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.     

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.     

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.     

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.     

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.     

Inhibition of neuronal nitric oxide synthase by 7-nitroindazole attenuates acute lung injury in an ovine model

Nitric oxide (NO) has been shown to play a major role in acute lung injury (ALI) after smoke inhalation. In the present study, we developed an ovine sepsis model, created by exposing sheep to smoke inhalation followed by instillation of bacteria into the airway, that mimics human sepsis and pneumonia. We hypothesized that the inhibition of neuronal NO synthase (nNOS) might be beneficial in treating ALI associated with this model. Female sheep (n = 26) were surgically prepared for the study and given a tracheostomy. This was followed by insufflation of 48 breaths of cotton smoke (40 degrees C) into the airway of each animal and subsequent instillation of live Pseudomonas aeruginosa [5 x 10(11) colony forming units (CFU)] into each sheep's lung. All sheep were mechanically ventilated using 100% O2. Continuous infusion of 7-nitroindazole (7-NI), an nNOS inhibitor, NG-monomethyl-l-arginine (l-NMMA), a nonspecific NOS inhibitor, or aminoguanidine (AG), an inducible NOS inhibitor, was started 1 h after insult. The administration of 7-NI improved pulmonary gas exchange (PaO2/FiO2; where PaO2 is arterial PO2 and FiO2 is fractional inspired oxygen concentration) and pulmonary shunt fraction and attenuated the increase in lung wet-to-dry weight ratio seen in the nontreated sheep. Histologically, 7-NI prevented airway obstruction. The increase in airway blood flow after injury in the nontreated group was significantly inhibited by 7-NI. The increase in plasma concentration of nitrate and nitrite (NOx) was inhibited by 7-NI as well. Posttreatment with l-NMMA improved the pulmonary gas exchange, but AG did not. The results of the present study show that nNOS may be involved in the pathogenesis of ALI after smoke inhalation injury followed by bacterial instillation in the airway.     

脊髓水平nNOS和iNOS在吗啡戒断反应中的作用差异

目的:观察鞘内注射选择性一氧化氮合酶(nNOS)和诱导型一氧化氮合酶(iNOS)抑制剂对吗啡依赖大鼠纳洛酮催促戒断反应、脊髓Fos蛋白表达和脊髓神经元nNOS和iNOS表达的影响,以探讨nNOS和iNOS在吗啡依赖和戒断反应中的作用。方法:在大鼠吗啡依赖和戒断模型上,采用行为学、免疫组织化学和Western blot方法观察鞘内应用nNOS抑制剂7-硝基吲哚(7-Ni)和iNOS抑制剂氨基胍(AG)对吗啡依赖大鼠纳洛酮催促戒断反应、脊髓Fos蛋白表达和脊髓神经元nNOS和iNOS表达的影响。结果:①鞘内注射7-Ni、AG可明显减轻吗啡依赖大鼠戒断症状,戒断组戒断症状评分为28.6±4.89,7-Ni组为16.2±3.99(P<0.01),AG组为22.94±4.0(P<0.05);戒断组TEA评分为13.5±2.55,7-Ni、AG组分别为7.5±2.56、10.5±2.71(P<0.05);②鞘内注射7-Ni、AG可减少脊髓背角Fos阳性神经元的数目,7-Ni、AG组为228.2±49.5、296.8±50.6,低于戒断组(380±71,P<0.05);③7-Ni、AG组nNOS和iNOS阳性神经元的数目分别为169±32、10.2±2.85,均低于戒断组(239±45,16.8±5.1,P<0.05),两给药组脊髓NOS蛋白的表达也显著减少。结论:nNOS和iNOS抑制剂能减轻吗啡依赖及戒断大鼠的戒断症状和在脊髓水平抑制nNOS和iNOS的表达,nNOS起主要作用而iNOS可能起辅助作用。     

