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The adipocyte-derived hormone, leptin, plays a key role in the maintenance of energy homeostasis. Leptin binds to the long form of its receptor, which is predominantly expressed in various hypothalamic regions, including the lateral hypothalamic area (LH) and supraoptic nucleus (SO). Several studies have suggested that leptin directly activates neuronal nitric oxide synthase, leading to increased nitric oxide production. We used histochemistry for nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) as a marker for nitric oxide synthase activity and assessed the effect of leptin on nitrergic neurons in the LH and SO of rats. We found that intraperitoneal administration of leptin led to a significant increase in the number of NADPH-d-positive neurons in the LH and SO. In addition, the intensity (optical density) of NADPH-d staining in LH and SO neurons was significantly elevated in rats that received leptin compared with saline-treated rats. These findings suggest that nitrergic neurons in the LH and SO may be implicated in mediating the central effects of leptin.

Xenin25 has a variety of physiological functions in the Gastrointestinal (GI) tract, including ion transport and motility. However, the motility responses in the colon induced by Xenin25 remain poorly understood. Therefore, the effect of Xenin25 on the spontaneous circular muscle contractions of the rat distal colon was investigated using organ bath chambers and immunohistochemistry. Xenin25 induced the inhibition followed by postinhibitory spontaneous contractions with a higher frequency in the rat distal colon. This inhibitory effect of Xenin25 was significantly suppressed by TTX but not by atropine. The inhibitory time (the duration of inhibition) caused by Xenin25 was shortened by the NTSR1 antagonist SR48692, the NK1R antagonist CP96345, the VPAC2 receptor antagonist PG99-465, the nitric oxide-sensitive guanylate-cyclase inhibitor ODQ, and the Ca 2+ -dependent K + channel blocker apamin. The higher frequency of postinhibitory spontaneous contractions induced by Xenin25 was also attenuated by ODQ and apamin. SP-, NOS-, and VIP-immunoreactive neurons were detected in the myenteric plexus (MP) of the rat distal colon. Small subsets of the SP-positive neurons were also Calbindin positive. Most of the VIP-positive neurons were also NOS positive, and small subsets of the NK1R-positive neurons were also VIP positive. Based on the present results, we propose the following mechanism. Xenin25 activates neuronal NTSR1 on the SP neurons of IPANs, and transmitters from the VIP and apamin-sensitive NO neurons synergistically inhibit the spontaneous circular muscle contractions via NK1R. Subsequently, the postinhibitory spontaneous contractions are induced by the offset of apamin-sensitive NO neuron activation via the interstitial cells of Cajal. In addition, Xenin25 also activates the muscular NTSR1 to induce relaxation. Thus, Xenin25 is considered to be an important modulator of post prandial circular muscle contraction of distal colon since the release of Xenin25 from enteroendocrine cells is stimulated by food intake., (© 2021 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Bisphenol A (BPA) is one of the most common environmental pollutants among endocrine disruptors. Due to its similarity to estrogen, BPA may affect estrogen receptors and show adverse effects on many internal organs. The reproductive system is particularly vulnerable to the impact of BPA, but knowledge about BPA-induced changes in the innervation of the uterus is relatively scarce. Therefore, this study aimed to investigate the influence of various doses of BPA on nitrergic nerves supplying the uterus with the double immunofluorescence method. It has been shown that even low doses of BPA caused an increase in the number of nitrergic nerves in the uterine wall and changed their neurochemical characterization. During the present study, changes in the number of nitrergic nerves simultaneously immunoreactive to substance P, vasoactive intestinal polypeptide, pituitary adenylate cyclase-activating peptide, and/or cocaine- and amphetamine-regulated transcript were found under the influence of BPA. The obtained results strongly suggest that nitrergic nerves in the uterine wall participate in adaptive and/or protective processes aimed at homeostasis maintenance in the uterine activity under the impact of BPA.

Given the well-known antioxidant and neuroprotective properties of quercetin, the aim of this work was to evaluate the effects of quercetin stabilized by microencapsulation at two doses (10 mg kg -1 and 100 mg kg -1 ) on the oxidative/antioxidant status, number and morphological features of ICC, nitrergic neurons and M2-like macrophages in jejunum of diabetic rats. The rats were randomly distributed into six groups: normoglycemic control (N), diabetic control (D) and either normoglycemic or diabetic groups treated with quercetin-loaded microcapsules at a dose of 10 mg kg -1 (NQ10 and DQ10, respectively) or 100 mg kg -1 (NQ100 and DQ100, respectively). After 60 days, the jejunum was collected. Whole mounts were immunostained for Ano1, nNOS and CD206, and oxidative stress levels and total antioxidant capacity of the jejunum were measured. Diabetes led to a loss of ICC and nitrergic neurons, but increased numbers of M2-like macrophages and elevated levels of oxidative stress were seen in diabetic animals. High-dose administration of quercetin (100 mg kg -1 ) further aggravated the diabetic condition (DQ100) but this treatment resulted in harmful effects on healthy rats (NQ100), pointing to a pro-oxidant activity. However, low-dose administration of quercetin (10 mg kg -1 ) gave rise to antioxidant and protective effects on ICC, nNOS, macrophages and oxidative/antioxidant status in DQ100, but NQ100 displayed infrequent negative outcomes in normoglycemic animals. Microencapsulation of the quercetin may become promising alternatives to reduce diabetes-induced oxidative stress but antioxidant therapies should be careful used under healthy status to avoid toxic effects., Competing Interests: Declaration of Competing Interest No potential conflicts of interests to declare., (Copyright © 2020. Published by Elsevier B.V.)

Nicotinic acetylcholine receptor activation on the perivascular sympathetic nerves via axo-axonal interaction mechanism causes norepinephrine release, which triggers the neurogenic nitrergic relaxation in basilar arteries to meet the need of a brain. Donepezil and huperzine A, which are the cholinesterase inhibitors used for Alzheimer's disease therapy, exert controversial effects on nicotinic acetylcholine receptors. Therefore, we investigated how donepezil and huperzine A via the axo-axonal interaction regulate the neurogenic vasodilation of isolated porcine basilar arteries and define their action on different subtypes of the nicotinic acetylcholine receptor by using blood vessel myography, calcium imaging, and electrophysiological techniques. Both nicotine (100 μM) and transmural nerve stimulation (TNS, 8 Hz) induce NO-mediated dilation in the arteries. Nicotine-induced vasodilations were concentration-dependently inhibited by huperzine A and donepezil, with the former being 30 fold less potent than the latter. Both cholinesterase inhibitors weakly and equally decreased TNS-elicited nitrergic vasodilations. Neither huperzine A nor donepezil affected isoproterenol (a β adrenoceptor-agonist)- or sodium nitroprusside (a NO donor)-induced vasodilation. Further, huperzine A was less potent than donepezil in inhibiting nicotine-elicited calcium influxes in rodent superior cervical ganglionic neurons and inward currents in α7- and α3β2-nicotinic acetylcholine receptor-expressing Xenopus oocytes. In conclusion, huperzine A may exert less harmful effect over donepezil on maintaining brainstem circulation and on the nicotinic acetylcholine receptor-associated cognition deficits during treatment for Alzheimer's disease., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Ethnopharmacological Relevance: Perivascular neuropathy was reported to involve in the vascular disorders associated with diabetes. The dried rhizomes of Coptis chinensis Franch. (Latin name: Coptidis Rhizoma; common name: Huang Lian in China), used frequently in Traditional Chinese medicine to treat diabetes (Xiaoke), have been confirmed to possess beneficial effects on diabetic peripheral neuropathy by modern clinical and pharmacological studies. Berberine (BBR), the main effective component of Huang Lian in the treatment of diabetes, is reported to ameliorate diabetic central and peripheral neuropathy. However, the effects of BBR on nerve function of mesenteric and iliac arteries are unclear., Aim of the Study: To investigate the effects of BBR on the diabetes-induced changes in nitrergic and adrenergic function in mesenteric and iliac arteries., Materials and Methods: In this study, the animals were randomized into three groups: control rats, diabetic rats, and diabetic rats gavaged with BBR. We established diabetic rat model using intraperitoneal injection of streptozotocin (STZ, 55 mg kg -1 ). Two weeks after model establishment, those in the BBR-treated groups were gavaged with berberine chloride (Sichuan Xieli Fharmaceutical. Co., Ltd; 200 mg·kg -1 ·day -1 ) diluted in distilled water for another 2 weeks. The superior mesenteric artery and iliac artery were excised. Electric field stimulation (EFS) was used to induce arterial vasoconstriction and explore (1) the diabetes-induced changes in neurogenic function of the superior mesenteric artery and iliac artery; (2) the effects of BBR on neurovascular dysfunction in the early stage of STZ-induced diabetic rats. Nitric oxide (NO) and noradrenaline (NA) released from the nitrergic and adrenergic nerves were quantified using fluorescence assays and ELISA, respectively., Results: EFS induced frequency-dependent vasoconstrictions in both superior mesenteric and iliac artery, and the contractile responses of arteries were abolished by 0.1 μmol·L -1 tetrodotoxin (TTX), or inhibited by 1 μmol·L -1 phentolamine or increased by 0.1 mmol·L -1 N ω -Nitro-L-arginine methyl ester hydrochloride (L-NAME). In superior mesenteric artery, but not in iliac artery, the changes of contractile responses with L-NAME were significantly decreased in diabetic rats, and NO release was less also. In contrast, in iliac artery of diabetic rats, but not in superior mesenteric artery, the changes of contractile responses with phentolamine were increased, and NA release was increased significantly. All these changes in diabetic rats on both superior mesenteric artery and iliac artery were reversed by treated with BBR., Conclusions: In the STZ-induced early diabetic rats, neural control of mesenteric and iliac vasomotor tone are altered differently. The diminished nitrergic nerve in superior mesenteric artery and enhanced adrenergic nerve in iliac artery both contributed to increased vasocontrictor responses. All these changes in diabetic rats were reversed by BBR, suggesting a novel mechanism of BBR in balance of neural regulation of vascular tone., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The enteric nervous system in the large intestine generates two important patterns relating to motility: 1 ) propagating rhythmic peristaltic smooth muscle contractions referred to as colonic migrating motor complexes (CMMCs) and 2 ) tonic inhibition, during which colonic smooth muscle contractions are suppressed. The precise neurobiological substrates underlying each of these patterns are unclear. Using transgenic animals expressing the genetically encoded calcium indicator GCaMP3 to monitor activity or the optogenetic actuator channelrhodopsin (ChR2) to drive activity in defined enteric neuronal subpopulations, we provide evidence that cholinergic and nitrergic neurons play significant roles in mediating CMMCs and tonic inhibition, respectively. Nitrergic neurons [neuronal nitric oxide synthase (nNOS)-positive neurons] expressing GCaMP3 exhibited higher levels of activity during periods of tonic inhibition than during CMMCs. Consistent with these findings, optogenetic activation of ChR2 in nitrergic neurons depressed ongoing CMMCs. Conversely, cholinergic neurons [choline acetyltransferase (ChAT)-positive neurons] expressing GCaMP3 markedly increased their activity during the CMMC. Treatment with the NO synthesis inhibitor N ω -nitro-l-arginine also augmented the activity of ChAT-GCaMP3 neurons, suggesting that the reciprocal patterns of activity exhibited by nitrergic and cholinergic enteric neurons during distinct phases of colonic motility may be related. NEW & NOTEWORTHY Correlating the activity of neuronal populations in the myenteric plexus to distinct periods of gastrointestinal motility is complicated by the difficulty of measuring the activity of specific neuronal subtypes. Here, using mice expressing genetically encoded calcium indicators or the optical actuator channelrhodopsin-2, we provide compelling evidence that cholinergic and nitrergic neurons play important roles in mediating coordinated propagating peristaltic contractions or tonic inhibition, respectively, in the murine colon.

