nNOS mRNA Research Articles

Change in the Interstitial Cells of Cajal and nNOS Positive Neuronal Cells with Aging in the Stomach of F344 Rats

The gastric accommodation reflex is an important mechanism in gastric physiology. However, the aging-associated structural and functional changes in gastric relaxation have not yet been established. Thus, we evaluated the molecular changes of interstitial cell of Cajal (ICC) and neuronal nitric oxide synthase (nNOS) and the function changes in the corpus of F344 rats at different ages (6-, 31-, 74-wk and 2-yr). The proportion of the c-Kit-positive area in the submucosal border (SMB) and myenteric plexus (MP) layer was significantly lower in the older rats, as indicated by immunohistochemistry. The density of the nNOS-positive immunoreactive area also decreased with age in the SMB, circular muscle (CM), and MP. Similarly, the percent of nNOS-positive neuronal cells per total neuronal cells and the proportion of nNOS immunoreactive area of MP also decreased in aged rats. In addition, the mRNA and protein expression of c-Kit and nNOS significantly decreased with age. Expression of stem cell factor (SCF) and the pan-neuronal marker PGP 9.5 mRNA was significantly lower in the older rats than in the younger rats. Barostat studies showed no difference depending on age. Instead, the change of volume was significantly decreased by L-NG63-nitroarginine methyl ester in the 2-yr-old rats compared with the 6-wk-old rats (P = 0.003). Taken together, the quantitative and molecular nNOS changes in the stomach might play a role in the decrease of gastric accommodation with age.

The Effects of Auricular Electro-Acupuncture on Ameliorating the Dysfunction of Interstitial Cells of Cajal Networks and nNOSmRNA Expression in Antrum of STZ-Induced Diabetic Rats.

BackgroudInterstitial cells of Cajal (ICCs) and nNOS play a crucial role in diabetic gastrointestinal dysmotility(DGD). Our previous study found that electro-acupuncture(EA) on ear point ‘stomach’ could repair the gastric dysrhythmias in rats induced by rectal distention(RD) after meal. However, little were known about the possible effect of auricular electro-acupuncture (AEA) on diabetic rats. Thus, we designed this study to investigate the effect of AEA on streptozotocin(STZ)-induced diabetic rats.MethodForty male Sprague_Dawley (SD) rats were injected with STZ, at the end of 8th week after injection, animals were randomly divided into four groups and received 2 weeks-treatment(10 times) respectively: control group(CON,n = 10, no stimulation), sham auricular electro-acupuncture group(SEA,n = 10, low frequency EA on earlobes), auricular eletro-acupuncture group(AEA,n = 10, low frequency EA on ear point ‘stomach’), and ST-36 group(ST-36,n = 10, low frequency EA on ST-36). Gastrointestinal (GI) motility was measured by GI transit rate. ICCs(c-kit+ expression) in antrum were analyzed by Immunohistochemistry and western blotting. NO level in blood serum were detected by Griess Reagent, and nNOSmRNA expression in antrum were determined by Real-time PCR.ResultsGI transit rate and ICCs(c-kit+ expression) in antrum of AEA group have the tendency to increase compared with CON group, but had no statistics difference (P>0.05). nNOSmRNA expression in antrum of AEA group was dramatically increased compared with CON group (P = 0.037).ConclusionsLow frequency EA on ear ‘stomach’ point could significantly up-regulate nNOS mRNA expression and ameliorate the ICCs networks partly in gastric antrum of STZ -induced diabetic rats, which may has benefits on regulating the GI motility.

Investigation of effects of albiflorin and paeoniflorin on hippocampal BDNF and NO in chronic restraint stress rats

