Constitutive Nitric Oxide Synthase Research Articles

Roles of nNOS and eNOS in rat heart ; comparison between the left and right ventricle myocytes

Right ventricle (RV) has physiological as well as anatomical differences compared with left ventricle (LV). There are constitutive NO synthase isoforms, neuronal NOS (nNOS) and endothelial NOS (eNOS) in myocardium, regulating the calcium dynamics and contractility. Here we compared the roles of nNOS and eNOS in the RV and LV myocytes from S‐D rats. After isolating the myocytes using Langendorff perfusion system, their sarcomere shortening and [Ca2+]i were simultaneously measured using the Ionoptix system while paced by 2 Hz field stimulation. nNOS‐specific inhibitor, S‐methyl‐L‐thiocitrulline (SMTC) and the non‐specific NOS inhibitor, L‐NG‐Nitroarginine methyl ester (L‐NAME) were used to pharmacologically dissect the roles of NOS isoforms.According to the immunoblot assay, the protein amounts of nNOS and its phosphorylated form were not different between RV and LV. Although the amount of eNOS was lower in RV than LV, its phosphorylated form was similar. RV myocytes showed slower contraction (longer TPeak) and slower relaxation (longer Trelax) than LV myocytes. The treatment with SMTC or L‐NAME shortened TPeak and Trelax of RV myocytes, abolishing the kinetic differences of ΔSL. It was notable that the Trelax of LV myocytes became slightly longer by SMTC. Paradoxically, the length of sarcomere shortening (ΔSL) was increased while the amplitude of calcium transient (Δ[Ca2+]i) was decreased by SMTC in RV myocytes. However, SMTC did not affect the ΔSL and Δ[Ca2+]i of LV myocytes. Our study suggest that the slower contraction and relaxation of RV myocytes might be partly due to differential responses to the nNOS‐dependent intracellular signaling. The opposite direction of changes in the amplitudes of ΔSL and Δ[Ca2+]i by SMTC suggest that the nNOS in RV myocytes is responsible for negative inotropy despite the positive effects on the calcium transient, which is not evident in LV myocytes.

Isoform profile of NOS enzyme in structure of rats’ solitary-vagal complex in arterial hypertension of various origin

The aim of this study was to characterize the nitric oxide isoforms profile in rats’ nucleus tractus solitarii (NTS) and dorsal motor nucleus (DMN) of vagus nerve in arterial hypertension (AH) of various origin (essential (EAH) – rats of SHR line, and endocrine-salt AH). Materials and methods. The study was performed on 30 aged male rats. Among them, 20 Wistar rats were divided into two groups – control (10 rats) and 10 rats with simulated endocrine-salt AH (ESAH) and 10 SHR rats. An immunohistochemical method was used to study the of nitric oxide synthase isoforms expression features in DMN and NTS. The following parameters were determined: the content of immunoreactive material (IRM) for the studied peptides (U if ), the relative area of the IRM (%) and the IRM concentration in 1 μm 2 (U if /μm 2 ). Results. It was found that in rats with both models of AH, the expression indices of all three nitric oxide synthase (NOS) isoforms in the studied structures increased. In our opinion, this is due to the activation of the studied structures in AH conditions. This must be considered as an important element of high blood pressure compensating. It is achieved through the implementation by a complex of mechanisms like reducing of the sympathetic tone and increasing of the parasympathetic tone by activating a system of secondary messengers; improving neurotrophy due to the high activity of constitutive NOS isoforms; iNOS-mediated NO overproduction, as a factor in compensating for its bioavailability in conditions of local ischemia in AH. Conclusions. Regardless of the AH etiopathogenesis in both experimental groups, the expression of all three NOS isoforms increases in the DMN and NTS structures. In rats with EAH in the DMN and NTS structures, the expression indices of NOS isoforms have their own characteristics. So in the first structure, the largest changes in the indices of the immunoreactive material content and concentration are observed for eNOS, and the relative area for iNOS. At the same time, in the NTS structure, the largest changes in the content indices are observed for iNOS, and the concentration and relative area for eNOS. In rats with ESAH in the DMN structure, the highest changes in the indices of IRM content and concentration are observed for the endothelial isoform of NOS, and the relative area – for inducible. In the NTS structure, the IRM content changed the most for nNOS, and the concentration and relative area for iNOS.