The present study was designed to investigate the effect of ascorbic acid (AA) treatment on the anxiety related behavioral and neurochemical alterations. AA (50, 100 and 200 mg/kg, i.p.) was administered to the mice and anxiety related behavior and levels of glutamate and nitrite in the brain of mice were determined. The results obtained revealed that the administration of AA (100 mg/kg, i.p.) significantly reduced the anxiety related behavior and the levels of nitrite in the brain of mice. Nitrergic interactions were further determined by the pretreatment of mice with nitric oxide (NO) modulator and AA treatment followed by behavioral and neurochemical measurements. The results obtained suggested that NO inhibition potentiated the anxiolytic like activity of AA in mice. It was also observed that the glutamate and nitrite level in the brain of mice were significantly reduced by the NO inhibitor pretreatment. Thus, the present study demonstrated the possible nitrergic pathways modulation in the anxiolytic like activity of AA in mice., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background and Purpose: The aim of the present study was to assess the interaction of nitrergic neurotransmission within the bed nucleus of the stria terminalis (BNST) with local glutamatergic and noradrenergic neurotransmission in the control of cardiovascular responses to acute restraint stress in rats., Experimental Approach: Interaction with local noradrenergic neurotransmission was evaluated using local pretreatment with the selective α 1 -adrenoceptor antagonist WB4101 before microinjection of the NO donor NOC-9 into the BNST. Interaction with glutamatergic neurotransmission was assessed by pretreating the BNST with a selective inhibitor of neuronal NOS (nNOS), Nω-propyl-L-arginine (NPLA) before local microinjection of NMDA. The effect of intra-BNST NPLA microinjection in animals locally pretreated with WB4101 was also evaluated., Key Results: NOC-9 reduced the heart rate (HR) and blood pressure increases evoked by restraint stress. These effects of NOC-9 on HR, but not in blood pressure, was inhibited by pretreatment of BNST with WB4101. NMDA enhanced the restraint-evoked HR increase, and this effect was abolished following BNST pretreatment with NPLA. Administration of NPLA to the BNST of animals pretreated locally with WB4101 decreased the HR and blood pressure increases induced by restraint., Conclusion and Implications: These results indicate that inhibitory control of stress-evoked cardiovascular responses by nitrergic signalling in the BNST is mediated by a facilitation of local noradrenergic neurotransmission. The present data also provide evidence of an involvement of local nNOS in facilitatory control of tachycardia during stress by NMDA receptors within the BNST., (© 2018 The British Pharmacological Society.)

We aimed to determine the influence of nitrergic innervation function on the decreased mesenteric arterial tone induced by high levels of triiodothyronine (T3), as a model of acute thyroiditis, as well as the mechanism/s implicated. We analysed in mesenteric segments from male Wistar rats the effect of 10 nmol/L T3 (2 h) on the vasomotor response to electrical field stimulation (EFS) in the presence/absence of specific neuronal NOS (nNOS) inhibitor L-NPA, or superoxide anion scavenger tempol. Nitric oxide (NO) release was measured in the presence/absence of tempol or PI3K inhibitor LY294002. Superoxide anion and peroxynitrite releases, nNOS, PI3K, AKT and superoxide dismutase (SOD) 1 and 2 expressions, nNOS and AKT phosphorylation, and SOD activity were analysed. T3 decreased EFS-induced vasoconstriction. L-NPA increased EFS-induced vasoconstriction more markedly in T3-incubated segments. T3 increased NO release. Tempol decreased EFS-induced vasoconstriction and augmented NO release only in segments without T3. LY294002 decreased NO release in T3-incubated segments. T3 diminished superoxide anion and peroxynitrite formation, enhanced SOD-2 expression, nNOS and AKT phosphorylations and SOD activity, and did not modify nNOS, PI3K, AKT and SOD-1 expressions. In conclusion, these results show a compensatory mechanism aimed at reducing the enhanced blood pressure that appears during acute thyroiditis., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Colitis, a colonic inflammatory condition, showed a linkage with hepatobiliary disorders such as cirrhosis. It has been reported that both endogenous opioids and nitric oxide (NO) play critical roles in colitis pathogenesis. Moreover, opioid and NO levels showed elevation in patients with cirrhosis. The aim of this study was to evaluate the effect of cirrhosis on the experimental model of colitis and the possible involvement of opioidergic/nitrergic systems in rats., Methods: Colitis was induced by acetic acid 28days after bile duct ligation (BDL). L-NAME, as an inhibitor of NO synthase and naltrexone, as an antagonist of opioid receptors were administered intraperitoneally to animals during 3days after induction of colitis. Macroscopic colitis lesion area, inflammatory mediators change, NO metabolite levels, and colon microscopic injuries were assessed 3days after induction., Results: Cirrhosis significantly reduced the severity of damages to the colon. Administration of L-NAME (10mg/kg), naltrexone (10mg/kg) and co-administration of L-NAME (1mg/kg) and naltrexone (5mg/kg) significantly decreased the protective effect of BDL on colitis. Nitrite elevated levels in BDL rats were significantly diminished in L-NAME- and naltrexone-treated animals. Histopathology parameters and cytokines level alterations in the colon of acetic acid-treated animals after BDL was reversed after injection of L-NAME, naltrexone, and co-administration of L-NAME (1mg/kg) + naltrexone (5mg/kg)., Conclusion: Cirrhosis improved the intestinal damages induced by acetic acid in rats which may be mediated through interaction of nitrergic and opioidergic systems., (Copyright © 2017 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier B.V. All rights reserved.)

Toll-like receptors (TLRs) are expressed in enteric neurons, glia, gastrointestinal (GI) smooth muscle and mucosa, yet their functional roles in the GI tract are not fully understood. TLRs have been linked to many of the undesirable central effects of chronic opioid administration including hyperalgesia and dependence via activation of central microglia. Opioid-induced bowel dysfunction (OIBD) remains a primary reason for the reduction or withdrawal of opioid analgesics. Morphine-induced inhibition of colonic motility was assessed in vivo by GI transit studies and in vitro using isolated colons from wildtype (WT) and TLR deficient mice. Morphine slowed movement of ingested content in WT but this retardation effect was attenuated in TLR4 -/- and TLR2/4 -/- . In isolated colons, morphine reduced amplitude and frequency colonic migrating motor contractions in both WT and TLR2/4 -/- . Electrical field stimulation elicited distal colon relaxation that was potentiated by morphine in WT but not in TLR2/4 -/- . Inhibitory junction potentials were of similar amplitude and kinetics in WT and TLR2/4 -/- distal colon and not altered by morphine. Enteric nerve density and proportion of nitrergic nerves were similar in WT and TLR2/4 -/- distal colon. These data suggest an involvement of TLRs in opioid pharmacodynamics and thus a potential interventional target for OIBD.

