Number Of Nitric Oxide Synthase Research Articles

Nitrergic Enteric Neurons in Health and Disease-Focus on Animal Models.

Nitrergic enteric neurons are key players of the descending inhibitory reflex of intestinal peristalsis, therefore loss or damage of these neurons can contribute to developing gastrointestinal motility disturbances suffered by patients worldwide. There is accumulating evidence that the vulnerability of nitrergic enteric neurons to neuropathy is strictly region-specific and that the two main enteric plexuses display different nitrergic neuronal damage. Alterations both in the proportion of the nitrergic subpopulation and in the total number of enteric neurons suggest that modification of the neurochemical character or neuronal death occurs in the investigated gut segments. This review aims to summarize the gastrointestinal region and/or plexus-dependent pathological changes in the number of nitric oxide synthase (NOS)-containing neurons, the NO release and the cellular and subcellular expression of different NOS isoforms. Additionally, some of the underlying mechanisms associated with the nitrergic pathway in the background of different diseases, e.g., type 1 diabetes, chronic alcoholism, intestinal inflammation or ischaemia, will be discussed.

Tenuigenin ameliorates learning and memory impairments induced by ovariectomy

Estrogen deficiency is associated with cognitive impairment. Hormone replacement therapy (HRT) has proven to be effective in preventing and reversing the memory and learning deficiencies. However, conventional estrogenic treatment could increase the risks of breast cancer and venous thromboembolism. Tenuigenin (TEN) is putatively believed as the active component extracted from a Chinese herb Polygala tenuifolia root. Although TEN has been shown to enhance learning and memory in healthy mice, it remains unknown whether or not TEN could ameliorate learning and memory impairments. In the present study, mice were divided into four groups: sham-operated (sham), ovariectomized (OVX), OVX+estradiol benzoate (EB) and OVX+TEN groups. Step-through passive avoidance and Y-maze tests were used to assess learning and memory abilities, and the number of nitric oxide synthase (NOS) positive neurons and the synaptic measurement of hippocampal CA1 area were examined. The results showed that TEN was given orally to OVX mice, leading to the improvement of learning and memory in step-through passive avoidance and Y-maze tests. TEN could reduce the loss of NOS positive neurons and prevent the synaptic morphological changes induced by ovariectomy. Our results suggest that TEN may exert a potential therapeutic value for menopause cognitive dysfunction.

Delayed testosterone replacement restores nitric oxide synthase-containing nerve fibres and the erectile response in rat penis.

To elucidate the effect of testosterone on penile innervation. Materials and methods Three groups of six rats each were assessed; two groups (1 and 2) were castrated and the third (group 3) underwent a sham operation (control). Eight weeks after castration, group 2 received a subcutaneous injection with testosterone. At 8 weeks, the rats in group 1 and 3 underwent a final functional analysis while those in group 2 did so at 12 weeks. The evaluation included a subcutaneous injection with apomorphine to study centrally mediated erection, and cavernosal nerve electrostimulation and papaverine injection to study peripherally mediated erection. At death a penile mid-shaft specimen was taken for NADPH-diaphorase staining. In the apomorphine study, castration resulted in significantly fewer yawns and erections than in the control, and those in group 2 significantly better central erectile function than in the controls. The mean (SEM) number of nitric oxide synthase (NOS)-containing nerve fibres in the corpora cavernosa and both dorsal nerves of castrated rats, at 46.2 (9.1) and 203 (32.1), respectively, were significantly lower than in rats in group 2, at 84.1 (11.2) and 300.6 (17.1), and than in the controls, at 88.6 (10.9) and 306.3 (22.9), respectively. The intracavernosal pressure decreased significantly in the absence of testosterone, both after electrostimulation and intracavernosal papaverine injection. However, there was no difference between the control and group 2 rats in either the number of NOS-containing nerve fibres or in the peripheral erectile functional study. Testosterone acts on the nervous system to mediate erection; when it is absent there may be down-regulation of both the production and activity of NO, thereby decreasing the response to peripheral stimulation via the NO pathway. The restoration of erectile function seen in rats in group 2 supports this phenomenon. Delayed testosterone replacement has no detrimental effect on the restoration of the erectile mechanism after castration.

Steroid synthesis and metabolism in the nervous system: trophic and protective effects.

