nNOS mRNA Levels Research Articles

Cigarette smoke exposure impairs early-stage recovery from lengthening contraction-induced muscle injury in male mice.

The use of tobacco cigarettes produces locomotor muscle weakness and fatigue intolerance. Also, smokers and chronic obstructive pulmonary disease patients have a greater incidence of muscle injury and a deficient myogenic response. However, the effects of smoke exposure on the recovery from eccentric exercise-induced muscle injuries are unknown. Mice were exposed daily to cigarette smoke (CS) or room air (Air) for 4 months; the anterior crural muscles from one limb were injured by a lengthening contractions protocol (LCP) and recovered for 7 days. Lung compliance was greater, and body weights were lower, in CS-exposed than in the Air group. In LCP-subjected limbs, CS exposure lowered tibialis anterior myofiber cross-sectional area, decreased the size of centrally nucleated myofibers, and decreased extensor digitorum longus (EDL) mass, but did not affect EDL force from both limbs. CS exposure upregulated the mRNA levels of several myogenic (Pax7, Myf5, nNOS) genes in the EDL. The combination of CS exposure and LCP decreased Myf5 and nNOS mRNA levels and exacerbated pro-inflammatory mRNA levels. These data suggest that smoke exposure leads to an excessive pro-inflammatory response in regenerating muscle that is associated with a lower muscle mass recovery from a type of injury that often occurs during strenuous exercise.

NNOS Increases Fiber Type-Specific Angiogenesis in Skeletal Muscle of Mice in Response to Endurance Exercise.

We studied the relationship between neuronal NO synthase (nNOS) expression and capillarity in the tibialis anterior (TA) muscle of mice subjected to treadmill training. The mRNA (+131%) and protein (+63%) levels of nNOS were higher (p ≤ 0.05) in the TA muscle of C57BL/6 mice undergoing treadmill training for 28 days than in those of littermates remaining sedentary, indicating an up-regulation of nNOS by endurance exercise. Both TA muscles of 16 C57BL/6 mice were subjected to gene electroporation with either the pIRES2-ZsGreen1 plasmid (control plasmid) or the pIRES2-ZsGreen1-nNOS gene-inserted plasmid (nNOS plasmid). Subsequently, one group of mice (n = 8) underwent treadmill training for seven days, while the second group of mice (n = 8) remained sedentary. At study end, 12-18% of TA muscle fibers expressed the fluorescent reporter gene ZsGreen1. Immunofluorescence for nNOS was 23% higher (p ≤ 0.05) in ZsGreen1-positive fibers than ZsGreen1-negative fibers from the nNOS-transfected TA muscle of mice subjected to treadmill training. Capillary contacts around myosin heavy-chain (MHC)-IIb immunoreactive fibers (14.2%; p ≤ 0.05) were only higher in ZsGreen1-positive fibers than ZsGreen1-negative fibers in the nNOS-plasmid-transfected TA muscles of trained mice. Our observations are in line with an angiogenic effect of quantitative increases in nNOS expression, specifically in type-IIb muscle fibers after treadmill training.

Differences of Transport Activity of Arginine and Regulation on Neuronal Nitric Oxide Synthase and Oxidative Stress in Amyotrophic Lateral Sclerosis Model Cell Lines.

L-Arginine, a semi-essential amino acid, was shown to delay dysfunction of motor neurons and to prolong the lifespan, upon analysis of transgenic mouse models of amyotrophic lateral sclerosis (ALS). We investigated the transport function of arginine and neuronal nitric oxide synthase (nNOS) expression after pretreatment with L-arginine in NSC-34 hSOD1WT (wild-type, WT) and hSOD1G93A (mutant-type, MT) cell lines. [3H]L-Arginine uptake was concentration-dependent, voltage-sensitive, and sodium-independent in both cell lines. Among the cationic amino acid transporters family, including system y+, b0,+, B0,+, and y+L, system y+ is mainly involved in [3H]L-arginine transport in ALS cell lines. System b0,+ accounted for 23% of the transport in both cell lines. System B0,+ was found only in MT, and whereas, system y+L was found only in WT. Lysine competitively inhibited [3H]L-arginine uptake in both cell lines. The nNOS mRNA expression was significantly lower in MT than in WT. Pretreatment with arginine elevated nNOS mRNA levels in MT. Oxidizing stressor, H2O2, significantly decreased their uptake; however, pretreatment with arginine restored the transport activity in both cell lines. In conclusion, arginine transport is associated with system y+, and neuroprotection by L-arginine may provide an edge as a possible therapeutic target in the treatment of ALS.

