Tetrachlorodibenzo-p-dioxin exposure alters radial arm maze performance and hippocampal morphology in female AhR mice

Perinatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been reported to alter spatial learning in rats tested on a radial arm maze (RAM). TCDD is believed to exert most of its effects through binding to the aryl hydrocarbon receptor (AhR). To determine whether the AhR mediates TCDD-induced alterations in spatial learning, we tested male and female AhR-knockout (AhR-/-), heterozygous (AhR+/-) and wild-type (AhR+/+) mice on the RAM. AhR+/- male and female mice were time mated, and treated dams were dosed with 5 microg TCDD/kg body weight on day 13 of gestation. When offspring reached adulthood, male and female AhR+/+, AhR+/- and AhR-/- mice from TCDD-exposed and unexposed litters were tested on the eight-arm RAM. After testing, we examined hippocampal morphology as visualized by the Timm's silver sulfide stain. TCDD-exposed female AhR+/- mice made more errors than their respective controls on the RAM and exhibited a decrease in the size of the intra- and infrapyramidal mossy fiber (IIP-MF) field of the hippocampus. None of the other TCDD-exposed groups differed from their respective control groups with regard to maze performance or hippocampal morphology. The reduction of IIP-MF field indicates a possible morphological basis for the learning deficit that was observed in the female AhR+/- mice. It is hypothesized that the effect of TCDD exposure is AhR dependent and that TCDD may alter GABAergic activity in the hippocampus of female mice during development.     

Decreased activity and impaired induction of nitric oxide synthase by lipopolysaccharides in streptozotocin-induced diabetic rats

The effect of diabetes was determined on nitric oxide synthase (NOS) activity in rat heart and liver. The diabetes was induced by streptozotocin (STZ) and NOS activity was determined after 1 or 12 weeks post-STZ injection. In both tissues, the majority of NOS activity was associated with endothelial constitutive calcium-sensitive NOS (ecNOS) isoform and found in the particulate (100,000xg pellet) fraction in young rats. The diabetes as well as age reduced this activity significantly in heart, whereas only the age caused a decrease in ecNOS activity in liver tissue. Lipopolysaccharides (LPS) induced calcium-insensitive iNOS activity in both young and old rats. The induction was significantly higher (up to 10-fold) in liver as compared to heart. Although the maximum induction of iNOS in young rats was almost similar in diabetic tissues as compared to control animals, there was a lag period for induction of iNOS in diabetic tissues. In old diabetic rats, the induction by LPS was almost completely abolished. These results suggest that diabetes causes either no change or a decrease in ecNOS activity and impairment in the induction of iNOS by LPS in rat heart and liver.     

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.     

Neuronal nitric oxide synthase proteolysis limits the involvement of nitric oxide in kainate-induced neurotoxicity in hippocampal neurons

In this work, we investigated the role of nitric oxide (NO) in neurotoxicity triggered by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor activation in cultured hippocampal neurons. In the presence of cyclothiazide (CTZ), short-term exposures to kainate (KA; 5 and 15 min, followed by 24-h recovery) decreased cell viability. Both NBQX and d-AP-5 decreased the neurotoxicity caused by KA plus CTZ. Long-term exposures to KA plus CTZ (24 h) resulted in increased toxicity. In short-, but not in long-term exposures, the presence of NO synthase (NOS) inhibitors (l-NAME and 7-NI) decreased the toxicity induced by KA plus CTZ. We also found that KA plus CTZ (15-min exposure) significantly increased cGMP levels. Furthermore, short-term exposures lead to decreased intracellular ATP levels, which was prevented by NBQX, d-AP-5 and NOS inhibitors. Immunoblot analysis revealed that KA induced neuronal NOS (nNOS) proteolysis, gradually lowering the levels of nNOS according to the time of exposure. Calpain, but not caspase-3 inhibitors, prevented this effect. Overall, these results show that NO is involved in the neurotoxicity caused by activation of non-desensitizing AMPA receptors, although to a limited extent, since AMPA receptor activation triggers mechanisms that lead to nNOS proteolysis by calpains, preventing a further contribution of NO to the neurotoxic process.     