Steroids influence the activity and plasticity of neurons and glial cells during early development, and they continue to exert trophic and protective effects in the adult nervous system. Steroids are produced by the gonads and adrenal glands and reach the brain, the spinal cord and the peripheral nerves via the bloodstream. However, some of them, named "neurosteroids", can also be synthesized within the nervous system. They include pregnenolone, progesterone, dehydroepiandrosterone and their reduced metabolites and sulfate esters. Little is known concerning the regulation of steroid synthesis in the nervous system, which involves interactions between different cell types. For example, the synthesis of progesterone by Schwann cells in peripheral nerves is regulated by a diffusible neuronal signal. Neurotrophic and neuroprotective effects of steroids have been documented both in cell culture and in vivo. PROG plays an important role in the neurological recovery from traumatic injury of the brain and spinal cord by mechanisms involving protection from excitotoxic cell death, lipid peroxydation and the induction of specific enzymes. After transection of the rat spinal cord, PROG increases the number of nitric oxide synthase expressing astrocytes immediately above and below the lesion. PROG also plays an important role in the formation of new myelin sheaths. This has been shown in the regenerating mouse sciatic nerve after lesion and in cocultures of sensory neurons and Schwann cells. PROG promotes myelination by activating the expression of genes coding for myelin proteins. The modulation of neurostransmitter receptors, in particular the type A gamma-aminobutyric acid, the N-methyl-D-aspartate and the sigma 1 receptors, is involved in the psychopharmacological effects of steroids and allows to explain their anticonvulsant, anxiolytic, antidepressive and sedative effects as well as their influence on memory. Pregnenolone sulfate has been shown to reverse age-related deficits in spatial memory performance and to have protective effects on memory in different models of amnesia.

Colonic enteric nervous system in patients with familial amyloidotic neuropathy.

The colonic enteric nervous system was investigated in autopsy specimens from 12 patients with familial amyloidotic neuropathy (FAP) and 9 controls. The infiltration of amyloid deposits in the enteric nervous system was studied by double staining for amyloid and nerve elements. The myenteric plexus was immunostained for protein gene product (PGP) 9.5, vasoactive intestinal peptide (VIP), substance P and nitric oxide synthase (NOS). The immunostained nerve elements were quantified by computerised image analysis. Double staining revealed that there was no amyloid infiltration in the ganglia, or in the nerve fibres in the colonic enteric nervous system of FAP patients. The relative volume density of PGP 9.5-immunoreactive nerve fibres in both the circular and the longitudinal muscle layers in FAP patients did not differ significantly from that of controls. The relative volume density of VIP-immunoreactive nerve fibres in the circular muscle layer was significantly decreased in FAP patients compared with controls, but not in the longitudinal layer. The number of VIP-immunoreactive neurons/mm2 myenteric ganglia was significantly decreased in FAP patients. There were no statistical differences in the relative volume density for substance P- and NOS-immunoreactive nerve fibres between FAP patients and controls, nor was there any difference between FAP patients and controls regarding the number of NOS- and substance P-immunoreactive neurons/mm2 myenteric ganglia. It is concluded that the colonic enteric nervous system as a whole is intact and is not damaged by amyloid infiltration. The present observation of a reduction of VIP-immunoreactive nerve fibres and neurons in myenteric plexus of FAP patients might be one of the factors that contribute to the motility disorders seen in FAP patients.

EFFECT OF CAVERNOUS NERVE FREEZING ON PROTEIN AND GENE EXPRESSION OF NITRIC OXIDE SYNTHASE IN THE RAT PENIS AND PELVIC GANGLIA

Purpose Cryoablation of the prostate has been reported to induce impotence as a result of cavernous nerve injury. This study is designed to investigate the early and late effects of cavernosal nerve cryoablation on erectile function and nitric oxide synthase (NOS) containing nerves in the rat penis and pelvic ganglia. Materials and Methods Forty male rats were divided into two groups (n = 20 each). The first group underwent unilateral cavernous nerve freezing (experimental group). Before their euthanization at 1 and 3 months (10 rats each), erectile function was assessed by electrostimulation of the cavernous nerves. The second group served as a control and was euthanized at the same time points. Nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase staining was used to identify NOS in the penile nerve fibers of the mid-shaft segment and neurons of the pelvic ganglia. Western blot, RT-PCR and immunostaining techniques were used to identify protein and gene expression of eNOS, iNOS, and nNOS. Results One month after unilateral cavernosal nerve freezing, there was a significant decrease in NOS-containing nerve fibers in the dorsal and intracavernosal nerves and ipsilateral pelvic ganglia as compared with the intact side. At three months, the number of NOS containing nerve fibers in the above-mentioned nerves and ganglia showed a significant increase on the frozen side but not the intact side. Electrostimulation of the frozen nerve after three months revealed a significantly higher maximal intracavernosal pressure and a shorter latency period than the one-month group. At three months, immunoblot showed up-regulation of eNOS and nNOS protein expression in the penile tissue. RT-PCR showed up-regulation of nNOS gene expression in the pelvic ganglia of the frozen side. Immunostaining confirmed the results of western blot and showed significant increase of the nNOS positive staining in the frozen side of the penile tissue after three months. There was no difference in iNOS after three months between both sides. Our repeated eNOS immunostaining was not successful. Conclusions The results show that intracavernous pressure response to neurostimulation markedly decreased at one month and then partially recovered three months after cavernosal nerve freezing. A similar pattern of changes of the NOS-containing nerve fibers in dorsal nerves, intracavernosal nerves, and pelvic ganglia were observed. This alteration in erectile function and NOS-containing nerves was associated with differential gene and protein expression of the three subtypes of NOS.