This paper aimed to investigate the antidepressant-like effects and the mechanism of the albiflorin, paeoniflorin on rats with chronic restraint stress model. Fluoxetine and Xiaoyao group served as the positive control, body weight, sucrose preference test and the open field behavioral experiment were measured, the levels of nitric oxide (NO) in hippocampus were detected by Greg colorimetric method. Furthermore, the levels of brain derived neurotrophic factor (BDNF) in hippocampus were detected by ELISA. Finally, the expressions of nNOS mRNA in hippocampus detected by RT-qPCR, the protein levels of nNOS and BDNF in hippocampus were detected by Western blot. Compared with the model group,the pass counts of paeoniflorin group(30 mg•kg⁻¹) and albiflorin group(30 mg•kg⁻¹) were obviously increased(P<0.05,P<0.01). Furthermore, compared with the model group,the levels of NO in paeoniflorin groups(30 mg•kg⁻¹ and 15 mg•kg⁻¹) and albiflorin groups(30 mg•kg⁻¹ and 15 mg•kg⁻¹) were all significantly decreased(P<0.05,P<0.01; P<0.05, P<0.05). BDNF levels of paeoniflorin group(30 mg•kg⁻¹) and albiflorin group(30 mg•kg⁻¹) were obviously increased(P<0.05). Finally, compared with the model group, the expressions of nNOS mRNA of paeoniflorin groups(30 mg•kg⁻¹ and 15 mg•kg⁻¹) (P<0.01, P<0.01) and albiflorin groups(30 mg•kg⁻¹) were significantly decreased(P<0.05). Compared with the model group, the protein exprsssions of BDNF of paeoniflorin group(30 mg•kg⁻¹) and albiflorin group(30 mg•kg⁻¹) were obviously increased(P<0.05). Albiflorin and paeoniflorin have the effects of smooth the liver and dispel stagnation, the mechanism has the relevant with adjusting and controlling the expression of NO and BDNF in hippocampus.

Response of the Nitric Oxide System to Hypobaric Hypoxia in the Aged Striatum

Background: Nitric oxide (NO) appears to play a key role in the hypoxic injury to the brain. We have previously reported that hypoxia/reoxygenation downregulated NO synthases (NOS) in the adult striatum. Until now, no data were available concerning the influence of aging in conjunction with hypoxia/reoxygenation on the NO system in the striatum. Objective: The aim of this study was to assess the role of the NO pathway in the hypoxic aged striatum. Methods: Wistar rats 24-25 months old were submitted to hypobaric hypoxia (20 min)/reoxygenation (0 h, 24 h, 5 days). Expression (PCR, immunohistochemistry/image analysis) and activity (NADPH-diaphorase/image analysis) of NOS isoforms (neuronal NOS or nNOS, endothelial NOS or eNOS, inducible NOS or iNOS) were analyzed together with nitrated protein expression (immunohistochemistry/image analysis). NO levels were indirectly quantified as nitrates/nitrites (NOx). Results: The mRNA levels of NOS isoforms were undetectable at 0 h after hypoxia in the striatum compared to the control. At later reoxygenation times, nNOS mRNA decreased, while eNOS mRNA augmented. Protein levels of nNOS and eNOS rose at 24 h after hypoxia, and iNOS protein increased at 5 days. NOx levels remained unchanged, whereas in situ NOS activity and protein nitration diminished during reoxygenation in the aged striatum. Conclusion: The aged striatum may overexpress NOS isoforms as a neuroprotective-adaptive mechanism to hypoxia. However, this mechanism may not work properly in the aged striatum, since no changes in NO levels were detected after hypoxia. This may be related to the low activity of NOS isoforms in the hypoxic striatum.

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.

Altered Arginine Metabolism in Cells Transfected with Human Wild-Type Beta Amyloid Precursor Protein (βAPP).

Alterations of enzymes linked to arginine metabolism have been recently implicated in Alzheimer&#039;s disease (AD). Despite strong association of arginine changes with nitric oxide (NO) pathway, the impact of amyloid β (Aβ) peptides on arginine degradation and re-synthesis is unknown. In the present study we compared expression levels of arginases (ARG1, ARG2), neuronal, endothelial and inducible NO synthase isoforms (NNOS, ENOS, INOS), enzymes that metabolize arginine or resynthesize it from citrulline and the levels of corresponding amino acids in rat pheochromocytoma (PC12) cells overexpressing human Aβ precursor protein (APPwt cells). Moreover, we investigated the changes in miRNAs responsible for modulation of arginine metabolism in AD brains. Real-time PCR analysis revealed in APPwt cells significant decreases of ARG1 and ARG2 which are responsible for lysing arginine into ornithine and urea; this reduction was followed by significantly lower enzyme activity. NNOS and ENOS mRNAs were elevated in APPwt cells while iNOS was undetectable in both cell lines. The expression of argininosuccinate synthase (ASS) that metabolizes citrulline was down-regulated without changes in argininosuccinate lyase (ASL). Ornithine decarboxylase (ODC), which decarboxylates ornithine to form putrescine was also reduced. Arginine, the substrate for both arginases and NOS, was unchanged in APPwt cells. However, citrulline concentration was significantly higher. Elevated miRNA-9 and miRNA-128a found in AD brain tissues might modulate the expression of ASS and NOS, respectively. Our results indicate that Aβ affects arginine metabolism and this influence might have important role in the pathomechanism of AD.