Behavioral tactics among rats in a radial maze

The experiments were carried out on rats of Wistar (W) and Krushinsky-Molodkina (KM) lines, differing in the level of task performance in conditions of multiple choice of food sources in a 12ray radial symmetrical maze. It is shown that in the radial maze, the rats of both lines use a number of behavioural tactics. To determine the influence of changes of environmental spatial characteristics while preserving the main experimental strategy--never revisit the places of reinforcement,--on the peculiarities of tactics manifestations maze modifications were elaborated. Besides the environmental procedure was changed. Under all conditions the rats of W line solved the task. According to the situation, they either preserved former tactics or developed some new ones. The rats of KM line could not solve the task in changed conditions. In most sessions, appearance or absence of given tactics was not adequate to new conditions. The presence of the necessary tactics not always facilitated the task solving. Thus, for the realization of adequate reactions, the presence of necessary tactics is not sufficient: their "tuning" is needed depending on situation. The possibility is supposed of genetic determination of rats ability to some tactics. However, it is emphasized, that tactics are not strongly fixed, as their realization depends on environmental conditions.     

Melanin-concentrating hormone,hippocampal nitric oxide levels and memory retention

The present study attempts to determine, if the effect of melanin-concentrating hormone (MCH) upon memory retention is correlated with changes in nitric oxide synthase (NOS) activity and tissue levels of nitric oxide (NO) and cGMP. We used a behavioral experiment using a step-down inhibitory avoidance test, the biochemical determinations of NO and cGMP, and electrophysiological model. Results of behavioral studies (step-down test) showed that MCH administration reverts the amnesic effects induced by N(G)-nitro-L-arginine (L-NOArg). Moreover, electrophysiological studies demonstrated that L-NOArg did not block the potentiation induced by the peptide. Hippocampal NO and cGMP levels increased after MCH injection.     

Endothelial nitric oxide synthase modulates gastric ulcer healing in rats

Nitric oxide has been shown to be beneficial for gastric ulcer healing. We determined the relative effects of endothelial and inducible nitric oxide synthases on gastric ulcer healing in rats. Ulcers were induced by serosal application of acetic acid. Ulcer severity, angiogenesis, and nitric oxide synthase expression were assessed 3-10 days later. The effects of inhibitors of nitric oxide synthase were also examined. Inducible nitric oxide synthase mRNA was only detected in ulcerated tissue (maximal at day 3), whereas the endothelial isoform mRNA was detected in normal tissue and increased during ulcer healing. Inducible nitric oxide synthase was expressed in inflammatory cells in the ulcer bed, whereas endothelial nitric oxide synthase was found in the vascular endothelium and in some mucosal cells in both normal and ulcerated tissues. Angiogenesis changed in parallel with endothelial nitric oxide synthase expression. N(6)-(iminoethyl)-L-lysine did not affect angiogenesis or ulcer healing, while N(G)-nitro-L-arginine methyl ester significantly reduced both. In conclusion, endothelial nitric oxide synthase, but not the inducible isoform, plays a significant role in gastric ulcer healing.     

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.     

Upregulation of neuronal nitric oxide synthase in skeletal muscle by swim training

Exercise enhances cardiac output and blood flow to working skeletal muscles but decreases visceral perfusion. The alterations in nitric oxide synthase (NOS) activity and/or expression of the cardiopulmonary, skeletal muscle, and visceral organs induced by swim training are unknown. In sedentary and swim-trained rats (60 min twice/day for 3-4 wk), we studied the alterations in NOS in different tissues along with hindquarter vasoreactivity in vivo during rest and mesenteric vascular bed reactivity in vitro. Hindquarter blood flow and conductance were reduced by norepinephrine in both groups to a similar degree, whereas N(G)-nitro-L-arginine methyl ester reduced both indexes to a greater extent in swim-trained rats. Vasodilator responses to ACh, but not bradykinin or S-nitroso-N-acetyl-penicillamine, were increased in swim-trained rats. Ca(2+)-dependent NOS activity was enhanced in the hindquarter skeletal muscle, lung, aorta, and atria of swim-trained rats together with increased expression of neuronal NOS in the hindquarter skeletal muscle and endothelial NOS in the cardiopulmonary organs. Mesenteric arterial bed vasoreactivity was unaltered by swim training. Physiological adaptations to swim training are characterized by enhanced hindquarter ACh-induced vasodilation with upregulation of neuronal NOS in skeletal muscle and endothelial NOS in the lung, atria, and aorta.     