Effect of Nitrate on Gene and Protein Expression of Nitric Oxide Synthase Enzymes in Insulin-Sensitive Tissues of Type 2 Diabetic Male Rats

Decreased nitric oxide (NO) bioavailability contributes to the pathophysiology of type 2 diabetes mellitus (T2DM). This study aims to determine the effects of nitrate (NO3 -) on gene and protein expression of NO synthase (NOS) enzymes in the liver, soleus muscle (SM), and epididymal adipose tissue (eAT) of rats with T2DM. Twenty-eight male rats were divided into 4 groups: Control, diabetes, control+NO3 -, and diabetes+NO3 - (n = 7/each group). NO3 - was administered for 6 months, and mRNA and protein levels of NOS enzymes were measured at the end of the study. mRNA and protein levels of inducible NOS (iNOS) were higher in the liver (475% and 73%), SM (271% and 43%), and eAT (543% and 24%) of rats with T2DM. In the case of the endothelial NOS (eNOS), diabetic rats had lower mRNA and protein levels in the liver (26% and 24%) and SM (60% and 62%) and lower mRNA level (30%) in eAT. mRNA and protein levels of neural NOS (nNOS) were lower in SM (69% and 73%) and eAT (25% and 31%) of rats with T2DM. NO3 - administration restored disrupted iNOS and eNOS expressions to their near normal values in all the studied tissues; NO3 - also increased nNOS mRNA and protein levels in SM and eAT but decreased nNOS protein level in the liver. Long-term NO3 - administration restored disrupted expression of NOS enzymes in the liver, SM, and eAT of rats with T2DM; these findings partly explain the beneficial metabolic effects of nitrate in T2DM.

Adenosine A1 and A2a receptors modulate the nitrergic system in cell culture from dorsomedial medulla oblongata.

Adenosine and nitric oxide act on the fine-tuning regulation of neural cardiovascular control in the nucleus tractus solitarius (NTS). Although the interaction between adenosine and NO is well known in the periphery, the mechanisms by which adenosine interferes in the dynamics of nitrergic neurotransmission, related to neural control of circulation, are not completely understood and might be relevant for individuals predisposed to hypertension. In this study we evaluate the interaction between adenosinergic and nitrergic systems in cell culture from the dorsomedial medulla oblongata of Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). Using quantification of nitrite levels, RT-PCR analysis and RNA interference we demonstrate that adenosine A1 (A1R) and A2a receptor (A2aR) agonists induce a concentration-dependent decrease and increase of nitrite and nNOS mRNA levels in cultured cells from WKY and SHR, respectively. These effects in nitrite levels are attenuated by the administration of A1R and A2aR selective antagonists, CPT and ZM 241385. Furthermore, knockdown of A1R and A2aR show an increase and decrease of nNOS mRNA levels, respectively. Pretreatment with the nonselective inhibitor of NOS, L-NAME, abolishes nitrite-increased levels triggered by CGS 21680 in WKY and SHR cells. Finally, it is shown that the cAMP-PKA pathway is involved in A1R and A2aR-mediated decrease and increase in nitrite levels in SHR and WKY cells. Our results highlight the influence of adenosine on nitric oxide levels in cultured cells from dorsal medulla oblongata of neonate WKY and SHR rats. In part, the modulatory profile is different in the SHR strain.

Involvement of nNOS in the antinociceptive activity of melatonin in inflammatory pain at the level of sensory neurons.