Involvement of nitric oxide in pentylenetetrazole-induced kindling in rats

We investigated the role of nitric oxide (NO) and brain-derived neurotrophic factor (BDNF) in the pentylenetetrazole (PTZ)-induced kindling in rats. Seizures were induced by single administration of PTZ, which was associated with an increase in levels of NO metabolites (NOx) in the hippocampus. Pretreatment with a neuronal NO synthase inhibitor, 7-nitroindazole (7-NI), diminished the PTZ-induced increase in NOx levels without affecting the seizure intensity. Repeated administration of PTZ produced a gradual increase in the seizure intensity, leading to the development of kindling. In the kindled rats, PTZ at a dose of 40 mg/kg increased NOx levels in the hippocampus, whereas it had no effect in control animals. Cotreatment of 7-NI with PTZ blocked the development of kindling and attenuated the PTZ-induced increase in NOx levels. A significant increase in BDNF levels was observed in the hippocampus of the kindled rats, which returned to the control levels following seizures induced by PTZ. 7-NI reduced the hippocampal BDNF levels in control rats and suppressed the increase of BDNF levels in the kindled rats. Our findings suggest that NO plays a role in the development of PTZ-induced kindling and that BDNF may contribute to the NO-dependent plastic changes in neuronal excitability.     

缺血再灌注对小鼠肠神经丛nNOS 和iNOS表达的影响

目的观察缺血再灌注后小鼠回肠神经型一氧化氮合酶(neuron alnitric oxide synthase,nNOS)和诱导型一氧化氮合酶(induciblenitric oxide synthase,iNOS)的表达,探讨肠缺血再灌注损伤(ischemia-reperfusion injury,IRI)的发生机制。方法采用小鼠肠系膜上动脉缺血再灌注模型,根据不同再灌注时间对小鼠随机分1d组、3d组、5d组、7d组、对照组和假手术组,用SP法检测小鼠回肠nNOS和iNOS的表达情况。结果与对照组和假手术组相比较,nNOS在再灌注1d后开始在肌间神经丛持续高表达(P<0.01);而iNOS在再灌注3d后开始在肌间神经丛持续高表达(P<0.05)。结论nNOS和iNOS在肠缺血再灌注后的表达增强,提示一氧化氮及一氧化氮合酶与肠神经节细胞在缺血再灌注中的损伤有着密切关系。     

Nitric oxide has dual opposite roles during early and late phases of apoptosis in cerebellar granule neurons

The involvement and the role of nitric oxide (NO) as a signaling molecule in the course of neuronal apoptosis, whether unique or modulated during the progression of the apoptotic program, has been investigated in a cellular system consisting of cerebellar granule cells (CGCs) where apoptosis can be induced by lowering extracellular potassium. Several parameters involved in NO signaling pathway, such as NO production, neuronal nitric oxide synthase (nNOS) expression, and cyclic GMP (cGMP) production were examined in the presence or absence of different inhibitors. We provide evidence that nitric oxide has dual and opposite effects depending on time after induction of apoptosis. In an early phase, up to 3 h of apoptosis, nitric oxide supports survival of CGCs through a cGMP-dependent mechanism. After 3 h, nNOS expression and activity decreased resulting in shut down of NO and cGMP production. Residual NO then contributes to the apoptotic process by reacting with rising superoxide anions leading to peroxynitrite production and protein inactivation. We conclude that whilst NO over-production protects neurons from death in the early phase of neuronal damage, its subsequent reduction may contribute to neuronal degeneration and ultimate cell death.     

Regulation and function of the protein inhibitor of nitric oxide synthase (PIN)/dynein light chain 8 (LC8) in a human mast cell line

The protein inhibitor of nitric oxide synthase (PIN) was independently identified as an inhibitor of nitric oxide (NO) produced by neuronal nitric oxide synthase (nNOS), and as a member of the cellular dynein light chain family, dynein light chain 8 (LC8), responsible for intracellular protein trafficking. Mast cells (MC) are involved in several homeostatic and pathological processes and can be regulated by NO. This study describes the expression of PIN/LC8 in the human MC line HMC-1. We also studied if PIN/LC8 binds nNOS, and what role this might have in leukotriene (LT) production. We found that PIN/LC8 mRNA and protein was expressed in HMC-1. Using a GST-PIN construct, we showed PIN binds to nNOS, but not endothelial (e)NOS in HMC-1; in our studies HMC-1 did not express inducible (i)NOS. Intracellular delivery of anti-PIN/LC8 antibody enhanced ionophore (A23187)-induced LT production through an unknown mechanism. Thus we established for the first time expression of PIN/LC8 in human MC, its ability to bind nNOS, and the effect that blocking it has on LT production in a human MC lines.     

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.     