Background: Previously, we found that the neuropeptide galanin was strongly upregulated soon after bilateral cavernous nerve injury (BCNI) and that galanin and its receptors were expressed in nitrergic erectile innervation. Galanin has been observed to exert neuroregenerative effects in dorsal root ganglion neurons, but evidence for these effects in the major pelvic ganglion (MPG) after BCNI is lacking., Aim: To evaluate the neurotropic effects of galanin receptor agonists and antagonists in vitro in nitrergic neurons and MPG and in vivo in rats after BCNI., Methods: Male Sprague-Dawley rats underwent BCNI and sham surgery. Organ culture and single-cell neuron culture of the MPG were performed. Osmotic pump treatment with the galanin agonist in vivo and measurement of erectile response to electrostimulation after BCNI, immunohistochemical localization of galanin and receptors in the human neurovascular bundle, and myographic analysis of rat corpus cavernosum smooth muscle relaxation to galanin receptor agonists were investigated., Outcomes: Neurite outgrowth in vitro and erectile response to electrostimulation after BCNI in vivo, immunohistochemical localization of galanin and receptors, and penile muscle relaxation in vitro., Results: Galanin showed neurotrophic action in vitro and inhibition of endogenous galanin significantly impaired neurite outgrowth in nitrergic but not in sympathetic MPG neurons. In vivo administration of a selective galanin receptor-2 agonist, M1145, resulted in partial recovery of erectile function (EF) after BCNI. Galanin did not act as a direct vasodilator on corpus cavernosum muscle strips., Clinical Translation: Endogenous neurotrophins such as galanin could be used as a strategy to improve EF for patients after BCNI from radical prostatectomy., Strengths and Limitations: We evaluated the effect of galanin on nerve regeneration and EF recovery in vivo and in vitro. Limitations include the lack of washout period for the in vivo experiment and absence of differences in the expression of neuronal markers between treatment groups., Conclusions: We identified galanin as a potential endogenous mechanism for nerve regeneration after BCNI, which could play a physiologic role in EF recovery after radical prostatectomy. In vivo treatment with exogenous galanin was beneficial in enhancing EF recovery after BCNI, but further research is necessary to understand the underlying mechanisms. Weyne E, Hannan JL, Gevaert T, et al. Galanin Administration Partially Restores Erectile Function After Cavernous Nerve Injury and Mediates Endogenous Nitrergic Nerve Outgrowth In Vitro. J Sex Med 2018;15:480-491., (Copyright © 2018 International Society for Sexual Medicine. Published by Elsevier Inc. All rights reserved.)

The aim of the study was to examine the toxic effects of Monosodium glutamate (MSG), an extensively used food additive, on the contraction of uterine visceral smooth muscle (UVSM) in rat and to elucidate the probable neurocrine mechanism involved in it. MSG produced significant potentiation of the force and inhibition of frequency of uterus recorded ex vivo in chronic MSG exposure and in single dose acute experiments. MSG also produced significant potentiation of force of acetylcholine induced contraction and no alterations in atropine induced contraction of uterus. Further, MSG produced significant increase in force and frequency of contraction of neostigmine incubated uterus. We have found significant potentiation of the post pause force of contraction of uterus when MSG was applied in adrenaline incubated uterus. MSG also produced significant decrease in frequency of contraction of sodium nitroprusside incubated uterus; increase in frequency of N-ω-Nitro-l-Arginine Methyl Ester incubated uterus and no significant changes in frequency of contraction of methylene blue incubated uterus. These results indicate that MSG potentiates the force of contraction of UVSM predominantly by augmenting the activity of cholinergic intrinsic efferents and inhibits the frequency of contraction probably by augmenting the activity of nitrergic efferents. In conclusion, MSG potentiates the force and inhibits the frequency of contraction of UVSM, and the MSG induced effect is probably mediated through the augmentation of acetylcholine and nitric oxide signaling pathways., (Copyright © 2018 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.)

Neuropathic pain is a severe condition with unsatisfactory treatments. Melatonin, an indolamine, seems to be a promising molecule suitable for this purpose due to its well-known anti-inflammatory, analgesic, and antioxidant effects, as well as its modulation of the nitroxidergic system. Nevertheless, the data on its mechanism of action and potentialities are currently insufficient in this pathology, especially at the peripheral level. Thus, this work evaluated the effect of a single administration of melatonin in an established mononeuropathy pain model that monitors the behaviour and the changes in the nitroxidergic system in dorsal root ganglia and skin, which are affected by nervous impairment. Experiments were carried out on Sprague Dawley rats subdivided into the sham operated (control) and the chronic constriction injured animals, a model of peripheral neuropathic pain on sciatic nerve. Single administrations of melatonin (5-10 mg/kg) or vehicle were injected intraperitoneally on the 14th day after surgery, when the mononeuropathy was established. The animals were behaviourally tested for thermal hyperalgesia. The dorsal root ganglia and the plantar skin of the hind-paws were removed and processed for the immunohistochemical detection of neuronal and inducible nitric oxide synthases. The behavioural results showed an increase of withdrawal latency during the plantar test as early as 30 min after melatonin administration. The immunohistochemical results indicated a modulation of the nitroxidergic system both at dorsal root ganglia and skin level, permitting speculate on a possible mechanism of action. We showed that melatonin may be a possible therapeutic strategy in neuropathic pain., Competing Interests: The authors declare no conflict of interest.

Urinary bladder function consists in the storage and controlled voiding of urine. Translational studies require animal models that match human characteristics, such as Octodon degus, a diurnal rodent. This study aims to characterize the contractility of the detrusor muscle and the morphology and code of the vesical plexus from O. degus. Body temperature was measured by an intra-abdominal sensor, the contractility of detrusor strips was evaluated by isometric tension recording, and the vesical plexus was studied by electrical field stimulation (EFS) and immunofluorescence. The animals showed a diurnal chronotype as judged from core temperature. The myogenic contractile response of the detrusor muscle to increasing doses of KCl reached its maximum (31.04 mN/mm 2 ) at 60 mM. In the case of cumulative dose-response of bethanecol, the maximum response (37.42 mN/mm 2 ) was reached at 3.2 × 10 -4  M. The response to ATP was clearly smaller (3.8 mN/mm 2 ). The pharmacological dissection of the EFS-induced contraction identified ACh and sensory fibers as the main contributors to this response. The neurons of the vesical plexus were located mainly in the trigone area, grouped in big and small ganglia. Out of them, 48.1 % of the neurons were nitrergic and 62.7 % cholinergic. Our results show functional and morphological similarities between the urinary bladder of O. degus and that of humans.

The consequences of using aspirin (ASA) for the pathogenesis of Chagas disease are unclear. This study evaluated the effects of treatment of Chagas disease with ASA on the esophageal nitrergic myenteric neuron population and esophageal wall in mice. We observed that treatment of chagasic infection with ASA protects the esophageal myenteric neurons from the atrophy caused by the Trypanosoma cruzi infection. The mice were infected with 1300 trypomastigotes of Y strain T. cruzi intraperitoneally. Part of infected mice was treated with ASA from fifth to twelfth day after inoculation. Our data support the hypothesis that eicosanoids given during the acute phase of the chagasic infection may act as immunomodulators aiding the transition to and maintenance of the chronic phase of the disease. Besides, ASA treatment did not provoke alterations in the esophageal wall and the myenteric neurons in infected mice., (© 2015 International Society for Diseases of the Esophagus.)

Background: Hydrogen sulfide (H 2 S) is a gaseous signaling molecule that, similar to nitric oxide (NO), plays an important role as an inhibitor neurotransmitter in the digestive tract. This study aimed to investigate the effect of H 2 S and to identify neurogenic contraction responses dependent on the electrical field stimulation (EFS) in the isolated lower esophageal sphincters of rabbits., Methods: An isolated lower esophageal sphincter was placed in an organ bath system and mechanical responses were recorded using a force transducer. The nerve-evoked contractile responses were obtained by EFS. The contractile responses were obtained as biphasic "on" and "off" phases seen at the beginning and end of EFS, respectively., Results: Sodium hydrogen sulfide (NaHS) reduced the EFS-mediated "off" phase and the EFS-mediated non-adrenergic non-cholinergic (NANC) "off" phase. NaHS reduced the EFS-mediated "on" phase as well. l-Cysteine ​​reduced the EFS-mediated "off" phase and the EFS-mediated NANC "off" phase. l-Propargylglycine (PAG) did not affect the EFS-mediated "off" phase or the EFS-mediated NANC "off" phase. NaHS, l-cysteine, and PAG reduced the EFS-mediated, NO-independent "off" phase. The effect of NaHS in all of the experiments returned in time. Also, NaHS caused significant relaxation of 80-mM KCl-Krebs solution induced-contractions, while l-cysteine ​​and PAG did not cause a significant relaxation., Conclusion: These findings suggest that H 2 S has an inhibitory effect on the lower esophageal sphincter muscle. While the effect of H 2 S on EFS-mediated responses disappeared in time, the effect of H 2 S sustained the KCl-Krebs solution-induced contractions. This shows that H 2 S may have an effect on neurotransmission at the nerve terminal., (Copyright © 2016. Published by Elsevier Urban & Partner Sp. z o.o.)

Background: Oxytocin (OT) reduces rat duodenal tone and mouse intestinal transit; however, the underlying mechanisms are not totally understood. Consequently, this study was designed to investigate the influence of OT on spontaneous mechanical activity and neurally evoked responses, to characterize the mechanisms of the action, and to determine the distribution of the OT receptor (OTR) in rat proximal colonic muscle strips., Methods: The organ bath technique with electrical field stimulation, western blotting, and immunofluorescence were used., Key Results: In rat proximal colon, exogenous OT induced a concentration-dependent reduction of the spontaneous mechanical activity without affecting the resting basal tone, which was abolished by atosiban, an OTR antagonist, by tetrodotoxin (TTX), a neural blocker or by Nω-propyl-l-arginine hydrochloride, an inhibitor of neuronal nitric oxide synthase (nNOS). The inhibitory effects of OT were not affected by atropine or the vasoactive intestinal peptide (VIP) receptor antagonist [Lys1, Pro2,5, Arg3,4, Tyr6]-VIP (VIPHyb). Proximal colon responses to electrical field stimulation were characterized by nonadrenergic, noncholinergic (NANC) relaxation, which was followed by an off-contraction. Oxytocin enhanced only NANC relaxation. Oxytocin stimulated spontaneous NO release from the longitudinal muscle myenteric plexus preparation of rat proximal colon. Western blot and immunohistochemistry experiments showing the presence of the OTR in proximal colon, and its co-localization with nNOS established that myenteric nitrergic neurons express OTR., Conclusions & Inferences: The activation of OTR located on nitrergic neurons may negatively modulate colonic spontaneous contraction and enhance electrically evoked NANC relaxation through excitation of NO release., (© 2016 John Wiley & Sons Ltd.)

