Nitric Oxide Synthase Immunoreactivity Research Articles

Activated RhoA/Rho Kinase Impairs Erectile Function After Cavernous Nerve Injury in Rats

RhoA and rho kinase serve as key regulators of penile vascular homeostasis. The role of RhoA/rho kinase signaling in the penis after cavernous nerve injury has not been fully investigated. We characterized the molecular expression profiles of RhoA/rho kinase signaling that occur in the penis after cavernous nerve injury. We hypothesized that erectile dysfunction after bilateral cavernous nerve injury is accompanied by up-regulation of RhoA/rho kinase activity in the rat penis. We used 2 groups, including sham operation and bilateral cavernous nerve injury. At 14 days after nerve injury each group underwent cavernous nerve stimulation to determine erectile function at baseline and after intracavernous injection of the rho kinase inhibitor Y-27632 (Tocris Bioscience, Ellisville, Missouri). Penes were assessed at baseline for protein expression of neuronal nitric oxide synthase, RhoA, and rho kinase 1 and 2 by Western blot, immunoreactivity of neuronal nitric oxide synthase, rho kinase 1 and 2, RhoA-guanosine triphosphatase and rho kinase activity. Erectile function was decreased in nerve injured rats. Neuronal nitric oxide synthase protein was significantly decreased while RhoA and rho kinase 2 protein levels were significantly increased in rat penes with nerve injury. Rho kinase 1 protein expression was equivalent. Rho kinase immunoreactivity was qualitatively increased in the corporeal smooth muscle of nerve injured rats. RhoA-guanosine triphosphatase and rho kinase activity was significantly increased in injured rat penes compared to that in sham operated penes. Intracavernous injection of Y-27632 caused a significantly greater increase in intracavernous pressure in nerve injured rats compared to that in sham operated rats, suggesting increased rho kinase activity. Data suggest that RhoA/rho kinase up-regulation in response to cavernous nerve injury contributes to penile vasculature dysfunction after cavernous nerve injury. Thus, the RhoA/rho kinase pathway may be a suitable target for treating post-radical prostatectomy erectile dysfunction.

Fasudil protects hippocampal neurons against hypoxia‐reoxygenation injury by suppressing microglial inflammatory responses in mice

Rho kinase (ROCK) may play an important role in regulating biological events of cells, including proliferation, differentiation and survival/death. Blockade of ROCK promotes axonal regeneration and neuron survival in vivo and in vitro, thereby exhibiting potential clinical applications in spinal cord damage and stroke. Our previous studies have demonstrated that Fasudil, a selective ROCK inhibitor, induced neuroprotection in vitro. Here we used an in vivo model of hypoxia/reoxygenation (H/R) injury to examine the neuroprotective effect of Fasudil, and explore its possible mechanism(s) in vivo. H/R resulted in the loss of hippocampal neurons, accompanied by increased apoptosis of neurons in hippocampus. The expression of ROCK II and activity of ROCK in the brain were increased after H/R, and located only in microglia, but not in astrocytes and neurons. The administration of Fasudil inhibited the activity of ROCK in brain tissue and cultured microglia, and protected hippocampal neurons against H/R injury. Further immunohistochemical analysis and cytokine determination revealed that Fasudil inhibited inducible nitric oxide synthase immunoreactivity in microglia and pro-inflammatory factors in brain tissue after H/R, which is consistent with the observation wherein Fasudil reduced the pro-inflammatory factors nitric oxide, IL-1β, IL-6 and TNF-, and increased anti-inflammatory factor IL-10 in cultured microglia under normoxic or hypoxic conditions. Our results indicate that inhibition of ROCK by Fasudil may represent a useful therapeutic perspective by inhibiting microglial inflammatory responses in the CNS.