Inhibition of Monoamine Oxidase Contributes to the Protective Effect of 7-Nitroindazole Against MPTP Neurotoxicity

Abstract: The ability of 7-nitroindazole (7-NI) to protect against MPTP-induced neurotoxicity has been attributed to its inhibition of neuronal nitric oxide synthase. In the present study, 7-NI was found to counteract almost completely striatal dopamine depletion caused by a single subcutaneus injection of 20 mg/kg MPTP in mice. This effect, however, was accompanied by a significant reduction in the striatal levels of MPP⁺, the toxic metabolite generated via monoamine oxidase B-catalyzed MPTP oxidation. In the presence of 7-NI, a dose of 40 mg/kg MPTP produced MPP⁺ concentrations similar to those measured after treatment with 20 mg/kg MPTP alone. A comparison of neurotoxicity in these two experimental conditions (i.e., mice treated with 20 mg/kg alone versus 40 mg/kg MPTP plus 7-NI) revealed only a slight (20%), but statistically significant, protection of dopamine depletion with 7-NI. These data indicate that the mechanism by which 7-NI counteracts MPTP neurotoxicity in mice is not due solely to inhibition of neuronal nitric oxide synthase, but involves a reduction in MPP⁺ formation.     

Prostaglandin E2 inhibits vasotocin-induced osmotic water permeability in the frog urinary bladder by EP1-receptor-mediated activation of NO/cGMP pathway

PGE(2) is a well-known inhibitor of the antidiuretic hormone-induced increase of osmotic water permeability (OWP) in different osmoregulatory epithelia; however, the mechanisms underlying this effect of PGE(2) are not completely understood. Here, we report that, in the frog Rana temporaria urinary bladder, EP(1)-receptor-mediated inhibition of arginine-vasotocin (AVT)-induced OWP by PGE(2) is attributed to increased generation of nitric oxide (NO) in epithelial cells. It was shown that the inhibitory effect of 17-phenyl-trinor-PGE(2) (17-ph-PGE(2)), an EP(1) agonist, on AVT-induced OWP was significantly reduced in the presence of 7-nitroindazole (7-NI), a neuronal NO synthase (nNOS) inhibitor. NO synthase (NOS) activity in both lysed and intact epithelial cells measured as a rate of conversion of l-[(3)H]arginine to l-[(3)H]citrulline was Ca(2+) dependent and inhibited by 7-NI. PGE(2) and 17-ph-PGE(2), but not M&B-28767 (EP(3) agonist) or butaprost (EP(2) agonist), stimulated NOS activity in epithelial cells. The above effect of PGE(2) was abolished in the presence of SC-19220, an EP(1) antagonist. 7-NI reduced the stimulatory effect of 17-ph-PGE(2) on NOS activity. 17-ph-PGE(2) increased intracellular Ca(2+) concentration and cGMP in epithelial cells. Western blot analysis revealed an nNOS expression in epithelial cells. These results show that the inhibitory effect of PGE(2) on AVT-induced OWP in the frog urinary bladder is based at least partly on EP(1)-receptor-mediated activation of the NO/cGMP pathway, suggesting a novel cross talk between AVT, PGE(2), and nNOS that may be important in the regulation of water transport.     

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.     

Regional differences in mechanisms of cerebral circulatory response to neuronal activation

Vibrissal stimulation raises cerebral blood flow (CBF) in the ipsilateral spinal and principal sensory trigeminal nuclei and contralateral ventroposteromedial (VPM) thalamic nucleus and barrel cortex. To investigate possible roles of adenosine and nitric oxide (NO) in these increases, local CBF was determined during unilateral vibrissal stimulation in unanesthetized rats after adenosine receptor blockade with caffeine or NO synthase inhibition with N(G)-nitro-L-arginine methyl ester (L-NAME) or 7-nitroindazole (7-NI). Caffeine lowered baseline CBF in all structures but reduced the percent increase during stimulation only in the two trigeminal nuclei. L-NAME and 7-NI lowered baseline CBF but reduced the percent increase during stimulation only in the higher stations of this sensory pathway, i.e., L-NAME in the VPM nucleus and 7-NI in both the VPM nucleus and barrel cortex. Combinations of caffeine with 7-NI or L-NAME did not have additive effects, and none alone or in combination completely eliminated functional activation of CBF. These results suggest that caffeine-sensitive and NO-dependent mechanisms are involved but with different regional distributions, and neither fully accounts for the functional activation of CBF.     

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.