Modeling of Single-Walled Carbon Nanotube Binding to Nitric Oxide Synthase and Guanylate Cyclase Molecular Structures

Previously, we have demonstrated that water dispersible single-walled carbon nanotubes (SWCNTs) may be used in low therapeutic doses in antihypertensive therapy as promising agents capable of activating constitutive nitric oxide synthase (NOS) in spontaneously hypertensive rats, thus increasing the NO production in central and peripheral elements of the cardiovascular system [1]. Here we confirm this effect by docking and molecular dynamics simulations, clearly showing that SWCNTs may interact with NOS and guanylate cyclase molecular structures.

Systemic Administrations of Water-Dispersible Single-Walled Carbon Nanotubes: Activation of NOS in Spontaneously Hypertensive Rats

Priority data have been obtained on the effects of repeated systemic administrations of water-dispersible single-walled carbon nanotubes (SWCNTs) to spontaneously hypertensive rats with respect to constitutive NO-synthase (cNOS). As is known, NO is an inhibitory transmitter in the cardiovascular system. It was found that the systemic (i.p., subcutaneous, and i.m.) introductions of SWCNTs during two weeks resulted in considerable elevations of the NO2– level (a marker of NO bioavailability) in the blood of experimental hypertensive animals. Thus, SWCNTs may be used in the future for antihypertensive therapy as a novel agent capable of activating cNOS and, thus, increasing the NO production in central and peripheral elements of the cardiovascular system.

Design of Light-Sensitive Triggers for Endothelial NO-Synthase Activation.

A specific light trigger for activating endothelial Nitric Oxide-Synthase (eNOS) in real time would be of unique value to decipher cellular events associated with eNOS activation or to generate on demand cytotoxic levels of NO at specific sites for cancer research. We previously developed novel tools called nanotriggers (NT), which recognized constitutive NO-synthase, eNOS or neuronal NOS (nNOS), mainly via their 2’ phosphate group which is also present in NADPH in its binding site. Laser excitation of NT1 bound to eNOS triggered recombinant NOS activity and released NO. We recently generated new NTs carrying a 2’ or 3’ carboxylate group or two 2’ and 3’ carboxylate moieties replacing the 2’ phosphate group of NADPH. Among these new NT, only the 3’ carboxylate derivative released NO from endothelial cells upon laser activation. Here, Molecular Dynamics (MD) simulations showed that the 3’ carboxylate NT formed a folded structure with a hydrophobic hub, inducing a good stacking on FAD that likely drove efficient activation of nNOS. This NT also carried an additional small charged group which increased binding to e/nNOS; fluorescence measurements determined a 20-fold improved affinity upon binding to nNOS as compared to NT1 affinity. To gain in specificity for eNOS, we augmented a previous NT with a “hook” targeting variable residues in the NADPH site of eNOS. We discuss the potential of exploiting the chemical diversity within the NADPH site of eNOS for reversal of endothelial dysfunction in cells and for controlled generation of cytotoxic NO-derived species in cancer tissues.

Stimulation of the endogenous hydrogen sulfide synthesis suppresses oxidative-nitrosative stress and restores endothelial-dependent vasorelaxation in old rats.