The efficacy of melatonin as an analgesic agent has been well documented in animals and humans. However, the underlying mechanisms by which melatonin exerts antinociceptive effects on inflammatory pain are poorly understood. Here, we investigated the potential of melatonin to ameliorate inflammatory pain. In vitro, ND7/23 neurons were treated with capsaicin. We used PCR and Western blot analyses to detect the expression of neuronal nitric oxide synthase (nNOS) in response to melatonin. Orofacial inflammatory pain was induced by 4% formalin administration on the right whisker pad of Sprague Dawley (SD) rats. The analgesic effect of melatonin was evaluated using mechanical threshold analyses. The expression level of nNOS in the trigeminal ganglion (TG) and trigeminal nucleus caudalis (Vc) neurons was assessed by RNAscope and immunohistochemistry. In vitro, capsaicin upregulated the expression of nNOS, which was dose-dependently reversed by melatonin pretreatment (p < 0.001). In a rat model of orofacial inflammatory pain, melatonin pretreatment significantly attenuated mechanical allodynia in both the acute and chronic phases (p < 0.05). Furthermore, melatonin decreased the formalin-evoked elevated nNOS mRNA and protein levels in the TG and Vc neurons in the acute and chronic phases (p < 0.05). Taken together, these results suggest that nNOS may play an active role in both peripheral and central processing of nociceptive information following orofacial inflammatory pain induction. The regulatory effect of melatonin on nNOS in inflammatory pain may have potential implications for the development of novel analgesic strategies.

Optogenetic Stimulation Reduces Neuronal Nitric Oxide Synthase Expression After Stroke

Abstract INTRODUCTION Poststroke optogenetic stimulation has been shown to enhance neurovascular coupling and functional recovery. Neuronal nitric oxide synthase (nNOS) has been implicated as a key regulator of neurovascular response in acute stroke but its role in subacute recovery remains unclear. Here we investigate nNOS expression in stroke mice undergoing optogenetic stimulation of the contralesional lateral cerebellar nucleus (cLCN). We also examine the effects of nNOS inhibition on functional recovery using a pharmacological inhibitor targeting nNOS. METHODS Transgenic Thy1-ChR2-YFP male mice (10-12 wk) were used. Stereotaxic surgery was performed to implant a fiber cannula in the cLCN and animals underwent intraluminal middle cerebral artery suture occlusion (30 min). Optogenetic stimulation began at poststroke (PD) day 5 and continued until PD14. Sensorimotor tests were used to assess behavioral recovery at PD4, 7, 10, and 14. At PD15, primary motor cortex from both ipsi- and contralesional motor cortex (iM1, cM1) were dissected. nNOS mRNA and protein levels were examined using quantitative polymerase chain reaction and western blot. In another set of studies, nNOS inhibitor ARL 17477 dihydrochloride (10 mg/kg, intraperitoneally) was administered daily between PD5-14 and functional recovery was evaluated using sensorimotor tests. RESULTS cLCN stimulated stroke mice demonstrated significant improvement in speed (cm/s) on the rotating beam task at PD10 and 14 day (P &lt; .05, P &lt; .001 respectively). nNOS mRNA and protein expression was significantly and selectively decreased in cM1 of cLCN stimulated mice (P &lt; .05). The reduced nNOS expression in cM1 was negatively correlated with improved recovery (R2 = −0.839, Pearson P = .009). nNOS inhibitor-treated stroke mice exhibited a significant functional improvement in speed at PD10, when compared to stroke mice receiving vehicle (saline) (P &lt; .05). CONCLUSION Our results suggest that nNOS may play a maladaptive role in poststroke recovery. Optogenetic stimulation of cLCN and systemic nNOS inhibition produce functional benefits after stroke.

The response of nitric oxide system to high Altitude in Phrynocephalus erythrurus on Qinghai-Tibetan plateau.

Nitric oxide (NO), produced by nitric oxide synthases (NOS) from L-arginine, plays important roles in a wide range of physiological processes. However, little is known about ectothermic species. To investigate the response of NO/NOS system in adaptation to different altitudes in Phrynocephalus genus, the red tail toad-headed lizard Phrynocephalus erythrurus that live at 4500-5300 m on the Qinghai-Tibet Plateau and another low altitude living lizard Phrynocephalus przewalskii were selected in the present study. The results of mRNA expression and activity of NOSs, as well as NO metabolite levels in different tissues of the two lizards indicate that nNOS mRNA levels in cardiac and skeletal muscle were notably elevated in P. erythrurus, and iNOS expression was also increased markedly (up to 4-fold) in cardiac muscle. There was no significant difference in eNOS mRNA level in tested tissues between two species. However, the total NOS activity in skeletal muscle of P. erythrurus was slightly lower than that of P. przewalskii (p < .05) while no difference in other tissues. Similarly, lower iNOS activity (p < .01) was found in cardiac and skeletal muscle in P. erythrurus compared to P. przewalskii. In addition, the NO metabolite levels were dramatically lower in P. erythrurus in all tested tissues. We propose that higher nNOS and iNOS mRNA expression, lower iNOS activity and NO metabolite levels may represent physiological characteristics in nitric oxide system, which may contribute to high-altitude adaptation in P. erythrurus.

