nNOS mRNA Expression Research Articles

Effects of octreotide on nitric oxide synthase expression in the small intestine of high fat diet-induced obese rats

To investigate whether obesity induced by high fat diet is associated with expression of neuronal, endothelial, and inducible nitric oxide synthase (nNOS, eNOS, and iNOS) in the intestine, and to test the effects of the somatostatin analog octreotide on this expression. The study included high fat diet-induced obese and normal control rats. The obese rats were further separated into an obese control group and an octreotide intervention group. Rats in the intervention group were injected with 40 μg/kg octreotide every 12 h for 8 days. Expressions of nNOS, eNOS, and iNOS in the small intestine were analyzed by RT-PCR and immunohistochemistry. The NO level of small intestinal homogenate was measured with an ELISA kit. The body weight; Lee's index; small intestinal eNOS and iNOS mRNA and protein expression levels; nNOS protein expression levels; and small intestinal homogenate NO levels were all significantly higher in the obese control group than in the normal controls (p < 0.01); nNOS mRNA expression was also higher in the obese control group, but not significantly so. Octreotide intervention significantly reduced the body weight and small intestinal homogenate NO level of the obese rats relative to the obese control group (p < 0.05). The mRNA and protein expression levels of eNOS and iNOS; the protein expression level of nNOS in the small intestine were also significantly lower in the octreotide intervention group than in the obese control group (p < 0.01), while nNOS mRNA expression was lower but not significantly so. High fat diet-induced obesity is associated with elevated small intestinal nNOS, eNOS, and iNOS expression levels. Octreotide treatment can inhibit nNOS, eNOS, and iNOS expression and lead to weight loss.

Increased proportion of nitric oxide synthase immunoreactive neurons in rat ileal myenteric ganglia after severe acute pancreatitis

BackgroundSevere acute pancreatitis (SAP) remains a potentially life-threatening disease. Gastrointestinal motility disturbance such as intestinal ileus is seen in every case. By now, the mechanisms of pancreatitis-induced ileus are largely unknown. The main purpose of the present study was to observe changes of nitric oxide synthase-immunoreactive (NOS-IR) neurons in ileal myenteric ganglia in SAP rats with gastrointestinal dysmotility, trying to explore underlying nervous mechanisms of pancreatitis-induced ileus.MethodsTwenty Sprague Dawley rats were randomly divided into sham operated group and SAP group. SAP was induced by retrograde cholangiopancreatic duct injection of 5% sodium taurocholate. Abdominal X-ray and intestinal transit were performed to detect the existence of paralytic ileus and intestinal dysmotility. Pathological damage of pancreas was evaluated. Double-immunolabeling was employed for the whole-mount preparations of ileal myenteric ganglia. The morphology of NOS-IR neurons were observed and the percentage of NOS-IR neurons was calculated based on the total Hu-immunoreactive neurons. Total RNA of ileum was extracted according to Trizol reagent protocol. Neuronal NOS (nNOS) mRNA expression was evaluated by RT-PCR.ResultsThe small intestinal transit index in the SAP group was significantly lower compared with the sham operated group (29.21 ± 3.68% vs 52.48 ± 6.76%, P <0.01). The percentage of NOS-IR neurons in ileal myenteric ganglia in the SAP group was significantly higher than that in the sham operated group (37.5 ± 12.28% vs 26.32 ± 16.15%, P <0.01). nNOS mRNA expression in ileum of SAP group was significantly higher than that in the sham operated group (1.02 ± 0.10 vs 0.70 ± 0.06, P < 0.01).ConclusionsThe increased quantity of NOS-IR neurons in ileal myenteric ganglia and increased nNOS mRNA expression may suggest nNOS over expression as one of the nervous mechanisms of gastrointestinal dysmotility in SAP rat.