Hydrogen sulfide (H2S) is an endogenous gas transmitter with profound effects on the cardiovascular system. We hypothesized that stimulation of H2S synthesis might alleviate age-associated changes in vascular reactivity. Pyridoxal-5-phosphate (PLP), the coenzyme of H2S-synthesizing enzymes, was administrated to old male Wistar rats per os at a dose of 0.7 mg/kg body mass once a day for 2 weeks. H2S content in the aortic tissue, markers of oxidative stress, inducible nitric oxide synthase (iNOS) and constitutive nitric oxide synthase (cNOS), arginase activities, and endothelium-dependent vasorelaxation of the aortic rings were studied. Our results showed that PLP restored endogenous H2S and low molecular weight S-nitrosothiol levels in old rat aorta to the levels detected in adults. PLP significantly reduced diene conjugate content, hydrogen peroxide and peroxynitrite generation rates, and iNOS and arginase activity in the aortic tissue of old rats. PLP also greatly improved acetylcholine-induced relaxation of old rat aorta (47.7% ± 4.8% versus 18.4% ± 4.1% in old rats, P < 0.05) that was abolished by NO inhibition with N-nitro-l-arginine methyl ester hydrochloride (L-NAME) or H2S inhibition with O-carboxymethylhydroxylamine (O-CMH). Thus, PLP might be used for stimulation of endogenous H2S synthesis and correction of oxidative and nitrosative stress and vessel tone dysfunction in aging and age-associated diseases.

MECHANISMS OF NEUROPROTECTION BY MTOR INHIBITORS

The mammalian/mechanistic target-of-rapamycin (mTOR) inhibitor rapamycin, that delays aging in mice, halts and even reverses memory deficits, and restores cerebral blood flow (CBF), neuronal activation, and neurovascular coupling in models of Alzheimer’s disease (AD), cognitive dysfunction of atherosclerosis, and normative aging. Genetic reduction of TORC1 in neurons to levels similar to those achieved by rapamycin, promoted synaptic bouton remodeling, enhanced memory, and increased brain glucose metabolism. In AD mice, the restoration of CBF and neurovascular coupling by mTOR attenuation was dependent on the activation of both constitutive nitric oxide synthase (NOS) isoforms, possibly due to stabilization of their mRNAs. The mechanisms by which mTOR attenuation preserves brain healthspan may be common to different models of age-associated neurological disease. We singled out (a) ablation of NOS activity, and (b) synaptic bouton loss as key mechanisms by which TOR drives brain aging and contributes to the pathogenesis of dementias modeled in mice.

Therapeutic effects of iNOS inhibition against vitiligo in an animal model.

Nitric oxide (NO) is involved in several biological processes, but its role in human melanogenesis and vitiligo need further studies. Previous studies revealed that exposure to UVA and UVB were capable of the inducing nitric oxide production in keratinocytes and melanocytes through the activation of constitutive nitric oxide synthase, whereas inducible nitric oxide synthase overexpression has been reported to play an important role in hyperpigmentary disorders. The aim of this study was to evaluate iNOS inhibitor aminoguanidine (AG) as a therapeutic agent in our mouse model of vitiligo. In this study, male C57BL/6J Ler-vit/vit mice were purchased to evaluate the effect of iNOS inhibitor (aminoguanidine) (50 and 100 mg/kg) and L-arginine (100 mg/kg) in a mouse model of vitiligo induced by monobenzone 40%. Moreover, we used phototherapy device to treat the mice with NBUVB as a gold standard.The findings revealed that monobenzone was capable of inducing depigmentation after 6 weeks. However, aminoguanidine in combination with monobenzone was decrease the effect of monobenzone, while L-arginine play a key role in promoting the effect of monobenzone (P<0.001). Based on the phototherapy, the efficacy of phototherapy significantly increased by adding L-arginine (P<0.05). Taken together, we suggest that iNOS inhibitor can be a novel treatment for the prevention and treatment of vitiligo by combination of NBUVB therapy, furthermore; NO agents like L-arginine could also increase the effectiveness of phototherapy. Taken together, this pilot study showed significant repigmentation of vitiligous lesions treated with iNOS inhibitor plus NBUVB therapy, where other aspect including expression of an inducible iNOS, NO and TNF levels remained to be evaluated in mice model.