Origin and Characterization of Retrograde Labeled Neurons Supplying the Rat Urethra Using Fiberoptic Confocal Fluorescent Microscopy In Vivo and Immunohistochemistry

Autonomic innervation of urethral smooth muscle may influence urinary continence after prostatectomy. It is unclear whether the cavernous nerves carry fibers that influence continence. Using a retrograde axonal tracer combined with real-time in vivo imaging and ex vivo immunohistochemistry we determined the course and type of neurons supplying urethral smooth muscle distal to the prostate in the rat. We injected the retrograde axonal tracers cholera toxin B fragment-Alexa Fluor 488 and Fast Blue in the distal urethral smooth muscle in 10 rats each. Five days later the cavernous nerves and pelvic ganglion were imaged using fiberoptic confocal fluorescence microscopy (cholera toxin B fragment-Alexa Fluor 488) or harvested for immunohistochemistry (Fast Blue). Dual immunofluorescence of Fast Blue neurons with tyrosine hydroxylase or neuronal nitric oxide synthase was done to characterize neurons as noradrenergic or nitrergic. To ascertain whether the cavernous nerves contain fibers to the urethra that originate in the pelvic ganglia we cut the cavernous nerves with their ancillary branches in 3 rats and imaged them for Fast Blue. Fluorescent neurons and axons were detected in cavernous nerves and the pelvic ganglion. Few neurons were seen in rats with cavernous nerve section. Of urethral neurons 53.1% showed neuronal nitric oxide synthase positivity while 40.6% were immunoreactive for tyrosine hydroxylase. About 6.2% of urethral neurons failed to show tyrosine hydroxylase or neuronal nitric oxide synthase immunoreactivity. Most of the autonomic innervation to the urethra beyond the prostatic apex travels in the cavernous nerves. Many nerves may be parasympathetic based on neuronal nitric oxide synthase immunoreactivity. Nerves supplying the urethra outside the cavernous nerves may course posterior to the prostate. Along with afferent fibers, tyrosine hydroxylase immunoreactivity expressing neuron fibers, ie noradrenergic nerves, traveling in the cavernous nerves may increase urethral resistance or regulate the reflex mechanisms controlling continence.

Differential involvement of the periaqueductal gray in multiple system atrophy

The periaqueductal gray (PAG) consists of distinct columns that participate in the integrated control of autonomic function. We sought to determine whether the PAG is affected in multiple system atrophy (MSA), a disorder characterized by prominent autonomic failure. Brains were obtained at autopsy from 13 MSA patients (10 M, 3 F, age 61 ± 3 years) and 13 controls (8 M, 5 F, age 67 ± 4 years). Transverse formalin-fixed 50 μm sections were obtained throughout the PAG and immunostained for the vesicular transporter 2 (VGLUT-2), nitric oxide synthase (NOS), or α-synuclein and co-stained with thionin. Some sections were processed for myelin or astrocyte staining. Stereological quantitation was performed separately in the ventrolateral, lateral, dorsolateral, and dorsomedial columns of the PAG. In MSA cases, there was a decrease in the total estimated number of VGLUT-2 immunoreactive neurons in the ventrolateral, lateral, and dorsomedial and to a lesser extent dorsolateral PAG compared to controls (ventrolateral PAG: 16,299 ± 1612 vs. 27,906 ± 2480 respectively, p < 0.01; lateral PAG: 11,004 ± 1401 vs. 16,078 ± 1140 respectively, p < 0.05; and dorsomedial PAG: 8847 ± 1052 vs. 15,412 ± 1097 respectively, p < 0.001). The number of NOS immunoreactive neurons in the dorsolateral PAG was similar to controls. In all columns, the number of non-immunolabelled Nissl-stained cells was similar between groups. There was accumulation of glial cytoplasmic inclusions in all PAG columns in MSA. Our findings indicate involvement of the PAG columns in MSA, which may contribute to autonomic disturbances in this disorder.