Decreased Number and Expression of nNOS-Positive Interneurons in Basolateral Amygdala in Two Mouse Models of Autism.

The basolateral amygdala (BLA) controls socio-emotional behaviors and is involved in the etiology of autism. We have recently shown that virtually every neuronal nitric oxide synthase (nNOS) positive cell is a GABAergic inhibitory interneuron in the mouse BLA. Here, stereology was used to quantify the number of nNOS-expressing interneurons in valproic acid (VPA)-exposed C57BL/6J (B6) and BTBR T+Itpr3tf/J (BTBR) mice models of autism. Additionally, the protein and mRNA levels of nNOS in the BLA were quantitatively assessed by western blot and qRT-PCR analysis, respectively. Our results showed the decreased number of nNOS interneurons in the BLA of animal models relative to autism. Consistently, nNOS was significantly reduced in the VPA-exposed and BTBR mice at both protein and mRNA levels. Together, these preliminary findings suggest that down-regulation of nNOS may be an attractive target for the pharmacological intervention in autism.

The role of neuronal nitric oxide synthase in cocaine place preference and mu opioid receptor expression in the nucleus accumbens

There is evidence that central mu opioid receptors (MORs) are implicated in several aspects of cocaine addiction, and that MOR expression is elevated by cocaine in vitro and in the nucleus accumbens (NAc) when administered in vivo. To understand the cellular mechanisms involved in regulating MOR expression, this study explored whether neuronal nitric oxide synthase (nNOS) modulates the neurochemical and behavioral effects of acute and repeated cocaine administration. Male Sprague-Dawley rats received a single cocaine injection (20mg/kg, i.p.) in combination with the selective nNOS inhibitor 7-nitroindazole (7-NI) (0, 25, or 50mg/kg, i.p.), and the expression of MOR and nNOS messenger RNA (mRNA) and protein levels in the NAc were measured. In a separate conditioned place preference (CPP) experiment, 7-NI (0, 25, or 50mg/kg, i.p.) was administered prior to cocaine (0 or 20mg/kg, i.p.) conditioning sessions, and levels of MOR and nNOS mRNA and protein in the NAc were measured following CPP test. Acute cocaine administration significantly enhanced nNOS and MOR mRNA and protein expression in the NAc, and this increase in MOR expression was blocked by 7-NI. Furthermore, in 7-NI pre-treated rats, cocaine-induced CPP was not statistically significant and the increase in MOR mRNA expression in the NAc in these animals was attenuated. These findings suggest that nNOS modulates MOR expression following acute cocaine administration, and that cocaine CPP and associated upregulation of MOR expression involve both nNOS-dependent and independent mechanisms. Elucidation of these molecular events may identify useful therapeutic target for cocaine addiction.

Adenosine A1 receptor: A neuroprotective target in light induced retinal degeneration.