Oxidative Stress in Perinatal Asphyxia and Fetal Asphyctic Preconditioning

Background: Asphyctic preconditioning is thought to attenuate the cellular stress response which reduces brain damage. Objective: We evaluated the expression of stress related genes in pre- and neonatal pups after fetal preconditioning (FA) and perinatal asphyxia (PA). Methods: FA was induced on E17 by clamping the uterine circulation for 30'. On P0 PA was induced by placing the fetuses-containing uterine horns in water for 19'. Control and FA pups were sacrificed 96h after the FA insult. Control, FA, PA and FA+PA pups were sacrificed 6h (P0) and 96h (P4) after birth. mRNA expression of iNos, nNos and Sod 1 was evaluated in the right hemisphere with qRT-PCR. Results: Sod1 was significant down-regulated (p=0.0057) on E21 for the FA group compared to controls. On P4 both the PA and the FA+PA group were down-regulated when compared to controls (p= 0.0021, p=0.0132) and compared to the FA group (p=0.0007, p=0.0297). On P4 there was a significant difference between the PA and the FA+PA groups (p=0.0005) (fig. 1). nNos expression revealed a significant increase on P0 for the PA and FA+PA groups compared to the FA group (p=0.0012, p=0.0145). iNos showed no significant differences. Conclusions: Modulation of the expression of antioxidants like Sod1 by FA could contribute to neuroprotection at birth. Additional whole-genome expression will provide a complete overview of the preconditioned phenotype.

Exercise Training Improves the Defective Centrally Mediated Erectile Responses in Rats with Type I Diabetes

Erectile dysfunction is a serious and common complication of diabetes mellitus. Apart from the peripheral actions, central mechanisms are also responsible for the penile erection. The goal of the present study was to determine the impact of exercise training (ExT) on the centrally mediated erectile dysfunction in streptozotocin (STZ)-induced type I diabetic (T1D) rats. Male Sprague-Dawley rats were injected with STZ to induce diabetes mellitus. Three weeks after STZ or vehicle injections, rats were assigned to either ExT (treadmill running for 3-4 weeks) or sedentary groups to produce four experimental groups: control + sedentary, T1D + sedentary, control + ExT, and T1D + ExT. After 3-4 weeks ExT, central N-methyl-D-aspartic acid (NMDA) or sodium nitroprusside (SNP)-induced penile erectile responses were measured. Neuronal nitric oxide synthase (nNOS) expression in the paraventricular nucleus (PVN) of the hypothalamus was measured by using histochemistry, real time polymerase chain reaction (PCR) and Western blot approaches. In rats with T1D, ExT significantly improved the blunted erectile response, and the intracavernous pressure changes to NMDA (50 ng) microinjection within the PVN (T1D + ExT: 3.0 ± 0.6 penile erection/rat; T1D + sedentary: 0.5 ± 0.3 penile erection/rat within 20 minutes, P < 0.05). ExT improved erectile dysfunction induced by central administration of exogenous nitric oxide (NO) donor, SNP in T1D rats. Other behavior responses including yawning and stretching, induced by central NMDA and SNP microinjection were also significantly increased in T1D rats after ExT. Furthermore, we found that ExT restored the nNOS mRNA and protein expression in the PVN in T1D rats. These results suggest that ExT may have beneficial effects on the erectile dysfunction in diabetes through improvement of NO bioavailability within the PVN. Thus, ExT may be used as therapeutic modality to up-regulate nNOS within the PVN and improve the central component of the erectile dysfunction in diabetes mellitus.

Role of neuronal nitric oxide synthase in colonic distension-induced hyperalgesia in distal colon of neonatal maternal separated male rats

Nitric oxide (NO) is implicated in the pathogenesis of irritable bowel syndrome (IBS) but the underlying mechanism is unclear. Thus, the aim of the present study is to examine the role of NO synthase (NOS) expression in the distal colon of neonatal maternal separation (NMS) model rats employed in IBS studies. Male neonates of Sprague-Dawley rats were randomly assigned into NMS and normal control (N) groups. Rats of NMS group were subjected to 3 h daily maternal separation on postnatal day 2-21. Rats were administrated non-selective NOS inhibitor l-NAME (100 mg kg(-1) ), selective neuronal NOS (nNOS) inhibitor 7-NINA (10mgkg(-1) ), selective inducible NOS (iNOS) inhibitor, endothelial NOS (eNOS) inhibitor (10mgkg(-1) ) or Vehicle (Veh; distilled water) intraperitoneally 1h prior to the experiment for the test and control groups, respectively. The amount of NO was significantly higher in the NMS Veh rats compared with unseparated N rats. Western-blotting and real-time quantitative PCR studies showed that protein and mRNA expression of nNOS were higher in the NMS group than that in the N rats; whereas no significant change in iNOS and eNOS was found in either groups. Neonatal maternal separation Veh rats showed low pain threshold and increased electromyogram (EMG) activity in response to colonic distension stimuli. l-NAME and 7-Nitroindazole monosodium salt (7-NINA) increased pain threshold pressure and attenuated EMG activity in the NMS rats. In addition, l-NAME and 7-NINA substantially reduced oxidative marker malondialdehyde level in NMS rats. Neonatal maternal separation increased the NO generation by nNOS upregulation that interact with reactive oxygen species contributing to the visceral hypersensitivity in IBS.