The diverse signaling events following mitochondrial depolarization in neurons are not clear. We examined for the first time the effects of mitochondrial depolarization on mitochondrial function, intracellular calcium, neuronal nitric oxide synthase (nNOS) activation, and nitric oxide (NO) production in cultured neurons and perivascular nerves. Cultured rat primary cortical neurons were studied on 7-10 days in vitro, and endothelium-denuded cerebral arteries of adult Sprague-Dawley rats were studied ex vivo. Diazoxide and BMS-191095 (BMS), activators of mitochondrial KATP channels, depolarized mitochondria in cultured neurons and increased cytosolic calcium levels. However, the mitochondrial oxygen consumption rate was unaffected by mitochondrial depolarization. In addition, diazoxide and BMS not only increased the nNOS phosphorylation at positive regulatory serine 1417 but also decreased nNOS phosphorylation at negative regulatory serine 847. Furthermore, diazoxide and BMS increased NO production in cultured neurons measured with both fluorescence microscopy and electron spin resonance spectroscopy, which was sensitive to inhibition by the selective nNOS inhibitor 7-nitroindazole (7-NI). Diazoxide also protected cultured neurons against oxygen-glucose deprivation, which was blocked by NOS inhibition and rescued by NO donors. Finally, BMS induced vasodilation of endothelium denuded, freshly isolated cerebral arteries that was diminished by 7-NI and tetrodotoxin. Thus pharmacological depolarization of mitochondria promotes activation of nNOS leading to generation of NO in cultured neurons and endothelium-denuded arteries. Mitochondrial-induced NO production leads to increased cellular resistance to lethal stress by cultured neurons and to vasodilation of denuded cerebral arteries., (Copyright © 2016 the American Physiological Society.)

Background: Intestinal ischemia/reperfusion injury can be caused by surgical procedures and inflammatory bowel disease. It is normally associated with the increased production of free radicals and changes in the enteric nervous system., Aims: Given the antioxidant and neuroprotective properties of resveratrol, the present study assessed its influence on oxidative stress in the intestinal wall and the morphology of myenteric neurons in the ileum of rats subjected to ischemia/reperfusion., Methods: Resveratrol was orally administered daily at a dose of 10 mg/kg for 5 days. Changes in the ileum response to ischemia after 45 min were investigated followed by 3 h reperfusion. Lipoperoxide and carbonylated protein levels, and the activity of the antioxidant enzymes glutathione reductase, glutathione peroxidase, and glucose-6-phosphate dehydrogenase were measured following ischemia/reperfusion injury., Results: The density and morphometry of the general neuronal population, nitrergic neurons and glial cells, and morphometry of VIP varicosities in the ileum were also studied. Lipoperoxide and carbonylated protein levels were 171 and 40% higher during the ischemia/reperfusion, respectively, compared to control cohorts, and resveratrol attenuated these values. The glutathione ratio was 64% lower during ischemia/reperfusion, compared to control cohorts. Resveratrol increased the reduced/oxidized glutathione ratio, attenuated the changes in the activity of the antioxidant enzymes and the detrimental morphologic changes caused by ischemia/reperfusion in the general neuronal population and nitrergic neurons., Conclusions: Oral treatment with resveratrol reduced the oxidative stress in the ileum and attenuated the morphologic changes that occurred in the myenteric plexus of the ileum in rats subjected to ischemia/reperfusion.

Erectile dysfunction (ED) is a major health problem worldwide and affects approximately 75% of diabetic patients, likely due to severely damaged cavernous body. While screening for cytokines produced by adipose tissue-derived stem cells, we detected neuromedin B (NMB). To explore a potential treatment option for ED, we examined whether NMB was capable of restoring erectile function. We also examined the potential mechanism by which NMB could restore erectile function. Male Wistar rats were injected with streptozotocin (STZ) to induce diabetes. An adenovirus expressing NMB (AdNMB) was injected into the penis 6 weeks after STZ administration. Four weeks after the injection of AdNMB, erectile function, penile histology, and protein expression were analyzed. As assessed by the measurement of intracavernous pressure, AdNMB injection significantly restored erectile function compared with the injection of an adenovirus expressing green fluorescent protein. This restoration was associated with conservation of the cavernous body structure and neural nitric oxide synthase (nNOS)-expressing nerves, together with recovery of α-smooth muscle actin, vascular endothelial-cadherin, and nNOS expression. Furthermore, NMB significantly stimulated the survival of SH-SY5Y cells derived from human neuroblastoma tissue with characteristics similar to neurons. Collectively, these results suggested that NMB restored erectile function via protection of the cavernous body from injury and stimulation of the survival of the associated nerves. NMB may be useful to treat ED patients with a severely damaged cavernous body.

Intake of vitamin A is essential for correct embryonic development of the central nervous system (CNS). Its increased intake during gravidity can cause various malformations and dysfunctions of the CNS. In our work, we intended to investigate the effect of vitamin A on emotional behavior and morphology of nitrergic neurons in basolateral nucleus of the rat amygdala. For this purpose, we have administered retinoic acid (RA), a metabolite of vitamin A, to females on 14-16 days of pregnancy at a dose 1 mg RA/kg body weight. Adult progeny of these mothers were tested in elevated plus maze test, the most widely used test for measuring anxiety-like behavior. After the test, brains of the rats were processed for reduced nicotinamide adenine dinucleotide phosphate diaphorase histochemistry, which is commonly used to mark cells containing nitric oxide synthase. Our results have shown that RA applied during the sensitive phase of intrauterine development influences emotional behavior of adult rats. Animals exposed to RA had increased levels of fear and anxiety, which has been manifested by reducing the time spent in the open arms of plus maze test. Interestingly, detected behavioral changes do not correlate with the result of our morphological study. The number and morphology of nitrergic neurons in amygdala were very similar in experimental and control rats. Our results demonstrate that prenatal exposure to RA has no effect on morphological structure of amygdala, but influences its function.

We have previously shown that kaolinite slowed down gastric emptying and intestinal transit and induced changes in enteric mechanical activities. As gastric emptying and intestinal transit have been shown to be regulated by nitric oxide (NO), the effect of an imposed ingestion of kaolinite on enteric nitrergic innervation was determined. Kaolinite has also been shown to increase plasmatic levels of leptin. Therefore, the responses of enteric neurons in the presence of leptin after kaolinite ingestion were determined, and a possible role of nitrergic neurons was evaluated in rats using organ bath technique. Our results showed that kaolinite modulates activities of enteric nerves at 14 days of ingestion. Exogenous l-NNA produced a decrease in nerve stimulation (NS)-induced relaxation in both jejunum and colon of control groups. At 14 days of kaolinite ingestion, this effect of l-NNA was significantly reduced only in the jejunum. Although l-NNA did not affect NS-induced contraction in jejunum and colon of control animals, it increased the amplitude of the NS-induced contraction in the colon of rats at 14 days of kaolinite ingestion. Leptin inhibitory effects on ENS in the jejunum were also altered at 14 days of ingestion. These differences were masked in the presence of l-NNA. Our data give evidence that changes in mechanical activities induced by kaolinite might be due to alterations in inhibitory (nitrergic and/or other) innervation at 14 days of kaolinite ingestion and to modifications of leptin effects on the responses to intramural nerve stimulation., (© 2013 Société Française de Pharmacologie et de Thérapeutique. Published by John Wiley & Sons Ltd.)

Context: The rectal distension in dogs increases the rate of transitory lower esophageal sphincter relaxation considered the main factor causing gastroesophageal reflux., Objectives: The aim of this study was evaluate the participation of the nitrergic pathway in the increased transitory lower esophageal sphincter relaxation rate induced by rectal distension in anesthetized dogs., Methods: Male mongrel dogs (n = 21), weighing 10-15 kg, were fasted for 12 hours, with water ad libitum. Thereafter, they were anesthetized (ketamine 10 mg.Kg-1 + xylazine 20 mg.Kg-1), so as to carry out the esophageal motility evaluation protocol during 120 min. After a 30-minute basal period, the animals were randomly intravenous treated whith: saline solution 0.15M (1ml.Kg-1), L-NAME (3 mg.Kg-1), L-NAME (3 mg.Kg-1) + L-Arginine (200 mg.Kg-1), glibenclamide (1 mg.Kg-1) or methylene blue (3 mg.Kg-1). Forty-five min after these pre-treatments, the rectum was distended (rectal distension, 5 mL.Kg-1) or not (control) with a latex balloon, with changes in the esophageal motility recorded over 45 min. Data were analyzed using ANOVA followed by Student Newman-Keuls test., Results: In comparison to the respective control group, rectal distension induces an increase in transitory lower esophageal sphincter relaxation. Pre-treatment with L-NAME or methylene blue prevents (P<0.05) this phenomenon, which is reversible by L-Arginine plus L-NAME. However, pretreating with glibenclamide failed to abolish this process., Conclusions: Therefore, these experiments suggested, that rectal distension increases transitory lower esophageal sphincter relaxation in dogs via through nitrergic pathways.

Aim: To investigate the effect of quercetin supplementation on the myenteric neurons and glia in the cecum of diabetic rats., Methods: Total preparations of the muscular tunic were prepared from the ceca of twenty-four rats divided into the following groups: control (C), control supplemented with quercetin (200 mg/kg quercetin body weight) (CQ), diabetic (D) and diabetic supplemented with quercetin (DQ). Immunohistochemical double staining technique was performed with HuC/D (general population)/nitric oxide synthase (nNOS), HuC-D/S-100 and VIP. Density analysis of the general neuronal population HuC/D-IR, the nNOS-IR (nitrergic subpopulation) and the enteric glial cells (S-100) was performed, and the morphometry and the reduction in varicosity population (VIP-IR) in these populations were analyzed., Results: Diabetes promoted a significant reduction (25%) in the neuronal density of the HuC/D-IR (general population) and the nNOS-IR (nitrergic subpopulation) compared with the C group. Diabetes also significantly increased the areas of neurons, glial cells and VIP-IR varicosities. Supplementation with quercetin in the DQ group prevented neuronal loss in the general population and increased its area (P < 0.001) and the area of nitrergic subpopulation (P < 0.001), when compared to C group. Quercetin induced a VIP-IR and glial cells areas (P < 0.001) in DQ group when compared to C, CQ and D groups., Conclusion: In diabetes, quercetin exhibited a neuroprotective effect by maintaining the density of the general neuronal population but did not affect the density of the nNOS subpopulation.