Immunohistochemical characterization of pelvic neurons projecting to the bladder in rats with the artificial somatic-autonomic reflex arc

Objective Retrograde-tracing and immunohistochemical techniques were used in combination to investigate the types of putative transmitters in pelvic ganglia neurons projecting to the bladder of rats with the artificial somatic-autonomic reflex arc. Methods The artificial somatic-autonomic reflex arc was established in 8 rats by end-to-end microanastomosis of the proximal stump of the left L4 ventral root to the distal stump of L6 ventral root. The major pelvic ganglia (MPG) neurons innervating the urinary bladder were labeled by retrograde axonal tracing methods with the use of horseradish peroxidase (HRP) injected into the bladder wall and identified with TMB-HRP reaction. Then immunohistochemical (ChAT, TH, VIP, SP) and histochemical methods (NADPH-diaphorase activity and nitric oxide synthase immunoreac-tivity) were used to identify the neurotransmitters of HRP-labeled neurons projecting to the bladder. Results HRP-labeled neurons with spherical or oval shape mainly distributed in the MPG. The HRP-labeled neurons were positive for ChAT (112/279, 40. 1%), TH (50/245,20.4%), VIP(25/233, 10.7%), NOS ( 15/230, 6. 5% ) or SP (8/242, 3. 3% ) respectively. Conclusion These results suggest that ace-tylcholine and adrenaline are the predominant neurotransmitters of postganglionic neurons innervating bladder in rats with the artificial somatic-autonomic reflex arc. VIP, NO and SP may be as the neurotransmitters or neuromodulators to participate the procedure of urinary storage and voiding. Key words: Bladder; Pelvic ganglion; Neurotransmitter; Artificial reflex arc

Inhibitory neuromuscular transmission to ileal longitudinal muscle predominates in neonatal guinea pigs

Inhibitory neurotransmission to the longitudinal muscle is more prominent in the neonatal than in the adult guinea pig ileum. Inhibitory neuromuscular transmission was investigated using in vitro ileal longitudinal muscle myenteric plexus (LMMP) preparations made from neonatal (< or =48 h postnatal) and adult ( approximately 4 weeks postnatal) guinea pigs. Amperometric measurements of nicotine-induced nitric oxide (NO) release (measured as an oxidation current) from myenteric ganglia revealed larger currents in neonatal (379 +/- 24 pA) vs adult (119 +/- 39 pA, P < 0.05) tissues. Nicotine-induced oxidation currents were blocked by the nitric oxide synthase (NOS) inhibitor, nitro-l-arginine (NLA, 100 micromol L(-1)). Nicotine-induced, NLA-sensitive oxidation currents could be detected in the tertiary plexus of neonatal but not adult tissues. Immunohistochemistry demonstrated stronger NOS immunoreactivity in neonatal compared with adult myenteric ganglia. Western blot studies revealed higher levels of NOS in neonatal compared with adult LMMP. Cell counts revealed that the total number of myenteric neurons in the small intestine was greater in adults than in neonatal guinea pigs, however, the ratio of NOS : Calbindin neurons was significantly higher in neonatal compared with adult tissues. Nitric oxide signaling to the longitudinal muscle is stronger in neonatal compared with adult guinea pig ileum. Nitric oxide synthase-containing neurons are diluted postnatally by cholinergic and other, as yet unidentified neuronal subtypes.

Seasonal potentiation of gill muscle contraction in four species of bivalve molluscs