Leptin Induces Phosphorylation of Neuronal Nitric Oxide Synthase in Defined Hypothalamic Neurons

Studies have indicated that the neurotransmitter nitric oxide (NO) mediates leptin's effects in the neuroendocrine reproductive axis. However, the neurons involved in these effects and their regulation by leptin is still unknown. We aimed to determine whether NO neurons are direct targets of leptin and by which mechanisms leptin may influence neuronal NO synthase (nNOS) activity. Nicotinamide adenine dinucleotide phosphate diaphorase activity and leptin-induced phosphorylation of signal transducer and activator of transcription-3 immunoreactivity were coexpressed in subsets of neurons of the medial preoptic area, the paraventricular nucleus of the thalamus, the arcuate nucleus (Arc), the dorsomedial nucleus of the hypothalamus (DMH), the posterior hypothalamic area, the ventral premammillary nucleus (PMV), the parabrachial nucleus, and the dorsal motor nucleus of the vagus nerve. Fasting blunted nNOS mRNA expression in the medial preoptic area, Arc, DMH, PMV, and posterior hypothalamic area, and this effect was not restored by acute leptin administration. No difference in the number of neurons expressing nNOS immunoreactivity was noticed comparing hypothalamic sections of fed (wild type and ob/ob), fasted, and fasted leptin-treated mice. However, we found that in states of low leptin levels, as in fasting, or lack of leptin, as in ob/ob mice, the number of neurons expressing the phosphorylated form of nNOS is decreased in the Arc, DMH, and PMV. Notably, acute leptin administration to fasted wild-type mice restored the number of phosphorylated form of nNOS neurons to that observed in fed wild-type mice. Herein we identified the first-order neurons potentially involved in NO-mediated effects of leptin and demonstrate that leptin regulates nNOS activity predominantly through posttranslational mechanisms.

Nitric oxide synthase (NOS) in the trigeminal vascular system and other brain structures related to pain in rats

Nitric oxide (NO) is considered to be a key mediator in the pathophysiology of migraine but the localisation of NO synthesizing enzymes (NOS) throughout the pain pathways involved in migraine has not yet been fully investigated. We have used quantitative real-time PCR and Western blotting to measure the respective levels of mRNA and protein for nNOS and eNOS in peripheral and central tissues involved in migraine pain: dura mater, pial arteries, trigeminal ganglion (TG) trigeminal nucleus caudalis (TNC), periaqueductal grey (PAG), thalamus, hypothalamus, cortex, pituitary gland, hippocampus and cerebellum. iNOS was excluded from the present study because it was not induced. In the trigeminal vascular system we found the highest expression of nNOS mRNA in pial arteries. However, protein expression of nNOS was maximum in TNC. Among other brain structures, nNOS mRNA and protein expression was remarkably higher in the cerebellum than in any other tissues. Regarding eNOS in the trigeminovascular system, the highest mRNA expression was found in pial arteries. In the other brain structures, eNOS mRNA expression was similar but with lowest mRNA concentration in the pituitary gland and the highest concentration in cortex. The same pattern of expression was also observed with the eNOS protein. In conclusion, we found both nNOS and eNOS located to areas relevant to migraine supporting the involvement of NO in migraine mechanisms.