Effect of cavernous nerve freezing on protein and gene expression of nitric oxide synthase in the rat penis and pelvic ganglia.

Cryoablation of the prostate has been reported to induce impotence as a result of cavernous nerve injury. This study is designed to investigate the early and late effects of cavernosal nerve cryoablation on erectile function and nitric oxide synthase (NOS) containing nerves in the rat penis and pelvic ganglia. Forty male rats were divided into two groups (n = 20 each). The first group underwent unilateral cavernous nerve freezing (experimental group). Before their euthanization at 1 and 3 months (10 rats each), erectile function was assessed by electrostimulation of the cavernous nerves. The second group served as a control and was euthanized at the same time points. Nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase staining was used to identify NOS in the penile nerve fibers of the mid-shaft segment and neurons of the pelvic ganglia. Western blot, RT-PCR and immunostaining techniques were used to identify protein and gene expression of eNOS, iNOS, and nNOS. One month after unilateral cavernosal nerve freezing, there was a significant decrease in NOS-containing nerve fibers in the dorsal and intracavernosal nerves and ipsilateral pelvic ganglia as compared with the intact side. At three months, the number of NOS containing nerve fibers in the above-mentioned nerves and ganglia showed a significant increase on the frozen side but not the intact side. Electrostimulation of the frozen nerve after three months revealed a significantly higher maximal intracavernosal pressure and a shorter latency period than the one-month group. At three months, immunoblot showed up-regulation of eNOS and nNOS protein expression in the penile tissue. RT-PCR showed up-regulation of nNOS gene expression in the pelvic ganglia of the frozen side. Immunostaining confirmed the results of western blot and showed significant increase of the nNOS positive staining in the frozen side of the penile tissue after three months. There was no difference in iNOS after three months between both sides. Our repeated eNOS immunostaining was not successful. The results show that intracavernous pressure response to neurostimulation markedly decreased at one month and then partially recovered three months after cavernosal nerve freezing. A similar pattern of changes of the NOS-containing nerve fibers in dorsal nerves, intracavernosal nerves, and pelvic ganglia were observed. This alteration in erectile function and NOS-containing nerves was associated with differential gene and protein expression of the three subtypes of NOS.

Hippocampal nitric oxide synthase in the fetal guinea pig: effects of chronic prenatal ethanol exposure

The effects of chronic maternal administration of ethanol on nitric oxide synthase (NOS) activity and the numbers of NOS containing neurons, and CA1 and CA3 pyramidal neurons in the hippocampus of the near term fetal guinea pig at gestational day (GD) 62 were investigated. Pregnant guinea pigs received oral administration of 4 g ethanol/kg maternal body weight ( n=5), isocaloric sucrose/pair feeding ( n=5) or water ( n=5), or no treatment (NT; n=5) from GD 2 to GD 61. NOS activity in the 25,000× g supernatant of hippocampal homogenate was determined using a radiometric assay. The numbers of NOS containing neurons, and CA1 and CA3 pyramidal neurons were determined using NADPH diaphorase histochemistry and cresyl violet staining, respectively. The chronic ethanol regimen produced a maternal blood ethanol concentration of 193±13 mg/dl at 1 h after the second divided dose on GD 57. Chronic ethanol exposure produced fetal body, brain, and hippocampal growth restriction and decreased fetal hippocampal NOS activity compared with the isocaloric sucrose/pair feeding, water, and NT experimental groups, but did not affect the number of NOS containing and CA1 or CA3 pyramidal neurons. These data demonstrate that, in the near term fetus, chronic maternal administration of ethanol suppresses hippocampal NOS activity and consequent formation of NO, without loss of NOS containing neurons and prior to loss of CA1 pyramidal neurons that occurs in the adult.