Abstract 867: NAD + Repletion Reverses HFpEF by Attenuating Myocardial Metabolic Dysfunction

Background: Heart failure with preserved ejection fraction (HFpEF) is a highly prevalent clinical condition associated with significant morbidity, mortality and health care expenses. Yet, no effective treatment has been identified. We recently demonstrated that concomitant metabolic and hypertensive stress in mice elicited by a combination of high fat diet (HFD) and constitutive nitric oxide synthase inhibition by N [w] -nitro-l-arginine methyl ester (L-NAME) faithfully recapitulates the numerous and myriad features of human HFpEF (paper in press at Nature ). Methods: Wild type C57Bl6 mice were fed with HFD and L-NAME via drinking water for 5-8 weeks. Myocardial mitochondrial morphology was assessed by electron microscopy. Mitochondrial function was assessed by measuring oxygen consumption rates using an oxygen electrode. Protein expression and modification were assessed by Western blotting and immunoprecipitation. Results: Using this novel mouse model, we discovered significant impairment of mitochondrial fatty acid oxidation (FAO) associated with hyperacetylation of key FAO enzymes in HFpEF myocardium. Mechanistically, downregulation of Sirtuin3, the major mitochondrial deacetylase, and deficiency of its co-substrate nicotinamide adenine dinucleotide (NAD + ), culminated in mitochondrial protein hyperacetylation. Strikingly, supplementation with nicotinamide riboside (NR), a NAD + precursor, led to dramatic improvement of mitochondrial function, and importantly, amelioration of the HFpEF phenotype. Conclusion: In summary, we have unveiled that protein hyperacetylation-mediated mitochondrial dysfunction is a crucial mechanism of HFpEF pathogenesis. This is, to our knowledge, the first study identifying a specific signature of metabolic remodeling in HFpEF heart. We also demonstrated the therapeutic effect of NAD + repletion in a preclinical HFpEF model. In next steps, confirming this benefit in a clinical trial is warranted.

Gaseous transmitters in human retinogenesis

Endogenous gaseous transmitters (nitric oxide, carbon monoxide, and hydrogen sulphide) form a special neuromodulation system mediating the development and modification of nerve centers. Here, we examined the localization of key gaseous transmitter enzymes: cystathionine β-synthetase (CBS), cystathionine γ-lyase (CSE), heme oxygenase 2 (HO-2), and constitutive NO synthase (nNOS) in the fetal human retina at different stages of development. The number of CBS- and CSE-positive photoreceptors and intermediate retinal neurons was high in trimester I and gradually decreased to the end of trimester III. The number of HO-2-positive cells followed the same trend. The number of nNOS-positive intermediate retinal neurons and neurons within the ganglion cell layer showed the opposite dynamics with the peak in trimester III. The results are interpreted in terms of the role of gaseous transmitters in retinogenesis and cytoprotection.

Nitrosative stress drives heart failure with preserved ejection fraction.

Heart failure with preserved ejection fraction (HFpEF) is a common syndrome with high morbidity and mortality for which there are no evidence-based therapies. Here we report that concomitant metabolic and hypertensive stress in mice-elicited by a combination of high-fat diet and inhibition of constitutive nitric oxide synthase using Nω-nitro-L-arginine methyl ester (L-NAME)-recapitulates the numerous systemic and cardiovascular features of HFpEF in humans. Expression of one of the unfolded protein response effectors, the spliced form of X-box-binding protein 1 (XBP1s), was reduced in the myocardium of ourrodent model and in humans with HFpEF. Mechanistically, the decrease in XBP1s resulted from increased activity of inducible nitric oxide synthase (iNOS) and S-nitrosylation of theendonuclease inositol-requiring protein 1α (IRE1α), culminating in defective XBP1 splicing. Pharmacological or genetic suppression of iNOS, or cardiomyocyte-restricted overexpression of XBP1s, each ameliorated the HFpEF phenotype. We report that iNOS-driven dysregulation of the IRE1α-XBP1 pathway is a crucial mechanism of cardiomyocyte dysfunction in HFpEF.

Suppression of Oxygen Radicals Protects Diabetic Endothelium Damage and Tissue Perfusion in a Streptozotocin-Induced Diabetes Rodent Model.