When isolated gills of Mercenaria mercenaria are treated with dopamine (DA), the branchial musculature contracts. Moreover, a second application of the same concentration of DA produces a larger contraction. The mean ratio of the second to the first contractions (contraction ratio) was 2.18, and it is significantly greater than 1. This potentiation was seasonal and was mediated by a nitric oxide/cyclic GMP (NO/cGMP) signaling cascade. The responses of the branchial muscles in the gills of four other bivalves were examined to determine whether they too are potentiated. The contraction ratios of Mya arenaria (mean = 1.48; 95% CI of mean: 1.28–1.72; n = 39) and Spisula solidissima (mean = 2.04; 95% CI of mean: 1.71–2.43; n = 31) are both significantly greater than 1; i.e., they were potentiated. In contrast, the contraction ratios of Arctica islandica (mean = 0.94; 95%CI of mean: 0.79–1.14; n = 16) and Crassostrea virginica (mean = 0.91; 95% CI of mean: 0.73–1.13; n = 20), are equal to 1; i.e., they were not potentiated. The potentiation of the gill muscles in both Mya and Spisula was seasonal like that in Mercenaria: potentiation was present during the winter but absent during the summer. Spisula and Mya gills contain immunoreactive nitric oxide synthase and soluble guanylate cyclase during the winter but not the summer, whereas Arctica and Crassostrea gills do not contain either enzyme. While potentiation in Mya and Spisula was abolished by the inhibition of nitric oxide synthase, inhibition of guanylate cyclase had no effect on potentiation. These results are consistent with the location of the enzymes: both are present in the gill filaments but not the gill muscles. Thus, NO must diffuse from the filaments to the muscles to induce potentiation.

Choline acetyltransferase-and nitric oxide synthase-containing neurons of the major pelvic ganglion projecting to the external urethral sphincter in rats

Objective To observe the choline acetyltransferase (ChAT)-and nitric oxide synthase ( NOS) -containing neurons of the major pelvic ganglion ( MPG) projecting to the external urethral sphincter (EUS). Methods Twenty-four Sprague-Dawley rats were divided into two groups: group A (n = 12) and group B (n = 12). In group A, the pelvic ganglia neurons innervating the EUS were labeled by retrograde axonal tracing methods with the use of horseradish peroxidase (HRP) injected into the EUS and identified with TMB-HRP reaction. Then immunohistochemical (ChAT) and histochemical methods ( NADPH-diaphorase activity and nitric oxide synthase immunoreactivity) were used to identify the neurotransmitters of HRP-labeled neurons projecting to the EUS. In group B, Malt agglutinator binding horseradish peroxidase (WGA-HRP) was injected into the MPG for antegrade tracing. Results In group A, HRP-labeled neurons with spherical or oval shape mainly distributed in the MPG. The HRP-labeled neurons were positve for ChAT (155/358, 43.3%) and NOS (49/339, 14.5%), respectively. In group B, HRP positive axon terminals were observed in the EUS after WGA-HRP was injectec into MPG. Conclusion ChAT and NOS-containing neurons of the major pelvic ganglion project to the ELS. Acetylcholine and nitric oxide may be as neurotransmitters or modulators to participate the procedure of urinary storage and voiding. Key words: Pelvic ganglion; External urethral sphincter; Choline acetyltransferase; Nitric oxide svnthase

Three‐dimensional distribution of NO sources in a primary mechanosensory integration center in the locust and its implications for volume signaling

Nitric oxide (NO) is an evolutionarily conserved mediator of neural plasticity. Because NO is highly diffusible, signals from multiple sources might combine in space and time to affect the same target. Whether such cooperative effects occur will depend on the effective signaling range and on the distances of NO sources to one another and to their targets. These anatomical parameters have been quantified in only few systems. We analyzed the 3D architecture of NO synthase (NOS) expression in a sensory neuropil, the ventral association center (VAC) of the locust. High-resolution confocal microscopy revealed NOS immunoreactive fiber boutons in submicrometer proximity to both the axon terminals of sensory neurons and their postsynaptic target, interneuron A4I1. Pharmacological manipulation of NO signaling affected the response of A4I1 to individual wind-puff stimuli and the response decrement during repetitive stimulation. Mapping NOS immunoreactivity in defined volumes around dendrites of A4I1 revealed NOS-positive fiber boutons within 5 mum of nearly every surface point. The mean distances between neighboring NOS-boutons and between any point within the VAC and its nearest NOS-bouton were likewise about 5 mum. For an NO signal to convey the identity of its source, the effective signaling range would therefore have to be less than 5 mum, and shorter still when multiple boutons release NO simultaneously. The architecture is therefore well suited to support the cooperative generation of volume signals by interaction between the signals from multiple active boutons.