Endothelial nitric oxide synthase gene haplotypes and circulating nitric oxide levels significantly associate with risk of essential hypertension

Nitric oxide (NO), a potent vasodilator, plays a pivotal role in blood pressure regulation. Endothelial NO synthase gene (NOS3) polymorphisms influence NO levels. Here, we investigated the role of the – 922A/G, – 786T/C, 4b/4a, and 894G/T polymorphisms of the NOS3 and NO_x levels in 800 consecutive unrelated subjects comprising 455 patients of essential hypertension and 345 controls. The polymorphisms were investigated independently and as haplotypes. Plasma NO_x levels (nitrate and nitrite) were estimated by the Griess method. Genotype frequencies for the –786T/C, 4b/4a, and 894G/T polymorphisms differed significantly (P < 0.001) between patients and controls and were associated with an increased risk of hypertension (OR = 2.0, OR = 3.8, OR = 1.6, respectively). The 4-locus haplotypes ATaG (H1), ATaT (H2), and GCaG (H3) were significantly associated with essential hypertension and served as susceptible haplotypes (P ≤ 0.0001). On the other hand, haplotypes ATbG (H4) and GTbG (H5) were negatively associated with hypertension and served as protective haplotypes (P < 0.0001). NO_x levels were significantly lower in patients than controls (P < 0.0001). The individual polymorphisms showed marginal association with NO_x level; however, the susceptible haplotype H2 associated significantly with lower NO_x levels in patients (P < 0.001) and conversely the haplotype H4 with higher NO_x levels in controls (P < 0.001). In conclusion, the 4b/4a and likely – 786T/C polymorphisms were identified as the determinants modifying the risk of hypertension. This study identifies the NOS3 variants and haplotypes as genetic risk factors and as useful markers of increased susceptibility to the risk of essential hypertension.     

Insulin restores neuronal nitric oxide synthase expression in streptozotocin-induced diabetic rats

Nitric oxide (NO) is known to play an important role in the pathophysiology of insulin-dependent diabetic mellitus (IDDM). In an attempt to investigate the relation between insulin and NO in IDDM, the present study employed male Wistar rats to induce IDDM by intravenous injection of streptozotocin (STZ). Four groups of rats were used; untreated normal control group, insulin treated STZ group, vehicle-treated STZ control, and one group of age-matched rats which were orally supplied with glucose to increase plasma glucose (glucose-challenged rats). Changes of the activity and gene expression of neuronal nitric oxide synthase (nNOS) were examined in cerebellum and kidney of these groups. The activity of nNOS in cerebellum, determined by conversion of [3H] L-arginine to [3H] L-citrulline, in STZ-induced diabetic rats was markedly lower than normal rats. Insulin treatment reversed the nNOS activity. Similar reversion by insulin treatment was also obtained in the gene expression of nNOS. However, the activity and gene expression of nNOS in glucose-challenged rats were not different from those in normal rats. The role of hyperglycemia can thus be ruled out. These findings indicated that an impairment of nNOS in the brain of rats with IDDM is mainly due to the absence of insulin.     

昆虫一氧化氮及其合酶的研究进展

一氧化氮作为一种重要的信息分子 ,参与调节昆虫嗅觉、视觉、机械感受、发育、机体防御及学习行为。该文从生理、生化、形态定位以及信号转导几方面综述了有关昆虫一氧化氮及其合酶的最新研究进展。     

Nitric oxide and nitric oxide synthase activity in plants

Research on NO in plants has gained considerable attention in recent years mainly due to its function in plant growth and development and as a key signalling molecule in different intracellular processes in plants. The NO emission from plants is known since the 1970s, and now there is abundant information on the multiple effects of exogenously applied NO on different physiological and biochemical processes of plants. The physiological function of NO in plants mainly involves the induction of different processes, including the expression of defence-related genes against pathogens and apoptosis/programmed cell death (PCD), maturation and senescence, stomatal closure, seed germination, root development and the induction of ethylene emission. NO can be produced in plants by non-enzymatic and enzymatic systems. The NO-producing enzymes identified in plants are nitrate reductase, and several nitric oxide synthase-like activities, including one localized in peroxisomes which has been biochemically characterized. Recently, two genes of plant proteins with NOS activity have been isolated and characterized for the first time, and both proteins do not have sequence similarities to any mammalian NOS isoform. However, different evidence available indicate that there are other potential enzymatic sources of NO in plants, including xanthine oxidoreductase, peroxidase, cytochrome P450, and some hemeproteins. In plants, the enzymatic production of the signal molecule NO, either constitutive or induced by different biotic/abiotic stresses, may be a much more common event than was initially thought.     