Unlabelled: CONTEXT. Cenostigma macrophyllum Tul. var. acuminata Teles Freire (Leguminosae- Caesalpinioideae) is popularly known as "caneleiro". Previous studies showed antioxidant action and analgesic effects of the ethanol extract from the leaves of C. macrophyllum. The phytochemical evaluation of the stem bark revealed the presence of antinociceptive compounds., Objective: To investigate the antinociceptive actions of the ethanol extract and ethyl acetate fraction from C. macrophyllum stem bark in streptozotocin (STZ)-induced diabetic rats and the involvement of opioid and nitrergic mechanisms., Materials and Methods: STZ-rats received the ethanol extract (E.EtOH 200 and 300 mg/kg, p.o.) during 5 weeks. In acute experiments, untreated diabetic rats were treated with the ethyl acetate fraction (F.EtOAc 250 and 500 mg/kg, p.o.), on the 28th day of diabetes induction when the opioid and nitrergic mechanisms were investigated. The mechanical nociceptive threshold (MNT) was determined by application of von Frey filaments., Results: Data show that STZ-induced diabetic rats developed a significant tactile allodynia during 5 weeks. Diabetic rats that received E.EtOH (200 and 300 mg/kg) and F.EtOAc (250 and 500 mg/kg) had a pain threshold higher than those in the STZ-vehicle group. F.EtOAc effects were inhibited by pretreatment with naloxone and were not influenced by .-arginine., Discussion and Conclusion: The results suggest that the ethanol extract and ethyl acetate fraction of C. macrophyllum presented antinociceptive activity. Thus, F.EtOAc may be exerting its effect by affecting the opioid system, but nitrergic mechanisms are not detectable. The observed activity may be due to its gallic acid, lupeol and bergenin content.

Objectives: We analyzed whether mast cell stabilization by either ketotifen or tranilast could alter either sympathetic or nitrergic innervation function in rat mesenteric arteries., Methods: Electrical field stimulation (EFS)-induced contraction was analyzed in mesenteric segments from 6-month-old Wistar rats in three experimental groups: control, 3-hour ketotifen incubated (0.1 αmol/L), and 3-hour tranilast incubated (0.1 mmol/L). To assess the possible participation of nitrergic or sympathetic innervation, EFS contraction was analyzed in the presence of non-selective nitric oxide synthase (NOS) inhibitor L-NAME (0.1 mmol/L), α-adrenergic receptor antagonist phentolamine (0.1 µmol/L), or the neurotoxin 6-hydroxydopamine (6-OHDA, 1.46 mmol/L). Nitric oxide (NO) and superoxide anion (O2.(-) levels were measured, as were vasomotor responses to noradrenaline (NA) and to NO donor DEA-NO, in the presence and absence of 0.1 mmol/L tempol. Phosphorylated neuronal NOS (P-nNOS) expression was also analyzed., Results: EFS-induced contraction was increased by ketotifen and decreased by tranilast. L-NAME increased the vasoconstrictor response to EFS only in control segments. The vasodilator response to DEA-NO was higher in ketotifen- and tranilast-incubated segments, while tempol increased vasodilator response to DEA-NO only in control segments. Both NO and O2(-) release, and P-nNOS expression were diminished by ketotifen and by tranilast treatment. The decrease in EFS-induced contraction produced by phentolamine was lower in tranilast-incubated segments. NA vasomotor response was decreased only by tranilast. The remnant vasoconstriction observed in control and ketotifen-incubated segments was abolished by 6-OHDA., Conclusion: While both ketotifen and tranilast diminish nitrergic innervation function, only tranilast diminishes sympathetic innnervation function, thus they alter the vasoconstrictor response to EFS in opposing manners.

Crinum glaucum A. Chev (Amaryllidaceae) (CG) is a bulbous plant widely used in folk medicine in the treatment of cough, asthma and convulsions. This study was carried out to investigate the anticonvulsant, anxiolytic and hypnotic effects of the aqueous bulb extract of C. glaucum and its possible mechanism (s) of action. The anticonvulsant activity of C. glaucum extract (400-1200 mg kg(-1) p.o.) was investigated using picrotoxin, strychnine, isoniazid, pentylenetetrazol and N-methyl-D-aspartate (NMDA)-induced seizures in mice while the elevated plus maze test (EPM) and hexobarbitone-induced sleeping time (HIST) were used to evaluate the anxiolytic and hypnotic effects, respectively. Animals were pretreated with flumazenil (3 mg kg(-1); i.p. GABA(A) receptor antagonist), cyproheptadine (4 mg kg(-1); i.p. 5-HT2 receptor antagonist), L-arginine (500 mg kg(-1); p.o. Nitric Oxide (NO) precursor) and L-Nitroarginine (L-NNA) (10 mg kg(-1) i.p. Nitric Oxide Synthase (NOS) inhibitor) were used to investigate the probable mechanism (s) of anticonvulsant activity. Oral administration of CG significantly (p < 0.001) delayed the onset of seizures induced by picrotoxin, strychnine, isoniazid and pentylenetetrazol with peak effect at 1200 mg kg(-1) in comparison to control groups. CG (800 and 1200 mg kg(-1)) strongly antagonized NMDA-induced turning behavior. Pretreatment of mice with cyproheptadine could not reverse the anticonvulsant effect of CG. However, pretreatment with flumazenil and L-NNA significantly (p < 0.05) reversed the anticonvulsant effect of CG while L-arginine pretreatment significantly (p < 0.001) delayed the onset of seizures when compared with control and extract (1200 mg kg(-1) only). CG potentiated hexobarbitone-induced sleeping time with peak effect at 400 mg kg(-1) and also significantly (p < 0.05) increased open arm exploration in EPM and had its peak anxiolytic effect at 100 mg kg(-1). The data obtained suggests that aqueous bulb extract of Crinum glaucum possess anticonvulsant, anxiolytic and hypnotic activities which involve an interaction with GABAergic, nitrergic and glutaminergic systems to exert its effects.

Nitric oxide is known to relax the internal anal sphincter, but its effect on the external anal sphincter (EAS) is unknown. The aim of this study was to investigate whether there is a nitrergic nerve plexus that modulates the EAS, similar to that found in oesophageal striated muscle. An in vitro ring preparation of rat anal canal was used to evaluate the effects of the nitric oxide synthase inhibitor N(ω)-nitro-L-arginine (L-NNA) and the NO donor sodium nitroprusside (SNP) on the EAS in conditions of neuromuscular blockade and the effect of SNP on nerve-evoked contractions. Immunohistological experiments were conducted to determine whether the neuronal isoform of nitric oxide synthase (nNOS) is present in the EAS. During direct muscle stimulation neither L-NNA (P = 0.32) nor SNP (P = 0.19) significantly changed the EF(50), which is the frequency at which 50% of maximal contraction is reached, compared with a time-dependent control. Nerve-evoked contractions were also not altered by addition of SNP to the tissue bath. Immunohistohistological experiments clearly showed co-localization of nNOS-positive nerve fibres at motor endplates of the oesophagus but not in the EAS. The internal anal sphincter was richly innervated by nitrergic fibres, but these did not extend into the EAS. In conclusion, there are no nitrergic motor fibres innervating the EAS, neurotransmission at the motor endplates is not affected by NO, and NO does not affect muscle force directly in conditions of neuromuscular blockade. There is, therefore, no evidence that EAS contraction is directly modulated by NO or by pudendal nitrergic fibres or diffusion from neighbouring nitrergic plexuses of the anal canal.

A compelling body of evidence suggests that nitric oxide (NO), a unique gaseous neurotransmitter and neuromodulator plays a key role in the regulation of motor function. Recently, the interest of researchers concentrates on the NO - soluble guanylyl cyclase (sGC) - cyclic GMP (cGMP) signaling pathway in the striatum as a new target for the treatment of Parkinson's disease (PD). The aim of the study is to review the available literature referring to the role of NO in the integration of basal ganglia functions. First, attention has been focused on behavioral effects of NO donors and neuronal nitric oxide synthase (nNOS) inhibitors in the modulation of motor behavior. Then, disturbances in the nitrergic neurotransmission in PD and its 6-OHDA animal model have been presented. Moreover, the most current data demonstrating the contribution of both dopamine and glutamate to the regulation of NO biosynthesis in the striatum have been analyzed. Finally, the role of NO in the tonic and phasic dopamine release as well as in the regulation of striatal output pathways also has been discussed.

The purpose of this study was to investigate the effect of prenatal administration of retinoic acid (RA) on the development of nicotinamide adenine dinucleotide diaphorase (NADPH-d) positive structures in the rat Meibomian glands. One mg/kg of RA was applied to pregnant Wistar rats intraperitonaelly during the gestational period in each of the 12th-14th embryonic days (totally 3 mg/kg). Sections of the central upper eyelids were investigated in rat pups on the 14th postnatal day. They were processed histochemically for NADPH-d, to study the presence and distribution of nitric oxide synthase (NOS) positive nerve structures. NADPH-d staining of Meibomian glands was compared in two groups of rat pups. In the control group, eyelids of 14 day-old rats were studied with no experimental intervention. The second group consisted of rat pups which were prenatally administered the excess of RA. Histochemical analysis of control eyelids revealed numerous NADPH-d well-stained acini of Meibomian glands arranged tightly into groups. Intensively stained vessels and NADPH-d/NOS-positive nerve fibers bordered acini of Meibomian glands. These structures were present in the submucosal layer as well. The analysis of RA group showed less numerous, shrunken acini of Meibomian glands that were seen not only smaller in size, but also in density of their staining and the amount of nitrergic nerve fibers around acini were considerably lowered. In the submucosa differences were noticed compared to the control group, there were numerous NADPH-d stained vessels accompanied by NADPH-d/NOS-positive nerve fibers. The excess of RA during the prenatal period may influence on the development and morphology of NADPH-d positive structures of rat's Meibomian glands.