Light induced retinal degeneration (LIRD) is a useful model that resembles human retinal degenerative diseases. The modulation of adenosine A1 receptor is neuroprotective in different models of retinal injury. The aim of this work was to evaluate the potential neuroprotective effect of the modulation of A1 receptor in LIRD. The eyes of rats intravitreally injected with N6-cyclopentyladenosine (CPA), an A1 agonist, which were later subjected to continuous illumination (CI) for 24 h, showed retinas with a lower number of apoptotic nuclei and a decrease of Glial Fibrillary Acidic Protein (GFAP) immunoreactive area than controls. Lower levels of activated Caspase 3 and GFAP were demonstrated by Western Blot (WB) in treated animals. Also a decrease of iNOS, TNFα and GFAP mRNA was demonstrated by RT-PCR. A decrease of Iba 1+/MHC-II+ reactive microglial cells was shown by immunohistochemistry. Electroretinograms (ERG) showed higher amplitudes of a-wave, b-wave and oscillatory potentials after CI compared to controls. Conversely, the eyes of rats intravitreally injected with dipropylcyclopentylxanthine (DPCPX), an A1 antagonist, and subjected to CI for 24 h, showed retinas with a higher number of apoptotic nuclei and an increase of GFAP immunoreactive area compared to controls. Also, higher levels of activated Caspase 3 and GFAP were demonstrated by Western Blot. The mRNA levels of iNOS, nNOS and inflammatory cytokines (IL-1β and TNFα) were not modified by DPCPX treatment. An increase of Iba 1+/MHC-II+ reactive microglial cells was shown by immunohistochemistry. ERG showed that the amplitudes of a-wave, b-wave, and oscillatory potentials after CI were similar to control values. A single pharmacological intervention prior illumination stress was able to swing retinal fate in opposite directions: CPA was neuroprotective, while DPCPX worsened retinal damage. In summary, A1 receptor agonism is a plausible neuroprotective strategy in LIRD.

Lazaroid (U-74389G) ameliorates lung injury due to lipid peroxidation and nitric oxide synthase-dependent reactive oxygen species generation caused by remote systematic ischemia-reperfusion following thoracoabdominal aortic occlusion

IntroductionLung ischemia-reperfusion injury after thoracoabdominal aortic occlusion represents a major complication, which increases morbidity and mortality. In the present study we hypothesized that lazaroid U-74389G intravenous administration protects from lung ischemia-reperfusion injury through lipid peroxidation inhibition. Materials and methodsA total of 24 pigs were randomized in three groups. Group I (n = 8) underwent sham operation, group II (n = 8) underwent thoracoabdominal aortic occlusion for 45min and received placebo and group III (n = 8) received 3 doses of lazaroid (3 mg/kg) 60 and 30min before thoracoabdominal aortic occlusion and at 30min during thoracoabdominal aortic occlusion (duration 45min). Aortic occlusion was performed with aortic balloon-catheters under fluoroscopic guidance. All animals were sacrificed at the 7 t h postoperative day and lung specimens were harvested for molecular analysis. ResultsmRNA levels of leukotrienes LB4 (LTB4R2), LC4 (LTC4S) and nitric oxide synthase (NOS) isoforms including iNOS, nNOS and eNOS were determined with real-time RT-qPCR. Nitric oxide can either induce (iNOS) or inhibit (nNOS and eNOS) lipid peroxidation based on its specific isoform origin. Group III showed significantly reduced mRNA levels of LTB4R2 (−63.7%), LTC4S (−35.9%) and iNOS (−60.2%) when compared with group II (P < 0.05, for all). The mRNA levels of nNOS was significantly increased (+37.4%), while eNOS was slightly increased (+2.1%) in group III when compared with group II (P < 0.05 and P = 0.467 respectively). ConclusionLazaroid U-74389G may represent an effective pharmacologic intervention in reducing lung ischemia-reperfusion injury following thoracoabdominal aortic occlusion.

Neuronal nitric oxide synthase inhibition reduces brain damage by promoting collateral recruitment in a cerebral hypoxia-ischemia mice model.

The collateral circulation development is considered as a compensatory inherent mechanism to restore damaged blood perfusion after ischemia. We aimed to detect the collateral flow and the mean blood-flow velocities (mBFVs) level in the basilar trunk during or after cerebral hypoxia-ischemia in the mice brain and explore the effect of neuronal nitric oxide synthase (nNOS) inhibition on the collateral flow. C57BL/6J mice and the nNOS knockout (KO) mice were randomly divided into a sham-operated group (control) and the hypoxia-ischemia (HI) groups that were treated with the phosphate buffered solution (PBS) control or 7-nitroindazole (7-NI). Cortexes were harvested after the HI treatment for analysis of nNOS expression using Western blot and reverse transcription-polymerase chain reaction (RT-PCR). Ultrasound imaging experiments were performed to detect the collateral flow and the mBFVs level in the basilar trunk. After cerebral HI, the cortical nNOS mRNA and protein levels increased markedly compared with the sham-operated control mice. Besides, 7-NI treatment had no effect on the blood flow in the sham-operated control mice. What's more, either the 7-NI pretreatment or the nNOS gene knockdown before the HI procedure could attenuate the brain injury by the increased collateral flow and the decreased mBFVs level in the basilar trunk. nNOS inhibition protected hypoxic-ischemic-induced mice brain damage by the increased collateral flow and the decreased mBFVs level in the basilar trunk. Therefore, the 7-NI administration may have potential utility for the treatment of HI injury in human beings.