Three‐dimensional distribution of no sources in a primary mechanosensory integration center in the locust and its implications for volume signaling

AbstractNitric oxide (NO) is an evolutionarily conserved mediator of neural plasticity. Because NO is highly diffusible, signals from multiple sources might combine in space and time to affect the same target. Whether such cooperative effects occur will depend on the effective signaling range and on the distances of NO sources to one another and to their targets. These anatomical parameters have been quantified in only few systems. We analyzed the 3D architecture of NO synthase (NOS) expression in a sensory neuropil, the ventral association center (VAC) of the locust. High‐resolution confocal microscopy revealed NOS immunoreactive fiber boutons in submicrometer proximity to both the axon terminals of sensory neurons and their postsynaptic target, interneuron A4I1. Pharmacological manipulation of NO signaling affected the response of A4I1 to individual wind‐puff stimuli and the response decrement during repetitive stimulation. Mapping NOS immunoreactivity in defined volumes around dendrites of A4I1 revealed NOS‐positive fiber boutons within 5 μm of nearly every surface point. The mean distances between neighboring NOS‐boutons and between any point within the VAC and its nearest NOS‐bouton were likewise about 5 μm. For an NO signal to convey the identity of its source, the effective signaling range would therefore have to be less than 5 μm, and shorter still when multiple boutons release NO simultaneously. The architecture is therefore well suited to support the cooperative generation of volume signals by interaction between the signals from multiple active boutons. J. Comp. Neurol. 518:2903–2916, 2010. © 2010 Wiley‐Liss, Inc.

Vasoprotective and Atheroprotective Effects of Angiotensin (1-7) in Apolipoprotein E–Deficient Mice

To evaluate the effectiveness of long-term angiotensin (Ang) (1-7) treatment to inhibit the progression of atherosclerosis in apolipoprotein E-deficient (ApoE(-/-)) mice. Ang (1-7) is a heptapeptide fragment that has been proposed to counterregulate the Ang II proatherogenic effects. The effect of long-term 4-week Ang (1-7) treatment on both inhibition of atherosclerotic lesion development and improvement of endothelial function was examined in apolipoprotein E(-/-) mice that had been fed an atherogenic high-fat (21%) diet for 16 weeks. Chronic Ang (1-7) treatment significantly improved endothelial function, an effect reversed with either angiotensin type 2 (AT(2)) or Mas receptor blockade. In these vessels, Ang (1-7) treatment significantly decreased superoxide production and increased endothelial nitric oxide synthase immunoreactivity when compared with vehicle treatment. These effects were blocked by both AT(2) and Mas receptor antagonists. Lesion development, assessed as both fatty deposits (oil red O) and intima to media ratio, was also significantly decreased with Ang (1-7) treatment compared with respective controls. Cotreatment with either AT(2) or Mas receptor antagonists reversed Ang (1-7)-mediated reduction in lesion development. Long-term Ang (1-7) treatment caused both vasoprotection, via improvement in endothelial function, and atheroprotection, with a reduction in lesion progression in a model of atherosclerosis. These effects appear to be mediated by the restoration of nitric oxide bioavailability and involve a complex interaction of both Mas and AT(2) receptors.