The sympathetic and parasympathetic divisions of the autonomic nervous system modulate cardiac rhythm and the probability of arrhythmia occurrence. Both increased sympathetic drive and hypoxia increase the likelihood for ventricular fibrillation (VF). Vagus nerve stimulation (VNS) can protect from fatal arrhythmias via cholinergic and nitrergic action. We sought to determine boundary conditions for VF and defibrillation by autonomic manipulations accompanied or not by hypoxic changes in urethane-anesthetized rats. VF was induced with (1) vagotomy, (2) systemic high-dose (>15 mg/kg) isoproterenol, and (3) hypoxemia. When VNS (50 Hz) produced cardiac standstill, it converted every VF episode (59/59). A nitric oxide synthase inhibitor did not reduce VNS efficacy (13/14 episodes converted), but addition of atropine reduced VNS efficacy (11/27 episodes converted). VF can be induced by autonomic derangements only under constrained conditions, including sympathetic over-activation, reduced parasympathetic input, and hypoxemia. VNS can provide an alternative method to defibrillate via its cholinergic action.

Silicon is not generally considered an essential nutrient for mammals and, to date, whether it has a biological role or beneficial effects in humans is not known. The results of a number of studies suggest that dietary silicon supplementation might have a protective effect both for limiting aluminium absorption across the gut and for the removal of systemic aluminium via the urine, hence, preventing potential accumulation of aluminium in the brain. Since our previous studies demonstrated that aluminium exposure reduces the number of nitrergic neurons, the aim of the present study was to compare the distribution and the morphology of NO-containing neurons in brain cortex of mice exposed to aluminium sulphate dissolved in silicic acid-rich or poor drinking water to assess the potential protective role of silicon against aluminium toxicity in the brain. NADPH-d histochemistry and nNOS immunohistochemistry showed that high concentrations of silicon in drinking water were able to minimize the impairment of the function of nitrergic neurons induced by aluminium administration. We found that silicon protected against aluminium-induced damage to the nitrergic system: in particular, we demonstrated that silicon maintains the number of nitrergic neurons and their expression of nitrergic enzymes at physiological levels, even after a 12 and 15 month exposure to aluminium.

The ionic basis of nitrergic "slow'" inhibitory junction potential (sIJP) is not fully understood. The purpose of the present study was to determine the nature and the role of calmodulin-dependent protein kinase II (CaMKII)-dependent ion conductance in nitrergic neurotransmission at the intestinal smooth muscle neuromuscular junction. Studies were performed in guinea pig ileum. The modified Tomita bath technique was used to induce passive hyperpolarizing electrotonic potentials (ETP) and membrane potential change due to sIJP or drug treatment in the same cell. Changes in membrane potential and ETP were recorded in the same smooth muscle cell, using sharp microelectrode. Nitrergic IJP was elicited by electrical field stimulation in nonadrenergic, noncholinergic conditions and chemical block of purinergic IJP. Modification of ETP during hyperpolarization reflected active conductance change in the smooth muscle. Nitrergic IJP was associated with decreased membrane conductance. The CAMKII inhibitor KN93 but not KN92, the Cl(-) channel blocker niflumic acid (NFA), and the K(ATP)-channel opener cromakalim hyperpolarized the membrane. However, KN93 and NFA were associated with decreased and cromakalim was associated with increased membrane conductance. After maximal NFA-induced hyperpolarization, hyperpolarization associated with KN93 or sIJP was not seen, suggesting a saturation block of the Cl(-) channel signaling. These studies suggest that inhibition of CaMKII-dependent Cl(-) conductance mediates nitrergic sIJP by causing maximal closure of the Cl(-) conductance.

Fluoxetine-induced relaxation of the smooth muscle of small cerebral arteries is thought beneficial in treating mental disorders. The present study was designed to examine effect of fluoxetine on neurogenic nitrergic vasodilation in large cerebral arteries, using in vitro tissue myography, techniques of electrophysiology, calcium imaging and biochemistry. In isolated porcine endothelium-denuded basilar arteries in the presence of U-46619-induced active muscle tone, fluoxetine in low concentration (<0.03 μM) significantly enhanced nicotine- and choline-induced relaxations. The vasorelaxation, however, was blocked by higher concentration of fluoxetine (>0.3 μM) with maximum inhibition at 3 μM. At this concentration, fluoxetine did not affect the basal tone or vasorelaxations induced by transmural nerve stimulation, sodium nitroprusside, or isoproterenol. Furthermore, fluoxetine exclusively blocked nicotine-induced inward currents and calcium influx in cultured neurons of rat superior cervical ganglion and Xenopus oocytes expressing human α7-, α3β2-, or α4β2-nicotinic acetylcholine receptors (nAChRs). In addition, fluoxetine at 0.03 μM and 3 μM significantly enhanced and blocked, respectively, nicotine-induced norepinephrine (NE) release from cerebral perivascular sympathetic nerves. These results indicate that fluoxetine via axo-axonal interaction mechanism exhibits bimodal effects on nAChR-mediated neurogenic nitrergic dilation of basilar arteries. Fluoxetine in high concentrations decreases while in low concentrations it increases neurogenic vasodilation. These results from in vitro experimentation suggest that optimal concentrations of fluoxetine which increase or minimally affect neurogenic vasodilation indicative of regional cerebral blood flow may be important consideration in treating mental disorders., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The functional roles of the nitrergic nerves innervating the monkey cerebral artery were evaluated in a tension-response study examining isolated arteries in vitro and cerebral angiography in vivo. Nicotine produced relaxation of arteries by stimulation of nerve terminals innervating isolated monkey arteries irrigating the cerebrum, cerebellum and brain stem. Relaxation of arteries induced by nicotine was abolished by treatment with N(G)-nitro-L-arginine, a nitric oxide synthase inhibitor, and was restored by addition of L-arginine. Cerebral angiography showed that electrical stimulation of the unilateral greater petrosal nerve, which connects to the pterygopalatine ganglion via the parasympathetic ganglion synapse, produced vasodilatation of the anterior, middle and posterior cerebral arteries in the stimulated side. However, stimulation failed to produce vasodilatation of the superior and anterior-inferior cerebellar arteries and the basilar artery in anesthetized monkeys. Therefore, nitrergic nerves derived from the pterygopalatine ganglion appear to regulate cerebral vasomotor function. In contrast, circulation in the cerebellum and brain stem might be regulated by nitrergic nerves originating not from the pterygopalatine ganglion, but rather from an unknown ganglion (or ganglia).

Disturbances of enteric nerve-mediated anorectal evacuation mechanisms have medical and social impact. The study aimed at further eliciting the contribution of cholinergic and nitrergic neurotransmission systems to modular nerve networks in different regions of Wistar rat anorectum. Electrical field stimulation (EFS, 0.8 ms, 40 V, 2, 5 or 10 Hz, 20 s), computerized mechanographic on-line setup and drugs were used to evaluate the motor responses of isolated rings from circular muscle of rectum (proximal, middle, and distal part), internal anal sphincter, and anal canal. Twitch-like frequency-dependent contractions, more pronounced in rectal preparations, characterized the modular motor responses of rectal circular muscle rings and anal canal. Depending on the frequency of stimulation, the motor activity of internal anal sphincter varied from deep long-lasting relaxation to initial short-lasting relaxation, followed by a contraction. Electrically-evoked responses of anorectal preparations were tetrodotoxin (0.1 microM)-sensitive. In the presence of atropine (0.3 microM) the contractions of rectal rings decreased, relaxation of internal anal sphincter increased and inhibition of the contractions of the anal canal occurred, followed by relaxation. During atropine treatment, NG-nitro-L-arginine (0.5 microM) increased the contractile responses and suppressed internal anal sphincter relaxations. L-arginine (0.5 microM) decreased the contractions and extended the relaxations of internal anal sphincter and anal canal. Our results suggest that cholinergic and nitrergic systems are not equally involved in modular nerve networks of various regions of anorectum. Cholinergic transmission is more expressed in distal rectum, underlying its contractile potency, while nitric oxide-dependent transmission(s) control the relaxation ability of the internal anal sphincter and anal canal.

Aim: β-amyloid peptides (Aβs) have been shown to block cerebral nitrergic neurogenic vasodilation by blocking sympathetic α7-nAChRs, and that oestrogen prevents Aβ-induced neurotoxicity. We examined whether Aβ-inhibition of α7-nAChR-mediated cerebral nitrergic vasodilation was prevented by oestrogen., Methods: Effects of Aβ and 17β-oestradiol on neurogenic nitrergic vasodilation in isolated porcine basilar arteries were examined using wire-myography. Drug effects on nicotine- and choline-induced calcium influx and inward currents in porcine cultured superior cervical ganglion (SCG) were investigated using confocal microscopy and patch-clamp techniques respectively., Results: Precontracted endothelium-denuded basilar arteries relaxed exclusively upon transmural nerve stimulation (TNS, 8 Hz), and applications of nicotine (100 μm) or choline (1 mm), which was sensitive to nitro-L-arginine (L-NNA, 30 μm) and tetrodotoxin (0.3 μm). The relaxation induced by nicotine and choline but not that by TNS was blocked reversibly by Aβ(1-40) in a concentration-dependent manner. Aβ(1-40) also reversibly blocked nicotine- and choline-induced increase of calcium influx and inward currents in the SCG neurons. Aβ inhibition of nicotine- and choline-induced α7-nAChR-mediated nitrergic vasodilation and inward currents was prevented by 17β-oestradiol (10 μm), but not by α-oestradiol (10 μm) or testosterone (10 μm)., Conclusion: These results provide further evidence supporting that Aβ is an antagonist for the α7-nAChR found on post-ganglionic sympathetic adrenergic nerve terminals originating in the SCG. Aβ can cause constriction of cerebral arteries with possible decreased regional cerebral blood flow by blocking sympathetic nerve-mediated release of nitric oxide from the perivascular nitrergic nerves. This effect of Aβ can be prevented by endogenous oestrogen but not testosterone., (© 2010 The Authors. Acta Physiologica © 2010 Scandinavian Physiological Society.)