Expression of nNOS and correlation with clinical prognosis in renal clear cell carcinoma

Objective To investigate the expression of neural-nitric oxide synthase (nNOS) in renal clear cell carcinoma and its clinical significance. Methods The expression of nNOS mRNA in 533 samples of TCGA database was analyzed with Student t test, and statistical analysis was performed to assess the relationship between nNOS expression and clinical prognosis with Kapla-Meier test. Western blot analysis of nNOS protein expression in 10 cases of clear cell renal cell carcinoma(ccRCC) from department of urology of Wuhan union hospital with student t test. Results The mRNA levels of nNOS in 72 cases of ccRCC in tumor tissues and adjacent tissues and were 2.99±0.28 and -1.57±0.17, it is significantly lower than those in adjacent tissues (P<0.01). The mRNA levels of nNOS in 533 cases of ccRCC, in tumor tissues and adjacent tissues and were 2.99±0.28 and -1.76±0.05, it is significantly lower than those in adjacent tissues (P<0.01 ). A total of 533 sample studies showed a low correlation between nNOS expression and clinical T stage, T1 -1.59±0.08, T2 -1.96±0.13, T3 -1.90±0.09, T4 -2.38±0.28 (P=0.0029) and -1.63±0.06 and -2.16±0.13 between non-metastasis and no-metastasis (P=0.0009), and -1.57±0.08 and -2.03±0.11 between non-recurrence and recurrence (P=0.008). Survival analysis showed that the overall survival time were (40.3±5.6) months and (48.3±5.7) months in lower and higher nNOS expression, and disease free survival time were(37.1±2.1) months and (40.3±5.6) months in lower and higher nNOS expression, both with shorter time in low expression of nNOS (P<0.01). nNOS proteins were 1.02±0.16 and 0.61±0.11 in tumor tissues and adjacent tissues with significantly lower expression(P<0.05). Conclusions The mRNA and protein of nNOS are lower in ccRCC with a poor prognosis of ccRCC. Key words: Clear cell renal cell carcinoma; Neural-nitric oxide synthase; Prognosis

Activation of α1A -adrenoceptors desensitizes the rat aorta response to phenylephrine through a neuronal NOS pathway, a mechanism lost with ageing.

A NO-mediated desensitization of vasoconstrictor responses evoked by stimulation of α1 -adrenoceptors has been reported in different vessels. We investigated the involvement of each α1 -adrenoceptor subtype and constitutive NOS isoforms and the influence of ageing and hypertension on this process. Wistar and spontaneously hypertensive rats (SHR), 16, 32, 52 and 72weeks-old, were used to evaluate the desensitization process. Expression of α1 -adrenoceptor subtypes, endothelial NOS (eNOS) and neuronal NOS (nNOS) were determined in rat aorta and left ventricle (LV). Expression levels were also evaluated in LV of a group of heart failure patients with a wide age range. Repeated application of phenylephrine decreased subsequent α1 -adrenoceptor-mediated vasoconstriction by increasing nNOS protein expression in aorta, but not in tail or mesenteric resistance arteries, where mRNA levels of nNOS were undetectable. This desensitization process disappeared in the absence of endothelium or in the presence of L-NAME (100μM), nNOS inhibitors, SMTC (1μM) and TRIM (100μM), and 5-methylurapidil (100nM, α1A -antagonist), but not BMY7378 (10nM, α1D -antagonist). The α1A /nNOS-mediated desensitization was absent in aged SHR and Wistar animals, where the expression of α1A -adrenoceptors was reduced in aorta and LV. In human LV, a negative correlation was found between age and α1A -adrenoceptor expression. The α1A -adrenoceptor subtype, through endothelial nNOS-derived NO, may act as a physiological 'brake' against the detrimental effects of excessive α1 -adrenoceptor-mediated vasoconstriction. Reduced α1A -adrenoceptor- and nNOS-mediated desensitization in aged patients could be involved in the age-dependent elevation of adrenergic activity.