NADPH-Diaphorase Activity and NO Synthase Expression in the Olfactory Epithelium of the Bovine

NADPH-diaphorase (NADPH-d) staining of the bovine olfactory epithelium was compared with the immunohistochemical localization of nitric oxide synthase (NOS), soluble guanylyl cyclase, and cGMP (cyclic guanosine 3',5'-monophosphate). Out of the three isoforms, only the inducible NOS (NOS-II) was found at the epithelial surface correlating with the strong labelling for NADPH-d. In contrast, light diaphorase staining associated with deeper epithelial regions did not coincide with any NOS immunoreactivity. As there is overlapping expression of NOS-II, soluble guanylyl cyclase and cGMP at the luminal surface morphologically occupied by dendritic knobs of olfactory receptor neurons and microvillar endings of supporting cells, the nitric oxide (NO)/cGMP pathway is likely to be involved in modulating the odour signals during olfactory transduction.

Plasticity of nodose ganglion neurons after capsaicin- and vagotomy-induced nerve damage in adult rats

Previous reports show that vagal afferent innervation of the stomach eventually regenerates from surviving nodose ganglion (NG) neurons after subdiaphragmatic vagotomy. Systemic capsaicin treatment destroys gastric vagal afferent neurons expressing vanilloid receptor 1 (VR1). However, it is not known whether gastric innervation lost after neuronal destruction can be restored. Here, we report that capsaicin-induced damage of NG neurons innervating the stomach in adult rats is followed by restoration of vagal afferent projections. Specifically, we compared measures of neuronal plasticity in NG and vagi after subdiaphragmatic vagotomy or capsaicin treatment. The numbers of VR1-immunoreactive neurons projecting to the stomach were significantly reduced 10 days after either capsaicin treatment or vagotomy. However, the VR1-immunoreactive afferent innervation of the stomach was restored to levels exceeding those of vagotomized rats by 37 days after capsaicin, whereas neither total afferent innervation nor VR1-immunoreactive innervation reached control levels, even by 67 days after vagotomy. Capsaicin treatment significantly increased NG neuronal nitric oxide synthase (nNOS) immunoreactivity at 10 days after capsaicin, and this increase was sustained for the duration of the study, indicating higher nNOS demand in restoration of vagal projections. Vagotomy was associated with a much smaller increase in the number of nNOS-immunoreactive NG neurons, detectable only at 10 days after surgery. The number of nNOS-immunopositive gastric-projecting neurons was dramatically reduced 10 days after either capsaicin treatment or vagotomy but returned to the control level in both groups at 67 days. We found a significantly higher number of growth cones in capsaicin-treated animals compared with controls. Capsaicin significantly increased the number of nNOS-immunopositive and nNOS-immunonegative growth cones in NG at all time points. Vagotomy did not increase the number of nNOS− growth cones in NG. We conclude that capsaicin treatment may result in more significant restorative capacities than vagotomy, mainly because of sprouting of capsaicin-insensitive nerve fibers.

Acute electroacupuncture inhibits nitric oxide synthase expression in the spinal cord of neuropathic rats

Objectives: To examine the effects of electroacupuncture stimulation on behavioral changes and neuronal nitric oxide synthase expression in the rat spinal cord after nerve injury.Methods: Under pentobarbital anesthesia, male Sprague–Dawley rats were subjected to neuropathic surgery by tightly ligating and cutting the left tibial and sural nerves. Behavioral responses to mechanical stimulation were tested for 2 weeks post-operatively. At the end of behavioral testing, electroacupuncture stimulation was applied to ST36 (Choksamni) and SP9 (Eumleungcheon) acupoints. Immunocytochemical staining was performed to investigate changes in the expression of neuronal nitric oxide synthase-immunoreactive neurons in the L4–5 spinal cord.Results: Mechanical allodynia was observed by nerve injury. The mechanical allodynia was decreased after electroacupuncture stimulation. Neuronal nitric oxide synthase expression was also decreased in L4–5 spinal cord by electroacupuncture treatment.Discussion: These results suggest that electroacupuncture relieves mechanical allodynia in the neuropathic rats possibly by the inhibition of neuronal nitric oxide synthase expression in the spinal cord.