Undetectable levels of tumor necrosis factor-alpha,nitric oxide and inadequate expression of inducible nitric oxide synthase in congenital hypothyroidism

OBJECTIVE: To analyse the production of TNF-alpha and NO in euthyroid and hypothyroid newborns. PATIENTS: A cross-sectional study was conducted involving 10 newborns diagnosed with primary congenital hypothyroidism (CH; group A) and 10 euthyroid children (group B). RESULTS: There were undetectable plasma levels of TNF-alpha and NO in the hypothyroid children, however plasma levels of TNF-alpha (5.5 0.5 pg/ml) and NO (5.6 1.7 microM) were detected at normal levels in all euthyroid children. Moreover, expression of iNOS mRNA in PBMC, determined by RT-PCR, was negative in both groups of infants. CONCLUSION: TNF-alpha and NO production are both impaired in hypothyroid newborns. We report for the first time evidence of undetectable levels of TNF-alpha and NO in infants with CH.     

Effects of nitric oxide synthase inhibitors on systemic hypotension, cytokines and inducible nitric oxide synthase expression and lung injury following endotoxin administration in rats

Endotoxin shock is characterized by systemic hypotension, hyporeactiveness to vasoconstrictors and acute lung edema. A nitric oxide synthase (NOS) inhibitor, N^G-monomethyl-L-arginine (L-NMMA) has been shown to be effective in reversing acute lung injury. In the present study, we evaluated the effects of NOS blockade by different mechanisms on the endotoxin-induced changes. In anesthetized rats, lipopolysaccharide (LPS,Klebsiella pneumoniae) was administered intravenously in a dose of 10 mg/kg. LPS caused sustained systemic hypotension accompanied by an eightfold increase of exhaled NO during an observation period of 4 h. After the experiment, the lung weight was obtained and lung tissues were taken for the determination of mRNA expressions of inducible NOS (iNOS), interleukin-1 (IL-1) and tumor necrosis factor--(TNF-). Histological examination of the lungs was also performed. In the control group injected with saline solution, mRNA expressions of iNOS, IL-1 and TNF- were absent. Four hours after LPS, the mRNA expressions of iNOS and IL-1 were still significantly enhanced, but TNF- was not discernibly expressed. LPS also caused a twofold increase in lung weight. Pathological examination revealed endothelial damage and interstitial edema. Various NOS inhibitors were given 1 h after LPS administration. These agents included N-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg), a constitutive NOS and iNOS inhibitor; S,S-1,4-phenylene-bis-(1,2-ethanedinyl) bis-isothiourea dihydrobromide (1,4-PBIT, 10 mg/kg), a relatively specific iNOS inhibitor, and dexamethasone (3 mg/kg), an inhibitor of iNOS expression. These NOS inhibitors all effectively reversed the systemic hypotension, reduced the exhaled NO concentration and prevented acute lung injury. The LPS-induced mRNA expressions of iNOS and IL-1 were also significantly depressed by these NOS inhibitors. Our results suggest that NO production through the iNOS pathway is responsible for endotoxin-induced lung injury. Certain cytokines such as IL-1 are possibly involved. These changes are minimized by NOS inhibitors through different mechanisms.     

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.     

Antioxidant levels in the rat brain after nitric oxide synthase inhibition: a preliminary report

Protective effects of NOS inhibitors and free radical scavengers in cerebral ischemia are well documented. The present study was undertaken to determine the possible effects of NOS inhibition on brain antioxidants. Levels of both enzymatic [glutathione peroxidase (GPx), catalase and superoxide dismutase (SOD)] and non-enzymatic [reduced glutathione (GSH)] antioxidants following nitric oxide synthase (NOS) inhibition by N(G)-nitro-L-arginine methyl ester (L-NAME), D-NAME or 7-nitroindazole (7-NI) have been investigated. NOS activity and antioxidant levels in the rat cerebellum and medulla were estimated 1 h after treatment with L-NAME (10, 30 and 100 mg/kg, i.p.), D-NAME (100 mg/kg, i.p.) or 7-NI (25 mg/kg, i.p.). L-NAME and 7-NI inhibited NOS activity in a dose-dependent manner. D-NAME also exhibited significant NOS inhibition. The activity of SOD and the GSH level remained unaltered following NOS inhibition. However, L-NAME and D-NAME at 100 mg/kg attenuated GPx activity in the cerebellum, though 7-NI had no effect. L-NAME inhibited catalase activity in medulla only at 30 mg/kg, but had no effect in cerebellum. However, 7-NI (25 mg/kg), D-NAME and L-NAME at 100 mg/kg did not affect catalase activity in the rat brain. Thus, NOS inhibition by the three agents did not have major effects on brain antioxidant levels.