The Presence of the Neuronal Nitric Oxide Synthase Isoform in the Intervertebral Disk.

Intervertebral disk degeneration is a progressive and debilitating disease with multifactorial causes. Nitric oxide (NO) might contribute to the cell death pathway. We evaluated the presence of the constitutive form of the neuronal NOS (nNOS) in both health and degenerated intervertebral disk through qPCR and immunohistochemistry. We also analyzed the potential role of nNOS modulation in the tail needle puncture model of intervertebral disk degeneration. Male Wistar rats were submitted to percutaneous disk puncture with a 21-gauge needle of coccygeal vertebras. The selective nNOS pharmacological inhibitor N (ω)-propyl-L-arginine (NPLA) or a nNOS-target siRNA (siRNAnNOShum_4400) was injected immediately after the intervertebral disk puncture with a 30-gauge needle. Signs of disk degeneration were analyzed by in vivo magnetic resonance imaging and histological score. We found that intact intervertebral disks express low levels of nNOS mRNA. Disk injury caused a 4 fold increase in nNOS mRNA content at 5h post disk lesion. However, NPLA or nNOS-target siRNA slight mitigate the intervertebral disk degenerative progress. Our data show evidence of the nNOS presence in the intervertebral disk and its upregulation during degeneration. Further studies would disclose the nNOS role and its potential therapeutical value in the intervertebral disk degeneration.

Role of nitric oxide synthase in the development of bone cancer pain and effect of L-NMMA.

Spinal nitric oxide is involved in the mechanisms of pain generation and transmission during inflammatory and neuropathic pain. The aim of the present study was to explore the role of spinal nitric oxide in the development of bone cancer pain. 2×105 osteosarcoma cells were implanted into the intramedullary space of right femurs of C3H/HeJ mice to induce a model of ongoing bone cancer. Polymerase chain reaction and immunohistochemical analyses were performed to assess the expression of neuronal nitric oxide synthase (nNOS) and inducible (i)NOS in the spinal cord following inoculation. The results showed that inoculation of osteosarcoma cells induced progressive bone cancer, accompanied with pain-associated behavior. The levels of nNOS mRNA in the spinal cord of tumor mice began to increase at day 10 and then decreased to the level in sham mice at day 14, while iNOS mRNA markedly increased in the tumor group at days 10 and 14. Immunohistochemical analysis showed that nNOS- and iNOS-positive neurons were mainly located in the superficial dorsal horn and around the central canal of the L3-L5 spinal cord. Intrathecal injection of 50 µg NOS inhibitor NG-monomethyl-l-arginine (L-NMMA) attenuated cancer-evoked pain behaviors at day 14. These findings indicated that an upregulation of nNOS and iNOS in the spinal cord is associated with bone cancer pain and suggests that exogenously administered L-NMMA may have beneficial effects to alleviate bone cancer pain.

Interaction between NMDA glutamatergic and nitrergic enteric pathways during in vitro ischemia and reperfusion

Nitric oxide (NO) and glutamate, via N-methyl-d-aspartate (NMDA) receptors, participate to changes in neuromuscular responses after ischemic/reperfusion (I/R) injury in the gut. In the present study we investigated the existence of a possible interplay between nitrergic and NMDA receptor pathways in the guinea pig ileum after in vitro I/R injury, resorting to functional and biomolecular approaches. In normal metabolic conditions NMDA concentration-dependently enhanced both glutamate (analyzed by high performance liquid chromatography with fluorimetric detection) and NO (spectrophotometrically quantified as NO2− and NO3−) spontaneous overflow from isolated ileal segments. Both effects were reduced by the NMDA antagonists, (-)-AP5 (10µM) and 5,7-diCl-kynurenic acid (10µM, 5,7-diCl-KYN). Nω-propyl-l-arginine (1µM, NPLA) and 1400W (10µM), respectively, nNOS and iNOS inhibitors, reduced NMDA-stimulated glutamate overflow. After in vitro I/R, glutamate overflow increased, and returned to control values in the presence of NPLA and 1400W. NO2− and NO3− levels transiently increased during I/R and were reduced by both (-)-AP5 and 5,7-diCl-KYN. In longitudinal muscle myenteric plexus preparations, iNOS mRNA and protein levels increased after in vitro I/R; both parameters were reduced to control values by (-)-AP5 and 5,7-diCl-KYN. Both antagonists were also able to reduce ischemia-induced enhancement of nNOS mRNA levels. Protein levels of GluN1, the ubiquitary subunit of NMDA receptors, increased after I/R and were reduced by both NPLA and 1400W. On the whole, this data suggests the existence of a cross-talk between NMDA receptor and nitrergic pathways in guinea pig ileum myenteric plexus, which may participate to neuronal rearrangements occurring during I/R.