Electroacupuncture at Zusanli (ST36) ameliorates colonic neuronal nitric oxide synthase upregulation in rats with neurogenic bowel dysfunction following spinal cord injury

Study designExperimental study.ObjectiveTo determine the effects of electroacupuncture (EA) at Zusanli (ST36) on colonic motility and neuronal nitric oxide synthase (nNOS) expression in rats with neurogenic bowel dysfunction (NBD) after spinal cord injury (SCI).SettingSecond School of Clinical Medical, Nanjing University of Chinese Medicine, Jiangsu, China.MethodsWe divided 30 adult Sprague-Dawley rats into a sham group (10 rats), a model group (SCI alone, 10 rats) and a EA group (SCI+EA at ST36, 10 rats). Defecation time was recorded as the time from activated carbon administration (on day 15) to evacuation of the first black stool. Immunohistochemical, real-time PCR and western blot analyses were performed to assess changes in nNOS-immunoreactive cells, and nNOS messenger RNA (mRNA) and protein, respectively, after 14 experimental days.ResultsDefecation time was lower in the EA group than in the model group (P<0.01). On immunohistochemical analysis, nNOS was localized in the myenteric plexus of the colon. The number of nNOS-immunoreactive cells and the intensity of nNOS staining were greater in the model group than in the sham group and lesser in the EA group than in the model group. Consistent with the immunohistochemical findings, nNOS mRNA and protein expression was higher in the model group than in the sham group and lower in the EA group than in the model group (P<0.05 for both).ConclusionIncreased colonic nNOS expression can induce/aggravate NBD in SCI rats. EA at ST36 ameliorated NBD, possibly by downregulating colonic nNOS expression.

Loss of Myocardial nNOS Mediated by Upregulation of miR-31 in Human Atria Contributes to Begetting of Atrial Fibrillation

Rationale: Neuronal nitric oxide synthase (nNOS) expression and activity in atria are markedly reduced from patients and goats with atrial fibrillation (AF). Whether loss of nNOS contributes to AF-induced atrial electrical remodelling and its upstream mechanism remain unclear.Methodology: Whole-cell patch clamp was used to record action potentials (APs) and ion currents. N/n: number of patients or mice/ number of myocytes.Results: Inhibition of nNOS by S-methylthiocitrulline (SMTC, N/n: 12/45) induces a significant reduction in AP duration (APD) and APD rate-dependent adaptation in human right atrial myocytes with sinus rhythm (SR, N/n: 14/52). In mice, nNOS inhibition (N/n: 4/9) or nNOS-/- (N/n: 9/28) reduce APD50 and 90 by 53% and 35%, respectively.Ionic investigations show SMTC increases atrial IKur by 60%, Ito by 34% and IK1 by 27% with no change in ICa or IKr. Computer modelling shows mimicking SMTC-induced increases in IKur and IK1 successfully retrieves experimental phenotype. Furthermore blocking IKur abolishes the effect of nNOS inhibition on APD and APD rate-dependent adaptation (SMTC: N/n: 6/11 vs vehicle: N/n: 8/16). Additionally, In vivo atrial burst stimulation shows nNOS-/- mice exhibit 2 folds higher AF inducibility.In AF, upregulation of atrial specific miR-31 results in accelerating nNOS mRNA and protein decay. Inhibition of miR-31 recovers the SMTC suppressed APD90 (N/n: 3/12 in each group) and APD rate-dependent adaptation. These effects are NO-mediated as they are reversed by SMTC. Whereas in SR patients, increasing miR-31(N/n: 4/30) reduces nNOS, shortens APD and suppresses APD rate-dependent adaptation (N/n: 3/14).Conclusions: In human and mammalian atrial myocytes, a reduction in nNOS, mediating by upregulation of miR31, is an important factor retrieving key hallmarks of atrial electrical remodelling and contributing to the atrial phenotype begetting AF.

Naoxintong Protects Primary Neurons from Oxygen-Glucose Deprivation/Reoxygenation Induced Injury through PI3K-Akt Signaling Pathway.