Impaired expression of nitric oxide synthase in the gastric myenteric plexus of spontaneously diabetic rats

BACKGROUND & AIMS: The mechanism responsible for defective gastric accommodation in diabetes is unknown. The aim of this study was to investigate if this abnormality is due to a defective nitric oxide pathway secondary to impaired nitric oxide synthase (NOS) expression in the gastric myenteric plexus.METHODS: To test this hypothesis, we studied nonadrenergic, noncholinergic (NANC) relaxation, NOS activity, NADPH diaphorase histochemistry, NOS immunohistochemistry, NOS immunoblotting, and NOS messenger RNA expression in the gastric myenteric plexus of spontaneously diabetic biobreeding/Worcester (BB/W) rats. Age-matched nondiabetic Wistar rats were used as controls.RESULTS: Gastric neuromuscular preparations from control rats showed a frequency-dependent NANC relaxation in response to transmural stimulation. This relaxation was markedly antagonized by N(G)-nitro-L- arginine-methyl ester, indicating mediation by the neuronal release of NO. The NANC relaxation in gastric muscle preparations obtained from diabetic BB/W rats was significantly impaired. The number of NOS- immunoreactive cells in the gastric myenteric plexus and the NOS activity were significantly reduced in diabetic BB/W rats, suggesting that NOS synthesis is impaired in diabetes. Northern blot analysis showed that the density of NOS messenger RNA bands at 9.5 kilobases was significantly reduced in the gastric tissues of diabetic BB/W rats.CONCLUSIONS: These results indicate that gastric relaxation in diabetics is hampered mainly by impaired NOS expression in the gastric myenteric plexus.(Gastroenterology 1997 Nov;113(5):1535-44)

Distribution of nitric oxide synthase in rat dorsal vagal complex and effects of microinjection of nitric oxide compounds upon gastric motor function.

Nitric oxide (NO) has received attention as a vagal nonadrenergic-noncholinergic (NANC) mediator of gastrointestinal relaxation. The dorsal vagal complex (DVC) is the primary hindbrain site of vagal control of the gastrointestinal tract, and yet the subnuclear distribution of NO and its physiological effects have not been analyzed in this nucleus. Therefore, this study estimates the relative number of NO synthase (NOS)-containing neurons in subnuclear regions of the DVC, identifies NOS-containing vagal abdominal preganglionic neurons in the dorsal motor nucleus of the vagus, and defines a role of NO in the DVC in control of gastric motor function. The location of NADPH-diaphorase-positive staining (a marker of NOS activity) and NOS immunoreactivity overlap in the DVC. In the dorsal motor nucleus of the vagus there are positively stained cells caudal to the obex and at its most rostral extent, but not at the intermediate level. Intraperitoneal fluorogold combined with NADPH-diaphorase activity labels approximately 5% and 15% of fluorogold-immunoreactive cells in the caudal and rostral dorsal motor nucleus of the vagus, respectively. Thus, a portion of NOS-containing neurons are preganglionic vagal neurons projecting to the abdominal viscera. In the nucleus tractus solitarius, the majority of NADPH-diaphorase-positive cells are within the centralis, medial, and ventral/ventrolateral subnuclei. Fiber/terminal staining is present in the subnucleus centralis, subnucleus gelatinosus, subpostremal zone, and the medial nucleus tractus solitarius. The presence of NOS terminal staining implicates NO in afferent control of gastric function in the DVC (e.g., vago-vagal circuits in subnucleus gelatinosus). To determine a role of NO in the DVC, NO-related agents were microinjected into the DVC in alpha-chloralose-anesthetized rats while recording indices of gastric motor function. L-Arginine, microinjected into the DVC, significantly decreases intragastric pressure (-2.2 +/- 0.4 cm2, N = 12), and this effect is abolished by vagotomy. Microinjection of an NOS inhibitor, NG-nitro-L-arginine methyl ester, increases intragastric pressure (1.9 +/- 0.7 cm2, N = 10), with the greatest effect in the DVC rostral to the obex. Overall, it was concluded that tonic release of NO in the DVC mediates gastric relaxation, at least in anesthetized animals, and NOS-containing preganglionic neurons in the dorsal motor nucleus of the vagus may be "command" NANC neurons which control a variety of gastrointestinal functions.