Protective effects of cold spinoplegia with fasudil against ischemic spinal cord injury in rabbits

Paraplegia remains a serious complication after surgical repair of thoracoabdominal aortic aneurysms. The aim of this study was to evaluate the neuroprotective efficacy of fasudil, a Rho kinase (ROCK) inhibitor, by reducing the number of infiltrating cells in the ventral horn and increasing the induction of eNOS against ischemic spinal cord injury in rabbits. Eighteen Japanese white rabbits were divided into three groups: saline (group 1, n = 7, 4 degrees C) and fasudil (group 2, n = 6, 4 degrees C) were immediately infused into the isolated segmental lumbar arteries over 30 seconds after aortic clamping. Group 3 (n = 5) was the sham-operated group. Hind limb function was evaluated 4 and 8 hours, and 1 and 2 days after 15 minutes of transient ischemia. Cell damage was analyzed by hematoxylin and eosin staining and temporal profiles of endothelial nitric oxide synthase immunoreactivity were performed. The number of intact motor neuron cells and infiltrating cells in the ventral horn were compared. Two days after reperfusion, group 2 and group 3 showed better neurologic function, a greater number of intact motor neuron cells, and a smaller number of infiltrating cells in the ventral horn than group 1. The induction of endothelial nitric oxide synthase (eNOS) was prolonged up to 2 days after reperfusion in group 2. These results indicate that fasudil has neuroprotective effects against ischemic spinal cord injury in rabbits by reducing the number of infiltrating cells in the ventral horn and prolonging the expression of eNOS.

Changes in contractile and electrical activity in the ileum of DSS-induced colitis model W/Wv mutant mice

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. 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 (-)). 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. 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.

Extrinsic afferents supplying the ovine duodenum and ileum

The study aimed at establishing the distribution of primary sensory neurons by means of retrograde tracers Diamidino Yellow (DY) and Fast Blue (FB) injected into both the sheep duodenum and ileum, respectively. Many DY-labelled cells were found in both the distal vagal ganglia (DVG) and the spinal ganglia (SG) from T9-L3; on the contrary, the majority of the FB-labelled cells were found in the SG. In the SG, a double immunofluorescence stain was used to reveal Nitric Oxide Synthase-Immunoreactivity (NOS-IR) in association with: substance P (SP), calcitonin gene-related peptide (CGRP), neurofilament 200kDa (NF) and isolectin B(4) (IB4). The labelled neurons, both DY and FB generally ranged in size from medium to large. The majority of the SG duodenal projections were NOS negative; the majority of the SG ileal afferent neurons expressed NOS-IR. Both DY and FB NOS-IR neurons often co-localized IB4, CGRP and SP, but rarely NF.

Carotid body and cardiorespiratory alterations in intermittent hypoxia: the oxidative link

Intermittent hypoxia, a feature of obstructive sleep apnoea, potentiates ventilatory hypoxic responses, alters heart rate variability and produces hypertension, partially owing to an enhanced carotid body responsiveness to hypoxia. Since oxidative stress is a potential mediator of both chemosensory and cardiorespiratory alterations, we hypothesised that an antioxidant treatment may prevent these alterations. Accordingly, we studied the effects of ascorbic acid (1.25 g.L(-1) drinking water) on plasma lipid peroxidation, nitrotyrosine and inducible nitric oxide synthase (iNOS) immunoreactivity in the carotid body, ventilatory and carotid chemosensory responses to acute hypoxia, heart rate variability and arterial blood pressure in male Sprague-Dawley rats exposed to 5% O(2); 12 episodes.h(-1); 8 h.day(-1) or sham condition for 21 days. Intermittent hypoxia increased plasma lipid peroxidation, nitrotyrosine and iNOS expression in the carotid body, enhanced carotid chemosensory and ventilatory hypoxic responses, modified heart rate variability and produced hypertension. Ascorbic acid prevented the increased plasma lipid peroxidation and nitrotyrosine formation within the carotid body, and the enhanced carotid chemosensory and ventilatory responses to hypoxia, as well as heart rate variability alterations and hypertension. The present results support an essential role for oxidative stress in the generation of carotid body chemosensory potentiation and systemic cardiorespiratory alterations induced by intermittent hypoxia.