Oxygen free radicals play a central role in diabetic angiopathy. This study investigated whether suppression of oxygen radicals could decrease endothelial damage and increase peripheral tissue circulation in a diabetic rodent model. Sprague-Dawley rats were treated using streptozotocin to induce diabetes. The experiments were performed 4 weeks after diabetes induction: group 1: control, consisted of normal rats; group 2: diabetes, did not receive treatment; groups III (SOD10) and IV (SOD50): diabetes, received polyethylene glycol-conjugated superoxide dismutase (SOD), an antioxidant, 10 and 50 U/kg per day intraperitoneally for 4 weeks. Each subgroup consisted of 10 rats. Oxygen radicals in blood mononuclear cells were detected by flow cytometry. The blood lipid peroxidation byproduct malondialdehyde was measured. Tissue perfusion of hind limb was examined by laser Doppler. The expressions of oxygen radicals, as demonstrated by 8-hydroxyguanosine (8-OG), and constitutive endothelial nitric oxide synthase in distal femoral vessels were examined by immunohistochemical staining. Oxygen radicals, as demonstrated by H2O2 with 2',7'-dichlorofluorescin diacetate-conjugated expression, were significantly increased in diabetic rats. However, the SOD treatment groups significantly suppressed the H2O2 reaction. Diabetic-induced high malondialdehyde levels were significantly suppressed in the SOD50 group. The topical tissue blood perfusion was significantly increased as detected by laser Doppler in SOD10 and SOD50 groups, as compared with that in diabetes without treatment group (P < 0.05). The expression of 8-OG was markedly increased in the diabetic endothelium and subintima compared with that in normal vessels. Polyethylene glycol-conjugated SOD significantly suppressed 8-OG expression and protected endothelial nitric oxide synthase expression. Suppression of oxygen radicals, particularly with the higher dosage of polyethylene glycol-conjugated SOD at 50 U/kg per day, could have a positive effect to protect against endothelial damage and enhance peripheral perfusion in diabetes.

Effect of TRPV1 on Activity of Isoforms of Constitutive Nitric Oxide Synthase during Regulation of Bicarbonate Secretion in the Stomach.

Application of mild irritants (1 M NaCl; pH 2.0) on the gastric mucosa potentiates the protective secretion of bicarbonates by epithelial cells. This response is mainly mediated by capsaicin-sensitive afferent nerve endings located in the submucosa. It was shown that activation of vanilloid type 1 receptors (TRPV1) induced by exogenous acidification of GM is not sufficient to potentiate the production of HCO3, including production depending on neuronal NO synthase. However, the effect of exogenous acid on TRPV1 leads to activation of endothelial NO synthase that restrict the gastric secretion of [Formula: see text].

Apelin‑13 ameliorates metabolic and cardiovascular disorders in a rat model of type 2 diabetes with a high‑fat diet.

Apelin has been reported to be associated with multiple physiological processes in the cardiovascular system. The aim of the present study was to investigate the effects of Apelin‑13 administration on cardiac function, hyperglycemia, insulin resistance (IR), dyslipidemia, endothelial function, inflammation and glucose metabolism in type 2 diabetic Goto‑Kakizaki (GK) rats, and compare the protective effects of Apelin‑13 with metformin or atorvastatin. In the present study, type 2 diabetes was induced in male Goto‑Kakizaki (GK) rats fed with high‑fat diet (HFD). Simultaneously, the rats were treated with metformin (350mg/kg/d, by gavage), atorvastatin (50mg/kg/d, by gavage) or Apelin‑13 (200µg/kg/d, intraperitoneal injection) once daily for 4 consecutive weeks. Hemodynamic parameters were examined by RM6240BD multi‑channel physiological signal monitoring. Fasting plasma glucose (FPG), fasting insulin (FINS), homeostasis model assessment for insulin resistance (HOMA‑IR), total cholesterol (TC), triglyceride (TG), high density lipoprotein‑cholesterol (HDL‑C), low density lipoprotein‑cholesterol (LDL‑C), endothelin‑1 (ET‑1), nitric oxide (NO), constitutive nitric oxide synthase (cNOS) activity, tumor necrosis factor‑α (TNF‑α), leptin and Apelin‑12 levels were measured. Western blotting was performed to determine the levels of Apelin‑12, glucose transporter 4 (GLUT4) and phosphorylated (p)‑5'adenosine monophosphate‑activated protein kinase (AMPK) α2. It was demonstrated that Apelin‑13 decreased heart rate, left ventricular end‑diastolic pressure, FPG, FINS, HOMA‑IR, TC, TG, LDL‑C, ET‑1, TNF‑α and leptin, whereas it increased the rise and fall of maximum rate of left ventricular pressure, HDL‑C, NO, cNOS activity and Apelin‑12 compared with the GK‑HFD group. In addition, GLUT4 and p‑AMPKα2 levels in myocardial tissues were elevated by administration of Apelin‑13. This protective effect of Apelin‑13 was comparable to that of metformin or atorvastatin. Overall, the present study demonstrated that administration ofApelin‑13 may be a promising therapeutic agent for the treatment of type 2 diabetes and metabolic syndrome.