The α(7)-nicotinic ACh receptor (α(7)-nAChR) on sympathetic neurons innervating basilar arteries of pigs crossed bred between Landrace and Yorkshire (LY) is known to mediate nicotine-induced, β-amyloid (Aβ)-sensitive nitrergic neurogenic vasodilation. Preliminary studies, however, demonstrated that nicotine-induced cerebral vasodilation in pigs crossbred among Landrace, Yorkshire, and Duroc (LYD) was insensitive to Aβ and α-bungarotoxin (α-BGTX). We investigated nAChR subtype on sympathetic neurons innervating LYD basilar arteries. Nicotine-induced relaxation of porcine isolated basilar arteries was examined by tissue bath myography, inward currents on nAChR-expressing oocytes by two-electrode voltage recording, and mRNA and protein expression in the superior cervical ganglion (SCG) and middle cervical ganglion (MCG) by reverse transcription PCR and Western blotting. Nicotine-induced basilar arterial relaxation was not affected by Aβ, α-BGTX, and α-conotoxin IMI (α(7)-nAChR antagonists), or α-conotoxin AuIB (α(3)β(4)-nAChR antagonist) but was inhibited by tropinone and tropane (α(3)-containing nAChR antagonists) and α-conotoxin MII (selective α(3)β(2)-nAChR antagonist). Nicotine-induced inward currents in α(3)β(2)-nAChR-expressing oocytes were inhibited by α-conotoxin MII but not by α-BGTX, Aβ, or α-conotoxin AuIB. mRNAs of α(3)-, α(7)-, β(2)-, and β(4)-subunits were expressed in both SCGs and MCGs with significantly higher mRNAs of α(3)-, β(2)-, and β(4)-subunits than that of α(7)-subunit. The Aβ-insensitive sympathetic α(3)β(2)-nAChR mediates nicotine-induced cerebral nitrergic neurogenic vasodilation in LYD pigs. The different finding from Aβ-sensitive α(7)-nAChR in basilar arteries of LY pigs may offer a partial explanation for different sensitivities of individuals to Aβ in causing diminished cerebral nitrergic vasodilation in diseases involving Aβ.

Introduction: Coitus in snakes may last up to 28 hours; however, the mechanisms involved are unknown., Aim: To evaluate the relevance of the nitric oxide (NO)-cyclic guanosine monophosphate (cGMP)-phosphodiesterase type 5 (PDE5) system in snake corpus cavernosum reactivity., Methods: Hemipenes were removed from anesthetized South American rattlesnakes (Crotalus durissus terrificus) and studied by light and scanning electronic microscopy. Isolated Crotalus corpora cavernosa (CCC) were dissected from the non-spiny region of the hemipenises, and tissue reactivity was assessed in organ baths., Main Outcome Measures: Cumulative concentration-response curves were constructed for acetylcholine (ACh), sodium nitroprusside (SNP), 5-cyclopropyl-2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine-3-yl]pyrimidin-4-ylamine (BAY 41-2272), and tadalafil in CCC precontracted with phenylephrine. Relaxation induced by electrical field stimulation (EFS) was also done in the absence and presence of N(ω) nitro-L-arginine methyl ester (L-NAME; 100 µM), 1H-[1, 2, 4] oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 µM) and tetrodotoxin (TTX; 1 µM)., Results: The hemipenes consisted of two functionally concentric corpora cavernosa, one of them containing radiating bundles of smooth muscle fibers (confirmed by α-actin immunostaining). Endothelial and neural nitric oxide synthases were present in the endothelium and neural structures, respectively; whereas soluble guanylate cyclase and PDE5 were expressed in trabecular smooth muscle. ACh and SNP relaxed isolated CCC, with the relaxations being markedly reduced by L-NAME and ODQ, respectively. BAY 41-2272 and tadalafil caused sustained relaxations with potency (pEC(50) ) values of 5.84 ± 0.17 and 5.10 ± 0.08 (N=3-4), respectively. In precontracted CCC, EFS caused frequency-dependent relaxations that lasted three times longer than those in mammalian CC. Although these relaxations were almost abolished by either L-NAME or ODQ, they were unaffected by TTX. In contrast, EFS-induced relaxations in marmoset CC were abolished by TTX., Conclusions: Rattlesnake CC relaxation is mediated by the NO-cGMP-PDE5 pathway in a manner similar to mammals. The novel TTX-resistant Na channel identified here may be responsible for the slow response of smooth muscle following nerve stimulation and could explain the extraordinary duration of snake coitus., (© 2011 International Society for Sexual Medicine.)

Background: Neural activity can be affected by nitric oxide (NO) produced by spiking neurons. Can neural activity also be affected by NO produced in neurons in the absence of spiking?, Methodology/principal Findings: Applying an NO scavenger to quiescent Aplysia buccal ganglia initiated fictive feeding, indicating that NO production at rest inhibits feeding. The inhibition is in part via effects on neurons B31/B32, neurons initiating food consumption. Applying NO scavengers or nitric oxide synthase (NOS) blockers to B31/B32 neurons cultured in isolation caused inactive neurons to depolarize and fire, indicating that B31/B32 produce NO tonically without action potentials, and tonic NO production contributes to the B31/B32 resting potentials. Guanylyl cyclase blockers also caused depolarization and firing, indicating that the cGMP second messenger cascade, presumably activated by the tonic presence of NO, contributes to the B31/B32 resting potential. Blocking NO while voltage-clamping revealed an inward leak current, indicating that NO prevents this current from depolarizing the neuron. Blocking nitrergic transmission had no effect on a number of other cultured, isolated neurons. However, treatment with NO blockers did excite cerebral ganglion neuron C-PR, a command-like neuron initiating food-finding behavior, both in situ, and when the neuron was cultured in isolation, indicating that this neuron also inhibits itself by producing NO at rest., Conclusion/significance: Self-inhibitory, tonic NO production is a novel mechanism for the modulation of neural activity. Localization of this mechanism to critical neurons in different ganglia controlling different aspects of a behavior provides a mechanism by which a humeral signal affecting background NO production, such as the NO precursor L-arginine, could control multiple aspects of the behavior.

The rostral ventro-medial medullary reticular formation is a complex structure that is involved with a variety of motor functions. It contains glycinergic neurons that are activated during active (rapid eye movement (REM)) sleep (AS); these neurons appear to be responsible for the postsynaptic inhibition of motoneurons that occurs during this state. We have reported that neurons in this same region contain nitric oxide (NO) synthase and that they innervate brainstem motor pools. In the present study we examined the c-fos expression of these neurons after carbachol-induced active sleep (C-AS). Three control and four experimental cats were employed to identify c-fos expressing nitrergic neurons using immunocytochemical techniques to detect the Fos protein together with neuronal nitric oxide synthase (nNOS) or nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase activity. The classical neurotransmitter content of the nitrergic cells in this region was examined through the combination of immunocytochemical techniques for the detection of glutamate, glycine, choline acetyltransferase (ChAT), tyrosine hydroxilase (TH) or GABA together with nNOS. During C-AS, there was a 1074% increase in the number of nitrergic neurons that expressed c-fos. These neurons did not contain glycine, ChAT, TH or GABA, but a subpopulation (15%) of them displayed glutamate-like immunoreactivity. Therefore, some of these neurons contain both an excitatory neurotransmitter (glutamate) and an excitatory neuromodulator (NO); the neurotransmitter content of the rest of them remains to be determined. Because some of the nitrergic neurons innervate brainstem motoneurons it is possible that they participate in the generation of tonic and excitatory phasic motor events that occur during AS. We also suggest that these nitrergic neurons may be involved in autonomic regulation during this state. In addition, because NO has trophic effects on target neurons, the present findings represent the first, albeit indirect, evidence for a possible trophic function of this nature during AS., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

Cigarette smoking is a major risk factor for atherosclerosis, cerebral and coronary vascular diseases, hypertension, and diabetes mellitus. Chronic smoking impairs endothelial function by decreasing the formation of nitric oxide and increasing the degradation of nitric oxide via generation of oxygen free radicals. Nitric oxide liberated from efferent nitrergic nerves is also involved in vasodilatation, increased regional blood flow, and hypotension that are impaired through nitric oxide sequestering by smoking-induced factors. Influence of smoking on nitric oxide-induced blood flow regulation is not necessarily the same in all organs and tissues. However, human studies are limited mainly to the forearm blood flow measurement that assesses endothelial function under basal and stimulated conditions and also determination of penile tumescence and erection in response to endothelial and neuronal nitric oxide. Therefore, information about blood flow regulation in other organs, such as the brain and placenta, has been provided mainly from studies on experimental animals. Nicotine, a major constituent of cigarette smoke, acutely dilates cerebral arteries and arterioles through nitric oxide liberated from nitrergic neurons, but chronically interferes with endothelial function in various vasculatures, both being noted in studies on experimental animals. Cigarette smoke constituents other than nicotine also have some vascular actions. Not only active but also passive smoking is undoubtedly harmful for both the smokers themselves and their neighbors, who should bear in mind that they can face serious diseases in the future, which may result in lengthy hospitalization, and a shortened lifespan., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Arterial baroreflex, an important physiological regulatory system for buffering systemic blood pressure, is impaired in obesity. This study investigated whether the blunted baroreflex function in obesity is attributed to the altered nitroxidergic or N-methyl--aspartate (NMDA) mechanism. Baroreflex bradycardia responses, blood pressure and heart rate in 30 lean and 30 obese anesthetized Zucker rats (8-12 weeks of age) were assessed after injecting phenylephrine with intravenous preadministration of saline (control), dextromethorphan (DXM, NMDA receptor antagonist, 10 mg kg(-1)) or N(G)-nitro-L-arginine methyl ester (L-NAME, nitric oxide synthase inhibitor, 100 mg kg(-1)). Compared with lean rats (-2.00+/-0.29 b.p.m. mm Hg(-1)), the baroreflex sensitivity (BRS) in obese rats (-0.43+/-0.05 b.p.m. mm Hg(-1)) was significantly blunted. The BRS was significantly suppressed by DXM in lean rats but not in obese rats. After administration of L-NAME, BRS was significantly suppressed in lean Zucker rats but not in obese Zucker rats. The normal BRS was significantly suppressed in lean rats after administration of both DXM and L-NAME, and the blunted BRS in obesity was significantly blocked to nearly no BRS after administration of both DXM and L-NAME. This study suggests that BRS is blunted in obese rats and that blunted baroreflex is, at least in part, attributed to altered nitroxidergic or NMDA receptor-mediated modulation.