Rosuvastatin preconditioning provides neuroprotection against spinal cord ischemia in rats through modulating nitric oxide synthase expressions

The study was aimed to investigate the protective effects of rosuvastatin on spinal cord ischemia in rats and to determine the effects of this agent on the expressions of nitric oxide synthase (NOS). Spinal cord ischemia was induced in male Sprague–Dawley rats by occluding the descending thoracic aorta. Experimental groups ( n = 30 per group) were as follows: sham operation, control (receiving only normal saline), rosuvastatin (5 mg/kg/day for 10 days before occlusion), and rosuvastatin-mevalonate (5 mg/kg/day rosuvastatin and 5 mg/kg/day mevalonate for 10 days before occlusion). Neurological function was assessed at 6, 12, 24, 48, and 72 h after reperfusion. After 72 h reperfusion, spinal cords were harvested for 2,3,5,-triphenyltetrazolium chloride (TTC) staining, TUNEL staining, and nitric oxide (NO) assay. Immunohistochemistry, reverse transcription polymerase chain reaction and western blot were performed to determine the expressions of inducible, endothelial, and neuronal NOS (iNOS, eNOS, and nNOS) in rats with spinal cord ischemia. Spinal cord ischemia thus induced was marked by neurological dysfunction, spinal infarction, and neural cell apoptosis in animals. The results show that rosuvastatin significantly reduced the motor disturbance and the volume of infarctions and attenuated apoptotic neural cells death in the treated rats. Treatment with rosuvastatin remarkably decreased the NO level in spinal cord tissue. In addition, rosuvastatin inhibited iNOS mRNA and protein expression and increased eNOS mRNA and protein expression. However, rosuvastatin had no influence on nNOS mRNA and protein expression. Administrations of mevalonate completely reversed the changes caused by rosuvastatin. These results indicate that rosuvastatin can protect rat spinal cord against ischemia injury by modulation of NOS expressions.

Expression of Neuronal Nitric Oxide Synthase in Rabbit Carotid Body Glomus Cells Regulates Large-Conductance Ca2+-Activated Potassium Currents

Our previous studies show that a decrease in endogenous nitric oxide (NO) is involved in the blunted outward K(+) currents in carotid body (CB) glomus cells from chronic heart failure (CHF) rabbits. In the present study, we measured the effects of the neuronal nitric oxide synthase (nNOS) transgene on the K(+) currents in CB glomus cells from pacing-induced CHF rabbits. Using single-cell real-time RT-PCR and immunofluorescent techniques, we found that nNOS mRNA and protein are expressed in the rabbit CB glomus cells and CHF decreased the expression of nNOS mRNA and protein in CB glomus cells. After 3 days of an adenoviral nNOS (Ad.nNOS) gene transfection, the expression of nNOS protein was increased to the level found in sham CB glomus cells. In whole cell patch-clamp experiments, Ad.nNOS markedly reversed the attenuated K(+) currents in CB glomus cells from CHF rabbits. The specific nNOS inhibitor (S-methyl-l-thiocitrulline [SMTC]) and large-conductance Ca(2+)-activated K(+) (BK) channel blocker (iberiotoxin) fully abolished the effect of Ad.nNOS on the K(+) currents in the CB glomus cells from CHF rabbits. However, neither CHF nor Ad.nNOS altered the protein expression of BK channel alpha-subunit. These results suggest that a decrease of NO induced by an attenuated nNOS activity lowers the activation of the BK channels but not the protein expression of the BK channel alpha-subunit in the CB glomus cells during CHF.

The impact of simulated birth trauma and ovariectomy on the gene expression of detrusor muscarinic receptors in female rats

This study aims to investigate the effects of simulated birth trauma and ovariectomy on detrusor muscarinic receptors (M2 and M3), urethral neuronal nitric oxide synthase (nNOS), and estrogen receptor beta (ER beta). Forty primiparous rats were equally divided into five groups: group A--delivery, group B--delivery plus ovariectomy, group C--delivery plus balloon dilatation for 2 h, group D--delivery plus balloon dilatation for 4 h, and group E--delivery plus balloon dilatation for 2 h plus ovariectomy. The gene expression of M2, M3, nNOS, and ER beta were assessed by reverse transcription polymerase chain reaction. Significant decreases in mRNA expression of M2 receptors and nNOS (P < 0.05), and a significant increase in M3 mRNA expression (P < 0.05) were observed in groups D and E when compared with group A. Ovariectomy following birth trauma may synergistically impact the function of urinary tract, this being possibly related to the modification of the gene expression of muscarinic receptors.