Phosphodiesterase type 5 in men with vasculogenic and post-radical prostatectomy erectile dysfunction: is there a molecular difference?

To clarify the molecular basis of penile erection at the human level and distinguish the mechanisms underlying vasculogenic and post-radical prostatectomy (RP) erectile dysfunction (ED) subtypes. Erectile tissue was obtained from men without history of ED who underwent penile surgery for Peyronie's disease (control group, n = 5) and from men with ED who underwent penile prosthesis implantation (n = 17). ED was categorized into vasculogenic (n = 8) and post-RP (n = 9) subtypes. Penile erectile tissue samples were collected for molecular analyses of protein expressions of neuronal and endothelial isoforms of nitric oxide synthase (nNOS and eNOS, respectively), phospho-nNOS (Ser-1412), phospho-eNOS (Ser-1177), phospho-protein kinase B (Ser-473), phosphodiesterase type 5 (PDE5), α-smooth muscle actin, phospho-myosin phosphatase target subunit 1, RhoA/Rho-associated protein kinase (ROCK)-α, ROCK-β, 4-hydroxy-2-nonenal, and nNOS and eNOS uncoupling by Western blot. Vasculogenic ED was characterized by decreased eNOS protein expression and eNOS and nNOS phosphorylation on their activatory sites (Ser-1177 and Ser-1412, respectively), uncoupled eNOS, upregulated PDE5 protein expression, increased ROCK activity, and increased oxidative stress in erectile tissue. Post-RP ED was characterized by decreased nNOS protein expression, increased nNOS phosphorylation on its activatory site (Ser-1412), uncoupled nNOS, downregulated PDE5 protein expression, and increased oxidative stress in erectile tissue. The mechanisms of vasculogenic and post-RP ED in the human penis involve derangements in constitutive nitric oxide synthase function, PDE5 protein expression and ROCK activity, and increased oxidative stress, which conceivably provide a molecular basis for chronically reduced nitric oxide bioavailability and increased smooth muscle contraction contributing to erectile impairment. Selective differences in PDE5 protein expression suggest distinct molecular mechanisms are in play for these ED subtypes.

Ultrastructural Changes in the Frog Brain in the Presence of High Concentrations of Glutamate and an NO-Generating Compound

This review summarizes published data and original findings on the morphology of neurons and glial cells in normal conditions and model conditions that simulate those in the human brain during stroke. Ultrastructural changes that occur in the presence of high concentrations of glutamate (Glu, 0.1–5.0 M) and the NO-generating compound NaNO2 (0.1–5.0 mM) were studied in the cerebella (frog cerebellum), which is one of the simplest circuitries structurally. Such studies and data analyses are important because hyperstimulation of Glu receptors is a leading pathogenetic factor of neuronal damage during a stroke. High Glu concentrations exert a toxic effect and damage cerebellar neurons and glial cells. Mitochondria are de-energized, ionic homeostasis is distorted, the intracellular Ca2+ concentration increases, and constitutive NO synthases are activated in the process. The changes result in an increase in the contents of NO and its transformation products, which are involved in a negative feedback mechanism from postsynaptic neurons to presynaptic cells. Biochemical processes are consequently affected, and morphological changes are induced in neurons and glial cells, leading to their swelling. At the same time, ultrastructural compensatory adaptive mechanisms develop to reduce the damaging effect of high concentrations of Glu and NO-generating compounds.