Naoxintong capsule (NXT), developed from Buyang Huanwu Decoction, has shown the neuroprotective effects in cerebrovascular diseases, but the neuroprotection mechanisms of NXT on ischemia/reperfusion injured neurons have not yet been well known. In this study, we established the oxygen-glucose deprivation/reoxygenation (OGD/R) induced neurons injury model and treat the neurons with cerebrospinal fluid containing NXT (BNC) to investigate the effects of NXT on OGD/R induced neurons injury and potential mechanisms. BNC improved neuron viability and decreased apoptotic rate induced by OGD/R. BNC attenuated OGD/R induced cytosolic and mitochondrial Ca2+ overload, ROS generation, intracellular NO levels and nNOS mRNA increase, and cytochrome-c release when compared with OGD/R group. BNC significantly inhibited both mPTP opening and ΔΨm depolarization. BNC increased Bcl-2 expression and decreased Bax expression, upregulated the Bcl-2/Bax ratio, downregulated caspase-3 mRNA and caspase-9 mRNA expression, and decreased cleaved caspase-3 expression and caspase-3 activity. BNC increased phosphorylation of Akt following OGD/R, while LY294002 attenuated BNC induced increase of phosphorylated Akt expression. Our study demonstrated that NXT protected primary neurons from OGD/R induced injury by inhibiting calcium overload and ROS generation, protecting mitochondria, and inhibiting mitochondrial apoptotic pathway which was mediated partially by PI3K-Akt signaling pathway activation.

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.

Mitogen-activated protein kinases regulate expression of neuronal nitric oxide synthase and neurite outgrowth via non-classical retinoic acid receptor signaling in human neuroblastoma SH-SY5Y cells

We have previously shown that retinoic acid receptor (RAR) stimulation by an agonist Am80 recruits nitric oxide-dependent signaling via increased expression of neuronal nitric oxide synthase (nNOS) in rat midbrain slice cultures. Using neuroblastoma SH-SY5Y cells, here we investigated the mechanisms of RAR-induced nNOS expression, together with relationship between nNOS expression and neurite outgrowth. Am80 promoted neurite outgrowth, which was attenuated by inhibitors of phosphoinositide 3-kinase (PI3K; LY294002), c-Jun N-terminal kinase (JNK; SP600125) and p38 mitogen-activated protein kinase (p38 MAPK; SB203580). A selective nNOS inhibitor 3-bromo-nitroindazole also suppressed Am80-induced neurite outgrowth. Am80-induced increase in nNOS protein expression was attenuated by LY294002, SP600125 and SB203580, whereas increase in nNOS mRNA expression was attenuated only by LY294002. Am80-induced activation of JNK and p38 MAPK was blocked by LY294002, suggesting that these kinases acted downstream of PI3K. We also confirmed that DAX1, a nuclear receptor reported to regulate nNOS expression, was up-regulated in response to Am80. siRNA-mediated knockdown of DAX1 abrogated Am80-induced nNOS expression and neurite outgrowth. These results reveal for the first time that nNOS expression is crucial for RAR-mediated neurite outgrowth, and that non-genomic signaling such as JNK and p38 MAPK is involved in RAR-mediated nNOS expression.

Abstract 226: Neuronal Nitric Oxide Synthase Maintains Endothelial NO Release after Inhibition of NADPH Oxidase 4

The NADPH oxidase isoform NOX4 mainly produces H 2 O 2 . Previously we could show, that NOX4 is the major endothelial NOX isoform and constitutively active. Regulation of NOX4 and formation of reactive oxygen species is involved in NO release. The transcription factor NRF2 is a key mediator of cellular adaptation to redox stress. Therefore, regulation of NOX4 on the transcriptional level by NRF2 might be directly linked to NO release and endothelial function. Besides endothelial nitric oxide synthase (eNOS), a role of neuronal nitric oxide synthase (nNOS) in endothelial NO and H 2 O 2 release has been proposed. In this study, endothelial cells (HUVEC) were constantly exposed to high laminar shear stress (24 h, 30 dyn/cm°). Application of shear stress stimulated NO formation and induced elongation of endothelial cells in the direction of flow. Lentiviral overexpression of NOX4 strongly increased H 2 O 2 release, while downregulation using shNOX4 decreased H 2 O 2 release. Furthermore, application of shear stress caused downregulation of NOX4 as well as upregulation of eNOS and antioxidative response via NRF2 and its target genes NQO-1 and HMOX-1. H 2 O 2 can increase NO release by eNOS. We could show that downregulation of NOX4 leads to upregulation of eNOS mRNA and protein expression accompanied by attenuation of NRF2 pathway under flow conditions. Determination of NO release confirmed these results. This supports a compensatory mechanism maintaining a stable NO release after NOX4 inhibition in response to flow. Attenuation of NRF2 by shNRF2 inhibited shear stress-dependent induction of NRF2 and its target genes. In addition, shNRF2 enhanced the shear stress-dependent downregulation of NOX4. Finally, we detected a stable mRNA and protein expression of nNOS in endothelial cells, unaffected by flow. Interestingly, downregulation of NOX4 as well as of NRF2 resulted in strong induction of nNOS expression. In conclusion, our data suggest an important role of H 2 O 2 production by NADPH oxidase 4 in maintaining NO release and endothelial function in response to flow. Inactivation of NADPH oxidase 4 attenuates NRF2 pathway and increases neuronal nitric oxide synthase as compensatory mechanisms.