Cell death mechanisms in the early stages of acute glutamate neurotoxicity

The present study focused on the early stages of acute glutamate ( l-Glu)-induced neurotoxic mechanisms, both biochemical, e.g. intracellular reactive oxygen species (ROS) and associated parameters as well as gene expression of cell survival/death pathways, i.e. Bcl-2 and caspases. Stereotactic intracortical injections of l-Glu (1 μmol/1 μl) resulted in decreased size of pyramidal neurons in rat after 1 h. We also observed that intracellular ROS, calcium (Ca 2+) and peroxynitrite (ONOO −) production were significantly elevated, whereas, mitochondrial transmembrane potential (ΔΨm) and total glutathione were significantly decreased by l-Glu bolus. The Bcl-2/Bax ratio in the l-Glu-injected rats was found to be significantly lower than the controls. Moreover, acute l-Glu significantly induced mRNA expression of nNOS, iNOS, caspase-3 and caspase-9. It may be concluded from the present study that acute l-Glu administration, at an early stage, increases intracellular ROS accumulation, Ca 2+ levels and peroxynitrite production and decreases glutathione pool. Furthermore, it appears that decreased mitochondrial Bcl-2/Bax ratio might have upregulated the mRNA expression of caspase-3 and caspase-9 which launch cell death cascade. Regarding the chronology of the events, we presume that acute l-Glu increases ROS and decreases ΔΨm and glutathione rapidly and it is more likely that these events precede gene expression changes, ultimately resulting in neuronal damage/death.

Nitric Oxide Synthase Isoenzyme Expression and Activity in Peripheral Lung Tissue of Patients with Chronic Obstructive Pulmonary Disease

Nitric oxide (NO) is increased in the lung periphery of patients with chronic obstructive pulmonary disease (COPD). However, expression of the NO synthase(s) responsible for elevated NO has not been identified in the peripheral lung tissue of patients with COPD of varying severity. Protein and mRNA expression of nitric oxide synthase type I (neuronal NOS [nNOS]), type II (inducible NOS [iNOS]), and type III (endothelial NOS [eNOS]) were quantified by Western blotting and reverse transcription-polymerase chain reaction, respectively, in specimens of surgically resected lung tissue from nonsmoker control subjects, patients with COPD of varying severity, and smokers without COPD, and in a lung epithelial cell line (A549). The effects of nitrative/oxidative stress on NOS expression and activity were also evaluated in vitro in A549 cells. nNOS nitration was quantified by immunoprecipitation and dimerization of nNOS was detected by low-temperature SDS-PAGE/Western blot in the presence of the peroxynitrite generator, 3-morpholinosydnonimine-N-ethylcarbamide (SIN1), in vitro and in vivo. Lung tissue from patients with severe and very severe COPD had graded increases in nNOS (mRNA and protein) compared with nonsmokers and normal smokers. Hydrogen peroxide (H(2)O(2)) and SIN1 as well as the cytokine mixture (IFN-gamma, IL-1beta, and tumor necrosis factor-alpha) increased mRNA expression and activity of nNOS in A549 cells in a concentration-dependent manner compared with nontreated cells. Tyrosine nitration resulted in an increase in nNOS activity in vitro, but did not affect its dimerization. Patients with COPD have a significant increase in nNOS expression and activity that reflects the severity of the disease and may be secondary to oxidative stress.

Oxidative Stress and Compartment of Gene Expression Determine Proatherosclerotic Effects of Inducible Nitric Oxide Synthase