The Effects of Beta-blockers on Endothelial Nitric Oxide Synthase Immunoreactivity in the Rat Corpus Cavernosum

To explain the mechanism of the effects of beta-blockers on endothelial dysfunction and release of nitric oxide from the endothelium. A total of 72 Sprague-Dawley rats were divided into 9 different groups as follows: group 1: control (n = 10), group 2: metoprolol (Beloc) 100 mg/kg/d (n = 7), group 3: carvedilol (Dilatrend) 50 mg/kg/d (n = 7), group 4: nebivolol (Vasoxen) 10 mg/kg/d (n = 6), group 5: estrogen receptor (ER) antagonist ICI 182.780 (Fluvestrant) 50 microg/g (n = 10), group 6: nebivolol+ER antagonist (n = 8), group 7: androgen receptor (AR) antagonist (flutamide) 20 mg/kg (n = 7), group 8: nebivolol+AR antagonist (n = 7), and group 9: DMSO (solvent for ER antagonist) (n = 10). All beta-blockers were applied with gastric gavage after dilution with 5 mL of serum physiological; ER and AR were both applied intraperitoneally (i.p.) for 14 days. In the isolated rat cavernous tissues, endothelial nitric oxide synthase (eNOS) and ER and AR immunoreactivity were analyzed quantitatively. One-way analysis of variance and Tukey test were used for statistical analysis. Although increased eNOS immunoreactivity was observed with nebivolol and nebivolol-flutamide in endothelial cells laying cavernous tissue, a lower score was observed after ICI-182.780 application, when compared with control cases. AR immunoreactivity in cavernosal endothelium was clearly higher with nebivolol. Higher H score and ER immunoreactivity were observed in the cavernous endothelium and smooth muscles in the nebivolol, carvedilol, and metoprolol groups when compared with control cases. We showed that eNOS activity was increased in the nebivolol and nebivolol-flutamide groups, whereas it was decreased in the ICI 182.780 group. We believe that an ER-dependent mechanism triggered by nebivolol played a role in nitric oxide formation.

Melatonin and Vitamin C Attenuates Alcohol‐Induced Oxidative Stress in Aorta

Epidemiological studies have shown that low to moderate doses of alcohol consumption are beneficial to cardiac health. However, chronic high doses of alcohol ingestion cause cardiovascular complications. The aim of this study was to investigate both the effects of melatonin and vitamin C and expression of endothelial nitric oxide synthase in aorta of chronic alcoholic rats. Twenty-four adult male Wistar rats weighing 200-250 g were used in the study. Rats were divided into four equal groups. Group I (control): rats were not fed on alcohol; Group II: rats were fed on alcohol; Group III: rats were fed on alcohol and 40 mg/kg vitamin C were injected intraperitoneally and Group IV: rats were fed on alcohol and 4 mg/kg melatonin were injected intraperitoneally. At the end of the experiment, rats were killed and aorta tissues were removed. Some parts of the aorta tissues were used for biochemical analyses and the other parts were used at histological procedures. In the control group, endothelial nitric oxide synthase immunoreactivity was (++) in smooth muscle cells and endothelial cells. Expression of endothelial nitric oxide synthase in the alcohol group was stronger than control group. Chronic ethanol ingestion significantly increased (p < 0.05); and melatonin significantly decreased both the plasma and tissue malondialdehyde levels. The superoxide dismutase levels and catalase activity did not change significantly in the Vitamin C and melatonin groups (p > 0.05) compared to the control and alcohol groups. The present results indicate that chronic alcohol consumption increase lipid peroxidation and cause endothelial nitric oxide synthase expression in the aorta. However, melatonin and vitamin C administration provide partial protection against alcohol-induced damage.