Molecular characterization and hypoxia-induced upregulation of neuronal nitric oxide synthase in Atlantic croaker: Reversal by antioxidant and estrogen treatments

Neuronal nitric oxide synthase (nNOS) catalyzes production of nitric oxide in vertebrate brains. Recent findings indicate that endothelial NOS and reactive oxygen species (ROS) are significantly increased during hypoxic stress and are modulated by antioxidants. However, the influence of antioxidants and steroids on nNOS upregulation by hypoxia is largely unknown. In this study, we characterized nNOS cDNA and examined the effects of hypoxia and antioxidant and steroid treatments on nNOS expression in Atlantic croaker hypothalamus. Hypoxia exposure (dissolved oxygen, DO: 1.7mg/L for 2 and/or 4weeks) caused significant increases in hypothalamic nNOS mRNA, protein and its neuronal expression. Hypothalamic nNOS expression and superoxide radical (O2−, an index of ROS) production were increased by pharmacological treatment of fish exposed to normoxic conditions with N-ethylmaleimide, an alkene drug which covalently modifies sulfhydryl groups and inhibits aromatase activity. In contrast, treatments with Nω-nitro-l-arginine methyl ester, a competitive NOS-inhibitor, or vitamin E, an antioxidant, prevented the upregulation of O2− production and nNOS expression in hypoxia-exposed (DO: 1.7mg/L for 4weeks) fish. Moreover, treatment with 1,4,6-androstatrien-3,17-dione, an aromatase inhibitor, increased hypothalamic O2− production and nNOS expression in normoxic control fish; whereas estradiol-17β treatment significantly reduced O2− production and nNOS expression in hypoxia-exposed fish. Double-labeled immunohistochemical results showed that nNOS and aromatase proteins are co-expressed in the hypothalamus. Taken together, the results suggest that upregulation of nNOS and ROS in the croaker hypothalamus in response to hypoxia is influenced by antioxidant and overall estrogen status.

Estrogen-dependent post-translational change in the nitric oxide system may mediate the leptin action on LH and prolactin secretion

Gonadotrophin-releasing hormone (GnRH) neurons do not express the leptin receptor (OB-R) in the medial preoptic area (MPOA) and the medial basal hypothalamus (MBH). We assessed whether the effect of leptin on the secretion of luteinizing hormone (LH), follicle stimulating hormone (FSH) and prolactin (PRL) in proestrus could be mediated by nitric oxide (NO) under estrogen modulation. Female rats were treated with an estrogen antagonist (tamoxifen s.c. 3mg/rat) or vehicle during metestrus and diestrus. At proestrus, they received leptin (3 or 10μg/μl) or intracerebroventricular saline at 11:00am and were decapitated at 5:00pm. The following were analyzed in this work: plasma LH, FSH and PRL levels (radioimmunoassay); neuronal NO-synthase (nNOS) and OB-R transcription (RT-PCR); nNOS and phosphorylated nNOS (pnNOS) translation levels (western blotting); and pSTAT3 immunoreactivity. Tamoxifen reduced the plasma LH and PRL levels and decreased the nNOS mRNA and pnNOS expression in the MPOA. Three micrograms of leptin increased the LH secretion and pnNOS protein levels in the MPOA and MBH. Ten micrograms of leptin decreased the transcription, translation and phosphorylation of nNOS in the MPOA. In the MBH, 10μg of leptin increased the protein expression of nNOS but not the mRNA expression neither pnNOS protein. Tamoxifen did not change either the mRNA or protein expression of nNOS or the phosphorylation of nNOS but decreased the number of cells that contained pSTAT3 immunoreactivity in both areas. In conclusion, the stimulatory effect of leptin on the secretion of LH and PRL on the afternoon of proestrus may be mediated by estrogen-dependent post-translational changes in the nNOS in the MPOA and MBH.