Lactobacillus casei-01 facilitates the ameliorative effects of proanthocyanidins extracted from lotus seedpod on learning and memory impairment in scopolamine-induced amnesia mice.

Learning and memory abilities are associated with alterations in gut function. The two-way proanthocyanidins-microbiota interaction in vivo enhances the physiological activities of proanthocyanidins and promotes the regulation of gut function. Proanthocyanidins extracted from lotus seedpod (LSPC) have shown the memory-enhancing ability. However, there has been no literature about whether Lactobacillus casei-01 (LC) enhances the ameliorative effects of LSPC on learning and memory abilities. In this study, learning and memory abilities of scopolamine-induced amnesia mice were evaluated by Y-maze test after 20-day administration of LC (109 cfu/kg body weight (BW)), LSPC (low dose was 60 mg/kg BW (L-LSPC) and high dose was 90 mg/kg BW (H-LSPC)), or LSPC and LC combinations (L-LSPC+LC and H-LSPC+LC). Alterations in antioxidant defense ability and oxidative damage of brain, serum and colon, and brain cholinergic system were investigated as the possible mechanisms. As a result, the error times of H-LSPC+LC group were reduced by 41.59% and 68.75% relative to those of H-LSPC and LC groups respectively. LSPC and LC combinations ameliorated scopolamine-induced memory impairment by improving total antioxidant capacity (TAOC) level, glutathione peroxidase (GSH-Px) and total superoxide dismutase (T-SOD) activities of brain, serum and colon, suppressing malondialdehyde (MDA) level of brain, serum and colon, and inhibiting brain acetylcholinesterase (AchE), myeloperoxidase, total nitric oxide synthase and neural nitric oxide synthase (nNOS) activities, and nNOS mRNA level. Moreover, LC facilitated the ameliorative effects of H-LSPC on GSH-Px activity of colon, TAOC level, GSH-Px activity and ratio of T-SOD to MDA of brain and serum, and the inhibitory effects of H-LSPC on serum MDA level, brain nNOS mRNA level and AchE activity. These results indicated that LC promoted the memory-enhancing effect of LSPC in scopolamine-induced amnesia mice.

Drug‐induced constipation alters gut microbiota stability leading to physiological changes in the host (1118.2)

Gut microbes play a significant role in development and maintenance of intestinal functions, but can contribute to pathogenesis of gastrointestinal (GI) disorders when host‐microbiome relationship is perturbed. In this study we sought to examine how constipation affects microbiome, and how these alterations in turn affect the host. Adult C57Bl/6 mice were treated with 0.1% Loperamide in drinking water for 7 days to induce constipation. Loperamide‐treated mice had increased GI transit time, indicating constipation. Bacterial DNA analysis by HiSeq sequencing showed decreased Firmicutes and increased Bacteroidetes in constipated mice. Microbiome metabolic potential was measured with tetrazolium dye assay and showed shift toward increased sugar metabolism in Loperamide‐treated mice. Microbial metabolites were measured by GC‐MS, and showed lower butyrate concentration in constipated mice. To determine constipation effects on the host, we introduced microbiota from constipated donors to germ‐free recipients. These mice had delayed GI transit time. To determine mechanism for delayed GI transit we analyzed colon tissues by qRT‐PCR and western blotting analysis, and determined lower expression of nNos mRNA and protein levels in these mice. Overall, these results suggest that delayed GI transit shifts microbiome taxonomic and metabolic profile, and these changes further contribute to development of constipation.Grant Funding Source: NIH NIDDK T32 DK07074