Neuronal nitric oxide (NO) has been shown to modulate perivascular adrenergic neurotransmission by inhibiting noradrenaline release from terminals in rat mesenteric arteries. This study was conducted to investigate changes in the inhibitory function of NO-containing nerves (nitrergic nerves) in mesenteric vascular beds of 2-kidney, 1-clip renovascular hypertensive rats (2K1C-RHR). Rat mesenteric vascular beds without endothelium were perfused with Krebs solution and the perfusion pressure was measured. In preparations from sham-operated rats (control) and 2K1C-RHRs, vasoconstriction induced by periarterial nerve stimulation (PNS; 2-8 Hz), but not vasoconstriction induced by exogenously injected noradrenaline (0.5, 1.0 nmol), was markedly facilitated in the presence of a nonselective NO synthase (NOS) inhibitor, N-omega-nitro-L-arginine methyl ester (L-NAME) (100 microM). The facilitatory effect of L-NAME in preparations from 2K1C-RHR was smaller than that in control preparations. L-NAME augmented PNS-evoked noradrenaline release, which was smaller in 2K1C-RHRs than in controls. The expression of neuronal NO synthase (nNOS) measured by western blotting in mesenteric arteries from 2K1C-RHRs was significantly decreased compared with control arteries. Immunohistochemical staining of mesenteric arteries showed dense innervation of nNOS-immunopositive nerves that was significantly smaller in arteries from 2K1C-RHR than that in control arteries. Mesenteric arteries were densely innervated by tyrosine hydroxylase-immunopositive nerves, which coalesced with nNOS-immunopositive nerves. These results suggest that the inhibitory function of nitrergic nerves in adrenergic neurotransmission is significantly decreased in 2K1C-RHRs. This functional alteration based on the decrease in nNOS expression and nitrergic innervation leads to enhanced adrenergic neurotransmission and contributes to the initiation and development of renovascular hypertension.

1. Nitrergic neurons regulate gastrointestinal (GI) activity and their dysfunction has been associated with various GI diseases. Nitric oxide (NO) typically relaxes GI smooth muscle, but nitrergic contractions also occur. Although guanylate cyclase is well established as mediating nitrergic GI relaxation, its role in contraction remains uncertain. 2. We used electrical field stimulation (EFS; 0.3 msec pulses, three trains of 1.2 s width, 2 Hz, at 30 s intervals) to evoke biphasic contraction-relaxation responses in rat ileum strips (longitudinal muscle-myenteric plexus preparations), mediated by the endogenous nitrergic transmitter, under non-adrenergic, non-cholinergic (NANC) conditions (1 micromol/L atropine and 4 micromol/L guanethidine). 3. All EFS responses were abolished by tetrodotoxin (1 micromol/L). Inhibition of NO synthase with N(omega)-nitro-L-arginine-methyl-ester (l-NAME; 100 and 300 micromol/L) prevented both EFS-evoked contractions and relaxations. L-Arginine (3 mmol/L) reversed l-NAME inhibition, primarily restoring contractions and suggesting that these require lower nitrergic transmitter levels than relaxations. 4. Pretreatment of preparations with subrelaxant concentrations of sodium nitroprusside (1 micromol/L) selectively desensitized EFS-evoked contractions without affecting relaxations, suggesting different downstream mechanisms. Nevertheless, the selective guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (3 and 10 micromol/L) inhibited both nitrergic contractions and relaxations, indicating that guanylate cyclase activation is required for both responses. 5. The results of the present study support the hypothesis that the endogenous nitrergic transmitter differentially regulates guanylate cyclase, leading to either contractions or relaxations depending on its concentrations, thus providing additional insight into the regulation of ileum contractility by nitrergic activity.

Purpose: In the ileum of W/W(v) mutant mice (W/W(v)), the absence of interstitial cells of Cajal (ICC) in the myenteric region (ICC-MY), and cross-talk between ICC in the deep muscular region (ICC-DMP) and enteric nitrergic motor nerves leads to irregular spontaneous electrical and contractile activity. The aim of the present study was to reveal changes in this irregular spontaneous electrical and contractile activity in the ileum of dextran sodium sulfate (DSS: m.w. 40,000)-induced colitis model W/W(v) mice., Methods: Electrical and contractile activity was recorded with a suction electrode and with both an isometric force transducer and a pressure transducer in the ileum of W/W(v) mice either with DSS-induced colitis (DSS (+)) in the distal colon or without in controls (DSS (-)). Neuronal NO synthase (nNOS) and inducible NO synthase (iNOS) immunoreactivity in the ileum was compared between the following groups of mice: W/W(v) DSS (+), W/W(v) DSS (-), wild type (WT) with (DSS (+)) and WT without DSS-induced colitis (DSS (-))., Results: DSS induced colitis in the distal colon of W/W(v) mice is reduced compared with that in WT mice, despite the reduction in the number of mast cells in the W/W(v) mutants. Irregular contractions in the ileum without colitis were strongly suppressed in W/W(v) DSS (+) mice. The mean interval of irregular contractions in W/W(v) DSS (+) mice was 5-fold larger than that in W/W(v) DSS (-) mice. N-nitro-L-arginine methyl ester (L-NAME) facilitated the frequency of irregular contractions in the ileum without colitis in W/W(v) DSS (+) mice. L-NAME decreased the mean interval of contractions to one-fourth in the ileum of W/W(v) DSS (+) mice, where strong iNOS immunoreactivity in nitrergic motor nerves was found with unchanged nNOS immunoreactivity., Conclusions: The stronger suppression of irregular contractions of the ileum in DSS-induced colitis model W/W(v) mice was elicited and mediated by cross-talk between ICC-DMP and enteric nitrergic motor nerves expressing iNOS/NO, even though the ileum was not demonstrating colitis.

Nitric oxide (NO)-containing neurons are widely distributed within the central nervous system, including regions involved in the control of reproduction and sexual behavior. Nitrergic neurons may co-localize with gonadal hormone receptors and gonadal hormones may influence neuronal NO synthase expression in adulthood as well as during development. In rodents, the female, in physiological conditions, is exposed to short-term changes of gonadal hormones levels (estrous cycle). Our studies, performed in mouse hypothalamic and limbic systems, reveal that the expression of neuronal NO synthase may vary according to the rapid variations of hormonal levels that take place during the estrous cycle. This is in accordance with the hypothesis that gonadal hormone activation of NO-cGMP pathway is important for mating behavior. NO-producing system appears particularly sensitive to alterations of endocrine balance during development, as demonstrated by our experiments utilizing perinatal exposure to bisphenol A, an endocrine disrupting chemical. In fact, significant effects were detected in adulthood in the medial preoptic nucleus and in the ventromedial subdivision of the bed nucleus of the stria terminalis. Therefore, alteration of the neuronal NO synthase expression may be one of the causes of the important behavioral alterations observed in bisphenol-exposed animals.

The interaction of purinergic and nitrergic mechanisms was evaluated in the caudal nucleus tractus solitarii (cNTS) using awake animals and brainstem slices. In awake animals, ATP (1.25 nmol/50 nL) was microinjected into the cNTS before and after the microinjection of a selective neuronal nitric oxide synthase (nNOS) inhibitor N-propyl-l-arginine (NPLA, 3 pmoles/50 nL, n=8) or vehicle (saline, n=4), and cardiovascular and ventilatory parameters were recorded. In brainstem slices from a distinct group of rats, the effects of ATP on the NO concentration in the cNTS using the fluorescent dye DAF-2 DA were evaluated. For this purpose brainstem slices (150 microm) containing the cNTS were pre-incubated with ATP (500 microM; n=8) before and during DAF-2 DA loading. Microinjection of ATP into the cNTS increases the arterial pressure (AP), respiratory frequency (f(R)) and minute ventilation (V(E)), which were significantly reduced by pretreatment with N-PLA, a selective nNOS inhibitor (AP: 39+/-3 vs 16+/-14 mm Hg; f(R): 75+/-14 vs 4+/-3 cpm; V(E): 909+/-159 vs 77+/-39 mL kg(-1) m(-1)). The effects of ATP in the cNTS were not affected by microinjection of saline. ATP significantly increased the NO fluorescence in the cNTS (62+/-7 vs 101+/-10 AU). The data show that in the cNTS: a) the NO production is increased by ATP; b) NO formation by nNOS is involved in the cardiovascular and ventilatory responses to microinjection of ATP. Taken together, these data suggest an interaction of purinergic and nitrergic mechanisms in the cNTS.