regulation are needed and may lead to additional strategies to protect neuronal cells against hypoxic/reoxygenation insults.     

大鼠局灶性脑缺血再灌注中nNOS来源的NO对细胞凋亡的影响

目的探讨大鼠局灶性脑缺血再灌注早期nNOS来源的NO对细胞凋亡的影响.方法闭塞大鼠左侧大脑中动脉造成局灶性脑缺血模型,给予选择性nNOS抑制剂-7硝基吲唑,应用原位末端标记法及流式细胞术检测缺血2h再灌注6h细胞凋亡的变化.结果 50mg/kg、25mg/kg剂量的7硝基吲唑可使1、NO含量显著降低.2、NT阳性细胞荧光强度及阳性细胞百分比显著减少.3、TUNEL阳性细胞明显减少.4、细胞凋亡百分率降低,AP峰降低.结论 nNOS来源的NO参与介导脑缺血再灌注早期的细胞凋亡.     

Contributions of nitric oxide synthase isozymes to exhaled nitric oxide and hypoxic pulmonary vasoconstriction in rabbit lungs

We investigated the source(s) for exhaled nitric oxide (NO) in isolated, perfused rabbits lungs by using isozyme-specific nitric oxide synthase (NOS) inhibitors and antibodies. Each inhibitor was studied under normoxia and hypoxia. Only nitro-L-arginine methyl ester (L-NAME, a nonselective NOS inhibitor) reduced exhaled NO and increased hypoxic pulmonary vasoconstriction (HPV), in contrast to 1400W, an inhibitor of inducible NOS (iNOS), and 7-nitroindazole, an inhibitor of neuronal NOS (nNOS). Acetylcholine-mediated stimulation of vascular endothelial NOS (eNOS) increased exhaled NO and could only be inhibited by L-NAME. Selective inhibition of airway and alveolar epithelial NO production by nebulized L-NAME decreased exhaled NO and increased hypoxic pulmonary artery pressure. Immunohistochemistry demonstrated extensive staining for eNOS in the epithelia, vasculature, and lymphatic tissue. There was no staining for iNOS but moderate staining for nNOS in the ciliated cells of the epithelia, lymphoid tissue, and cartilage cells. Our findings show virtually all exhaled NO in the rabbit lung is produced by eNOS, which is present throughout the airways, alveoli, and vessels. Both vascular and epithelial-derived NO modulate HPV.