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.

Induced nitric oxide synthase and diabetic cardiomyopathy

Introduction: Nearly 80% of deaths associated with type 2 diabetes mellitus are attributed to cardiac complications. Diabetics tend to develop heart failure in the absence of risk factors such as hypertension and coronary artery disease. Nitric oxide (NO) is constitutively or inductively synthesized by nitric oxide synthase, which has three isoforms, endothelial (eNOS), neuronal (nNOS) and inducible (iNOS). eNOS has effects that include regulation of heart rate, contraction, diastolic relaxation and oxygen consumption. nNOS regulates the activity of the sarcoplasmic reticulum Ca2+ ATPase activator and the L-type Ca2+ channel. Both eNOS and nNOS are considered to be cardioprotective. In contrast, iNOS is induced by stress, including hyperglycaemia and oxidative stress, and causes nitration of skeletal proteins resulting in impairment of myofilament function. The high concentrations of NO produced also form peroxynitrite, an oxidant that causes tissue damage. Myocardial damage during ischaemia reperfusion in hyperglycaemia is abolished in iNOS-knockout mice, confirming the importance of iNOS in the development of cardiomyopathy. Objectives: To investigate expression of nNOS and iNOS in two models of type 2 diabetes mellitus in rats. Materials and methods: Expression of nNOS and iNOS mRNA were measured using fast real-time reverse transcription polymerase chain reaction in cardiac tissue from 8-month-old Goto– Kakizaki lean diabetic (GK) and Zucker diabetic fatty (ZDF) rats. Results: Expression of nNOS was modestly, but significantly, increased in GK (P <0.0001) and ZFD rats (P <0.001). In contrast, iNOS expression was massively increased by 15-fold in GK rats (P <0.0001). Conclusions: Modest up-regulation of nNOS in both models suggests a cardioprotective effect. However, the massive up-regulation of expression of cardiac iNOS in type 2 diabetes mellitus supports the concept that this inducible isoform is involved in the development of diabetic cardiomyopathy.

Abstract 17767: A MiR-31-dependent Loss of Dystrophin &amp; Nnos in the Human Atria Plays a Key Role in Atrial Fibrillation-induced Electrical Remodelling

Background: Management of atrial fibrillation (AF) remains a challenge. AF remodels the atrial electrical properties, thereby increasing resistance to treatment. Although remodeling has long been a target for therapeutic intervention in AF, the mechanisms driving this phenomenon are incompletely understood. Methods &amp; Results: Using atrial samples from 351 patients (73 AF), and 67 goats (34 AF), we show that atrial-specific upregulation of miR31 causes dystrophin (DYS) translational repression and accelerates mRNA degradation of neuronal nitric oxide synthase (nNOS) leading to a profound reduction in NO availability. Six prediction algorithms and reporter assays established DYS and nNOS as miR-31 targets. In atrial myocytes from patients with AF (hAFm), both DYS and nNOS bind to miR-31 within the RNA induced silencing complex (RISC). In actinomycin D-treated myocytes from patients in sinus rhythm (hSRm), miR31 accelerated nNOS (but not DYS) mRNA decay. mRNA of nNOS (but not DYS) &amp; protein content of nNOS, DYS &amp; DYS associated proteins were significantly reduced in hAFm. Inhibition of miR31 in hAFm restored both DYS &amp; nNOS protein. Protection of the nNOS miR31 binding site with a target site blocker (TSB) restored nNOS mRNA and protein (but not DYS), whereas, DYS-TSB increased both DYS and nNOS protein (but not mRNA), in keeping with an effect of DYS restoration on nNOS protein stability. Indeed, K48-linked polyubiquitination of nNOS was increased in hAFm &amp; prevented by proteasome inhibition with MG132. NOS: inhibition (SMTC, 100 nmol) or transfection with a miR31 mimic was employed to evaluate the impact of these findings on atrial electrical properties. Both interventions shortened action potential duration (APD90) and abolished rate-dependency of the APD90 in hSRm but not in hAFm. By contrast, miR31 inhibition restored APD &amp; APD rate-dependency in hAFm (both reversed by SMTC) but had no effect on hSRm. In mice (n=126), nNOS gene deletion or inhibition shortened atrial APD90 &amp; lead to a 2-fold increase in AF inducibility in response to atrial burst pacing. Conclusions: These findings identify atrial-specific upregulation of miR31 in human AF as a key mechanism causing atrial loss of dystrophin and nNOS, which, in turn, lead to the electrical phenotype begetting AF.

