The Influence of Bisphenol a on the Nitrergic Nervous Structures in the Domestic Porcine Uterus.

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.

Activity within specific enteric neurochemical subtypes is correlated with distinct patterns of gastrointestinal motility in the murine colon.

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 Nω-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.

Galanin Administration Partially Restores Erectile Function After Cavernous Nerve Injury and Mediates Endogenous Nitrergic Nerve Outgrowth In Vitro.

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.

Aspirin prevents atrophy of esophageal nitrergic myenteric neurons in a mouse model of chronic Chagas disease.

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.

Single Administration of Melatonin Modulates the Nitroxidergic System at the Peripheral Level and Reduces Thermal Nociceptive Hypersensitivity in Neuropathic Rats.

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.

Depolarization of mitochondria in neurons promotes activation of nitric oxide synthase and generation of nitric oxide.

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.

Resveratrol Reduces Morphologic Changes in the Myenteric Plexus and Oxidative Stress in the Ileum in Rats with Ischemia/Reperfusion Injury.

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.

Vitamin A and amygdala: functional and morphological consequences.

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.

Effect of leptin on nitrergic neurons in the lateral hypothalamic area and the supraoptic nucleus of rats.

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.

Is there a nitrergic modulation of the rat external anal sphincter?

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.

The stem bark extracts of Cenostigma macrophyllum attenuates tactile allodynia in streptozotocin-induced diabetic rats.

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.

CaMKII inhibition hyperpolarizes membrane and blocks nitrergic IJP by closing a Cl(-) conductance in intestinal smooth muscle.

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.

Sympathetic α3ß2-nAChRs mediate cerebral neurogenic nitrergic vasodilation in the swine.

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, ß-amyloid (Aß)-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 Aß 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 Aß, α-BGTX, and α-conotoxin IMI (α(7)-nAChR antagonists), or α-conotoxin AuIB (α(3)ß(4)-nAChR antagonist) but was inhibited by tropinone and tropane (α(3)-containing nAChR antagonists) and α-conotoxin MII (selective α(3)ß(2)-nAChR antagonist). Nicotine-induced inward currents in α(3)ß(2)-nAChR-expressing oocytes were inhibited by α-conotoxin MII but not by α-BGTX, Aß, or α-conotoxin AuIB. mRNAs of α(3)-, α(7)-, ß(2)-, and ß(4)-subunits were expressed in both SCGs and MCGs with significantly higher mRNAs of α(3)-, ß(2)-, and ß(4)-subunits than that of α(7)-subunit. The Aß-insensitive sympathetic α(3)ß(2)-nAChR mediates nicotine-induced cerebral nitrergic neurogenic vasodilation in LYD pigs. The different finding from Aß-sensitive α(7)-nAChR in basilar arteries of LY pigs may offer a partial explanation for different sensitivities of individuals to Aß in causing diminished cerebral nitrergic vasodilation in diseases involving Aß.

Role of a novel tetrodotoxin-resistant sodium channel in the nitrergic relaxation of corpus cavernosum from the South American rattlesnake Crotalus durissus terrificus.

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.

The effect of Xenin25 on spontaneous circular muscle contractions of rat distal colon in vitro.

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 Ca2+ -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.

Guanylate cyclase regulates ileal longitudinal muscle contractions induced by neurogenic nitrergic activity in the rat.

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.

Anti- and pro-oxidant effects of quercetin stabilized by microencapsulation on interstitial cells of Cajal, nitrergic neurons and M2-like macrophages in the jejunum of diabetic rats.

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.

Differential blockade by huperzine A and donepezil of sympathetic nicotinic acetylcholine receptor-mediated nitrergic neurogenic dilations in porcine basilar arteries.

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 ß 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 α3ß2-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.

Presynaptic prostaglandin E2 EP1-receptor facilitation of cerebral nitrergic neurogenic vasodilation.

BACKGROUND AND PURPOSE: Prostaglandin E(2) (PGE(2)) modulates autonomic transmission in the peripheral circulation. We investigated the role of endogenous PGE(2) and its presynaptic EP(1) receptor subtype in modulating the autonomic neurotransmission in cerebral vasculature. METHODS: The standard in vitro tissue-bath technique was used for measuring changes in isolated porcine basilar arterial tone. Calcium imaging and nitric oxide estimation along with immunohistochemical analysis for cyclo-oxygenase-1, cyclo-oxygenase-2, EP(1) receptor, PGE synthase, and neuronal nitric oxide synthase were done in cultured sphenopalatine ganglia and basilar artery. RESULTS: Selective EP(1) receptor antagonists (SC-19220 and SC-51322) inhibited relaxation of endothelium-denuded basilar arterial rings elicited by transmural nerve stimulation (2 and 8 Hz) without affecting that induced by nicotine or sodium nitroprusside (a nitric oxide donor). The SC-19220 inhibition of transmural nerve stimulation-elicited relaxation was blocked by cyclo-oxygenase inhibitors (salicylic acid and naproxen) but was not affected by guanethidine (a sympathetic neuronal blocker) or atropine. Perivascular cyclo-oxygenase-1- and cyclo-oxygenase-2-immunoreactive fibers were observed in basilar arteries. PGE synthase and EP(1) receptor immunoreactivities were coincident with neuronal nitric oxide synthase immunoreactivities in perivascular nerves of the basilar arteries and the sphenopalatine ganglia. omega-conotoxin (an N-type calcium channel blocker) significantly blocked transmural nerve stimulation-induced relaxation, which was further attenuated by SC-19220. In cultured sphenopalatine ganglia neurons, exogenous PGE(2) significantly increased calcium influx and diaminofluorescein fluorescence indicative of nitric oxide synthesis. Both responses were blocked by SC-19220. CONCLUSIONS: These results suggest that neuronal PGE(2) facilitates nitric oxide release from the cerebral perivascular parasympathetic nitrergic nerve terminals by increasing neuronal calcium influx through activation of presynaptic EP(1) receptors. PGE(2) may play an important role in regulating the nitrergic neurovascular transmission in the cerebral circulation.

Role of PSD95 in membrane association and catalytic activity of nNOSalpha in nitrergic varicosities in mice gut.

We have recently shown that membrane association of neuronal nitric oxide synthase-alpha (nNOSalpha) is critical in the regulation of synthesis of NO during nitrergic neurotransmission. The purpose of this study was to examine the role of the synapse-associated proteins (SAPs) in membrane association of nNOSalpha. Varicosities (swellings on terminal axons) were isolated from mice gastrointestinal tract and examined for nNOSalpha, postsynaptic density protein 95 (PSD95), and membrane interactions by coimmunoprecipitation and SDS-PAGE. Our results show that PSD95 protein was present in the membrane fraction of the nerve varicosity, whereas both PSD95 and SAP97 were present in the cytosol. nNOSalpha was associated with PSD95 but not SAP97. nNOSalpha-PSD95 complex was bound to the membrane via palmitoylation of PSD95. Depalmitoylation of PSD95 with 2-bromopalmitate dislocates nNOSalpha and PSD95 from the varicosity membrane and abolishes NO production. These studies show that palmitoylation of PSD95 anchors nNOSalpha to the varicosity membrane and that it is obligatory for NO production by the enzyme. Palmitoylation of PSD95 may provide a novel target for regulation of nitrergic neurotransmission.