Genetic and pharmacological inhibition of inducible nitric oxide synthase (iNOS) decreases atherosclerosis development. Potential proatherogenic effects of iNOS include iNOS mediated oxidative stress and iNOS expression in different cellular compartments. Lesional iNOS can potentially produce nitric oxide radicals (NO), superoxide radicals (O2(-)), or both; these radicals may then react to form peroxynitrite. Alternatively, O2(-) radicals from oxidases co-expressed with iNOS could react with NO to produce peroxynitrite. Therefore, the expression profiles of the genes that modulate the redox system in different iNOS-expressing cell compartments may determine the role of iNOS in atherosclerosis. We used apoE (apoE(-/-)) and apoE/iNOS double knockout (apoE(-/-)/ iNOS(-/-)) mice to assess vascular NO, O2(-), and peroxynitrite formation by electron spin resonance spectroscopy, high performance liquid chromatography, and 3-nitrotyrosine staining. The relevance of the iNOS expressing cell compartment was tested by bone marrow transplantation. We show that iNOS significantly contributes to vascular NO production and itself produces O2(-), leading to peroxynitrite formation in atherosclerotic lesions. Our bone marrow transplantation experiments show that bone marrow derived cells exclusively mediate the proatherosclerotic effects of iNOS in males, while both parenchymal and bone marrow derived iNOS equally contribute to atherosclerosis in females. Moreover, iNOS expression affects vascular remodeling. These findings establish for the first time that the proatherosclerotic effects of iNOS vary with sex in addition to the compartment of its expression.

Cellular and Vascular Changes in the Retina of Neonatal Rats after an Acute Exposure to Hypoxia

This study was undertaken to examine the effects of an acute hypoxic exposure on the retinal cells and production of vascular factors such as vascular endothelial growth factor (VEGF) and nitric oxide (NO), which may affect vascular permeability in the developing retina. Retinas of 1-day-old rats were examined at 3 hours to 14 days after hypoxic exposure. The mRNA and protein expression of hypoxia-inducible factor-1alpha (HIF-1alpha), VEGF, endothelial nitric oxide synthase (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS) were determined by real-time RT-PCR, Western blot analysis, and immunohistochemistry. Electron microscopy was used to examine the structural alterations in retinal cells, and rhodamine isothiocyanate (RhIC) or horseradish peroxidase (HRP) was administered intraperitoneally or intravenously to determine vascular permeability. The mRNA and protein expression of HIF-1alpha, VEGF, eNOS, nNOS, and iNOS, along with VEGF concentration and NO production, were increased in response to hypoxia. Swollen Müller cell processes, apoptotic and necrotic cells in the inner nuclear layer, and changes in ganglion cells such as swollen and disrupted mitochondria were observed in hypoxic animals. Increased leakage of RhIC and HRP from retinal and hyaloid vessels was seen after hypoxic exposure. The authors suggest that increased VEGF and NO production in hypoxia resulted in increased vascular permeability, leading to changes in Müller cells and degeneration of neural cells. Melatonin administration reduced VEGF and NO production, diminished leakage of RhIC and HRP, and promoted cell proliferation, suggesting this as a potential therapeutic agent in reducing hypoxia-associated damage in the developing retina.

Decreased nitric oxide markers and morphological changes in the brain of arsenic-exposed rats

Epidemiological studies demonstrate an association between chronic consumption of arsenic contaminated water and cognitive deficits, especially when the exposure takes place during childhood. This study documents structural changes and nitrergic deficits in the striatum of adult female Wistar rats exposed to arsenic in drinking water (3 ppm, approximately 0.4 mg/kg per day) from gestation, throughout lactation and development until the age of 4 months. Kainic acid injected animals (10 mg/kg, i.p.) were also analyzed as positive controls of neural cell damage. Morphological characteristics of cells, fiber tracts and axons were analyzed by means of light microscopy as well as immunoreactivity to neuronal nitric oxide synthase (nNOS). As nitrergic markers, nitrite/nitrate concentrations, nNOS levels and expression of nNOS-mRNA were quantified in striatal tissue. Reactive oxygen species (ROS) and lipid peroxidation (LPx) were determined as oxidative stress markers. Arsenic exposure resulted in moderate to severe alterations of thickness, organization, surrounding space and shape of fiber tracts and axons, while cell bodies remained healthy. These anomalies were not accompanied by ROS and/or LPx increases. By contrast, except the expression of nNOS-mRNA, all nitrergic markers including striatal nNOS immunoreactivity presented a significant decrease. These results indicate that arsenic targets the central nitrergic system and disturbs brain structural organization at low exposure levels.