Abstract 15396: Maintenance of Endothelial NO Release after Inhibition of NADPH Oxidase 4

The NADPH oxidase isoform NOX4 mainly produces H 2 O 2 . Previously we could show, that NOX4 is the major endothelial NOX isoform and constitutively active. Regulation of NOX4 and formation of reactive oxygen species are involved in NO release. The transcription factor NRF2 is a key mediator of cellular adaptation to redox stress. Therefore, regulation of NOX4 on the transcriptional level by NRF2 might be directly linked to NO release and endothelial function. Besides endothelial nitric oxide synthase (eNOS), a role of neuronal nitric oxide synthase (nNOS) in endothelial NO and H 2 O 2 release has been proposed. In this study, endothelial cells (HUVEC) were constantly exposed to high laminar shear stress (24 h, 30 dyn/cm 2 ). Application of shear stress stimulated NO formation and induced elongation of endothelial cells in the direction of flow. Lentiviral overexpression of NOX4 strongly increased endothelial H 2 O 2 release, while downregulation using shNOX4 decreased H 2 O 2 release. Furthermore, application of shear stress caused downregulation of NOX4 as well as upregulation of eNOS and antioxidative response via NRF2 and its target genes NQO-1 and HMOX-1. H 2 O 2 can affect NO release by eNOS. We could show that downregulation of NOX4 leads to upregulation of eNOS mRNA and protein expression accompanied by attenuation of NRF2 pathway under flow conditions. Determination of NO release confirmed these results. This supports a compensatory mechanism maintaining a stable NO release after NOX4 inhibition in response to flow. Attenuation of NRF2 by shNRF2 inhibited shear stress-dependent induction of NRF2 mRNA and protein expression and its target genes. In addition, shNRF2 enhanced the shear stress-dependent downregulation of NOX4. Finally, we detected a stable mRNA and protein expression of nNOS in endothelial cells, which was unaffected by flow. Interestingly, downregulation of NOX4 resulted in induction of nNOS expression. In conclusion, our data suggest an important role of H 2 O 2 production by NADPH oxidase 4 in maintaining NO release and endothelial function in response to flow. Inactivation of NADPH oxidase 4 attenuates NRF2 pathway and increases neuronal nitric oxide synthase as compensatory mechanisms.

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.

Role of nitric oxide synthase uncoupling at rostral ventrolateral medulla in redox-sensitive hypertension associated with metabolic syndrome.

Metabolic syndrome (MetS), which is rapidly becoming prevalent worldwide, is long known to be associated with hypertension and recently with oxidative stress. Of note is that oxidative stress in the rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons reside, contributes to sympathoexcitation and hypertension. This study sought to identify the source of tissue oxidative stress in RVLM and their roles in neural mechanism of hypertension associated with MetS. Adult normotensive rats subjected to a high-fructose diet for 8 weeks developed metabolic traits of MetS, alongside increases in sympathetic vasomotor activity and blood pressure. In RVLM of these MetS rats, the tissue level of reactive oxygen species was increased, nitric oxide (NO) was decreased, and mitochondrial electron transport capacity was reduced. Whereas the protein expression of neuronal NO synthase (nNOS) or protein inhibitor of nNOS was increased, the ratio of nNOS dimer/monomer was significantly decreased. Oral intake of pioglitazone or intracisternal infusion of tempol or coenzyme Q10 significantly abrogated all those molecular events in high-fructose diet-fed rats and ameliorated sympathoexcitation and hypertension. Gene silencing of protein inhibitor of nNOS mRNA in RVLM using lentivirus carrying small hairpin RNA inhibited protein inhibitor of nNOS expression, increased the ratio of nNOS dimer/monomer, restored NO content, and alleviated oxidative stress in RVLM of high-fructose diet-fed rats, alongside significantly reduced sympathoexcitation and hypertension. These results suggest that redox-sensitive and protein inhibitor of nNOS-mediated nNOS uncoupling is engaged in a vicious cycle that sustains the production of reactive oxygen species in RVLM, resulting in sympathoexcitation and hypertension associated with MetS.