Expression of endothelin, endothelin type A receptor, isoforms of nitric oxide synthase, and angiotensin receptors in endothelin type B receptor deficient mice

Endothelin‐1 (ET‐1) is involved in the control of renal vessel contractions by acting on endothelin type A (ETA) and type B (ETB) receptors. Several studies indicate an interaction of ET‐1 with the angiotensin system; which includes modulation of receptor expression. The aim of this study was to determine the influence of ETB receptor deficiency on the expression of ET‐1, ETA receptors, nitric oxide synthases (NOS) as well as angiotensin receptors (AT) in preglomerular vessels and kidneys of ETB(‐/‐) mice. ETA mRNA expression, determined by real time PCR, was decreased in preglomerular vessels compared to wild type mice (ETB(+/+)). ETA mRNA and protein were also diminished in whole kidney preparations in ETB(‐/‐) mice. The endothelial NOS (eNOS) mRNA was less expressed in preglomerular vessels. No significant differences were found for mRNA expression of eNOS, nNOS, and iNOS in whole kidney preparations. AT1 and AT2 receptor mRNA in the kidney did not differ between ETB(‐/‐) and ETB(+/+). As shown by western blotting analysis the ET‐1 protein was stronger expressed in the kidneys of ETB(‐/‐) The results of ET‐1 and ETA expression confirm earlier findings of a clearance function of the ETB receptor and modulation of ETA expression by ET‐1. Moreover, ET receptor expression/function seems to influence eNOS mRNA expression in renal arterioles.

Expression of neuronal nitric oxide synthase in corpus cavernosum with diabetic erectile dysfunction: experiment with rabbits

To investigate the expression characteristics of neuronal nitric oxide synthase (nNOS) in the corpus cavernosum of diabetic erectile dysfunction (ED). Fifty rabbits underwent single intravenous injection of alloxan (100 mg/kg). Blood glucose, system arterial pressure (SAP), and intracavemosal pressure (ICP) were measured 15, 30, and 45 days later. The rabbits with the blood glucose >10 mmol/L were used as diabetic models and those with the ICP lower than the normal control group by more than 25% were used as diabetic ED models. RT-PCR, immunohistochemistry, and Western-blotting were used to detect the expression of nNOS in the corpus cavemosum. Seven days after the alloxan injection the blood glucose levels of 35 rabbits (70%) were > 10 mmol/L. 40% rabbits (14/35) were were used as diabetic ED models. Compared with control group, the ICP 15, 30, and 45 days after alloxan injection were considerately decreased in diabetic ED rabbits (all P < 0.01), and there was no significant difference in SAP between the two groups (all P > 0.05). The relative values of nNOS mRNA expression 15, 30, and 45 days after alloxan injection of diabetic ED rabbits were 0.670 +/- 0.030, 0.451 +/- 0.012, and 0.206 +/- 0.023 respectively,all significantly lower than those of the control group (0.817 +/- 0.010, 0.814 +/- 0.020, and 0.802 +/- 0.007 respectively, all P < 0.05). Western blotting showed that the relative values of nNOS 15, 30, and 45 days after alloxan injection of the ED rabbits were 0.713 +/- 0.014, 0.424 +/- 0.007, and 0.337 +/- 0.009 respectively, all significantly lower than those of the control group (0.797 +/- 0.015, 0.706 +/- 0.020, and 0. 750 +/- 0.022 respectively, all P < 0.05). Decreased expression of nNOS in corpus cavernosum is one of possible pathway of diabetic ED.

EFFECT OF INHIBITION OF NEURONAL NITRIC OXIDE SYNTHASE AND l-ARGININE SUPPLEMENTATION ON RENAL ISCHAEMIA-REPERFUSION INJURY AND THE RENAL NITRIC OXIDE SYSTEM

The role of nitric oxide synthases (NOS) and the nitric oxide (NO) substrate l-arginine in renal ischaemia-reperfusion (I/R) has been studied extensively. However, the results reported are often controversial. In the present study, we examined the effects of the neuronal (n) NOS inhibitor 7-nitroindazole (7-NI) and L-arginine administration on renal I/R injury and the renal NO system in rats. Following 7 days pretreatment with 7-NI (50 mg/kg per day), L-arginine (2 g/kg per day) or vehicle (dimethylsulphoxide : sesame oil, 1 : 9), the left renal vascular pedicles were clamped for 50 min in male Sprague-Dawley rats and kidneys were removed 24 h after reperfusion (n = 7/group). Neither 7-NI nor L-arginine had any effect on parameters of renal function, the grade of tissue injury or the number of terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL)-positive tubular cells compared with vehicle-treated rats. 7-Nitroindazole decreased nNOS mRNA expression and inducible (i) NOS protein levels, but had no effect on endothelial NOS expression. L-arginine supplementation increased mRNA expression of all NOS isoforms, but only increased protein expression of iNOS. The results of the present study demonstrate that selective inhibition of nNOS has no effect on renal injury, indicating that nNOS does not play a central role in the pathophysiology of renal I/R. In addition, although L-arginine has no effect on renal I/R injury in the model used in the present study, its administration increases the mRNA expression of NOS isoforms.

Simvastatin Inhibits Central Sympathetic Outflow in Heart Failure by a Nitric-Oxide Synthase Mechanism

Our previous study demonstrated that oral treatment with simvastatin (SIM) suppressed renal sympathetic nerve activity (RSNA) in the rabbits with chronic heart failure (CHF). The purpose of this experiment was to determine the effects of direct application of SIM to the central nervous system on RSNA and its relevant mechanisms. Experiments were carried out on 21 male New Zealand White rabbits with pacing-induced CHF. The CHF rabbits received infusion of vehicle, SIM, or SIM + N(omega)-nitro-L-arginine methyl ester into the lateral cerebral ventricle via osmotic minipump for 7 days. We found that 1) in CHF rabbits, intracerebroventricular infusion of SIM significantly suppressed basal RSNA (1st day 69.5 +/- 8.9% maximum; 7th day 26.0 +/- 6.0% maximum; P < 0.05, n = 7) and enhanced arterial baroreflex function starting from the 2nd day and lasting through the following 5 days; 2) statin treatment significantly up-regulated neuronal nitric-oxide synthase (nNOS) protein expression in the rostral ventrolateral medulla (RVLM) (control, n = 6, 0.12 +/- 0.04; SIM-treated, n = 7, 0.31 +/- 0.05. P < 0.05); 3) in CATH.a neurons, incubation with SIM significantly up-regulated the nNOS mRNA expression, which was blocked by coincubation with mevalonate, farnesyl-pyrophosphate, or geranylgeranyl-pyrophosphate; and 4) incubation with Y-27632 [(R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide] significantly up-regulated nNOS mRNA expression in these neurons. These results suggest that central treatment with SIM decreased sympathetic outflow in CHF rabbits via up-regulation of nNOS expression in RVLM, which may be due to the inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase and a decrease in Rho kinase by SIM.

The role of nitric oxide in the mechanical repression of RANKL in bone stromal cells

Both mechanical loading and nitric oxide (NO) have positive influences on bone mass. NO production is induced by mechanical strain via upregulation of eNOS mRNA and protein, the predominant NOS in adult bone. At the same time, strain causes decreased expression of RANKL, a factor critical for osteoclastogenesis. In this study, we harvested primary stromal cells from wild-type (WT) and eNOS(−/−) mice to test whether induction of NO by mechanical strain was necessary for transducing mechanical inhibition of RANKL. We found that strain inhibition of RANKL expression was prevented by NOS inhibitors (L-NAME and L-NMMA) in WT stromal cells. Surprisingly, stromal cells from eNOS(−/−) mice showed significant mechanical repression of RANKL expression (p<0.05). Mechanical strain still increased NO production in the absence of eNOS, and was abolished by SMTC, a specific nNOS inhibitor. nNOS mRNA and protein expression were increased by strain in eNOS(−/−) but not in WT cells, revealing that nNOS was mechanically sensitive. When NO synthesis was blocked with either SMTC or siRNA targeting nNOS in eNOS(−/−) cells however, strain still was able to suppress RANKL expression by 34%. This indicated that strain suppression of RANKL can also occur through non-NO dependent pathways. While our results confirm the importance of NO in the mechanical control of skeletal remodeling, they also suggest alternative signaling pathways by which mechanical force can produce anti-catabolic effects on the skeleton.