Direct and specific inhibition of constitutive nitric oxide synthase uniquely regulates brush border membrane Na-absorptive pathways in intestinal epithelial cells

Pharmacological manipulations of constitutive nitric oxide (cNO) levels have been shown to have variable effects on Na absorption in vivo and in vitro in different tissues. Species differences, untoward in vivo effects (e.g. ENS, blood flow) and pharmacological non-specificity may account for these confounding observations. Thus, to directly and specifically determine the effect of cNO on brush border membrane Na/H exchange (NHE3) and Na-dependent glucose co-transport (SGLT-1), we inhibited cNO synthase (NOS3) with its siRNA in rat small intestinal epithelial cells (IEC-18) in vitro. As expected, intracellular cNO levels were reduced in siRNA NOS3 transfected cells. In these cells, SGLT-1 was significantly reduced compared to control. In contrast, NHE3 was significantly increased in siRNA NOS3 transfected cells. To determine if SGLT-1 changes were secondary to altered Na/K-ATPase, its activity was measured and found to be increased in NOS3 silenced cells. The mechanism of inhibition of SGLT-1 was secondary to diminished affinity of the co-transporter for glucose in NOS3 silenced cells. In contrast, the mechanism of stimulation of NHE3 is by increasing BBM exchanger numbers in siRNA NOS3 cells while the affinity was unaffected. Western blot studies of immunoreactive BBM proteins also confirmed the kinetic studies. All these data indicates that direct and specific inhibition of NOS3 with its siRNA inhibits SGLT-1 while stimulating NHE3 in the BBM. Thus, cNO uniquely and compensatorily regulates BBM NHE3 and SGLT-1 to maintain cellular Na homeostasis and these unique alterations by cNO are mediated by its intracellular 2nd messenger cGMP.

Enhancing Endogenous Nitric Oxide by Whole Body Periodic Acceleration Elicits Neuroprotective Effects in Dystrophic Neurons.

We have previously shown that inadequate dystrophin in cortical neurons in mdx mice is associated with age-dependent dyshomeostasis of resting intracellular Ca2+ ([Ca2+]i) and Na+ ([Na+]i), elevated reactive oxygen species (ROS) production, increase in neuronal damage and cognitive deficit. In this study, we assessed the potential therapeutic properties of the whole body periodic acceleration (pGz) to ameliorate the pathology observed in cortical neurons from the mdx mouse. pGz adds small pulses to the circulation, thereby increasing pulsatile shear stress to the vascular endothelium, which in turn increases production of nitric oxide (NO). We found [Ca2+]i and [Na+]i overload along with reactive oxygen species (ROS) overproduction in mdx neurons and cognitive dysfunction. mdx neurons showed increased activity of superoxide dismutase, glutathione peroxidase, malondialdehyde, and calpain as well as decreased cell viability. mdx neurons were more susceptible to hypoxia-reoxygenation injury than WT. pGz ameliorated the [Ca2+]i, and [Na+]i elevation and ROS overproduction and further increased the activities of superoxide dismutase, glutathione peroxidase and reduced the malondialdehyde and calpains. pGz diminished cell damage and elevated [Ca2+]i during hypoxia-reoxygenation and improved cognitive function in mdx mice. Moreover, pGz upregulated the expression of utrophin, dystroglycan-β and CAPON, constitutive nitric oxide synthases, prosaposin, brain-derived neurotrophic, and glial cell line-derived neurotrophic factors. The present study demonstrated that pGz is an effective therapeutic approach to improve mdx neurons function, including cognitive functions.

Improvement of penile neurogenic and endothelial relaxant responses by chronic administration of resveratrol in rabbits exposed to unpredictable chronic mild stress.

Chronic stress is an important public health problem known as a risk factor for depression, cognitive deficits, and also erectile dysfunction (ED). Resveratrol, a plant polyphenol, was reported to activate constitutive endothelial nitric oxide synthase (eNOS). Although resveratrol has been proven to exert beneficial effects on the unpredictable chronic mild stress (UCMS)-induced decline in cognitive functions, its potential protecting effect on the penile tissue subjected to UCMS was in fact not investigated. Therefore, restorative effects of resveratrol on neurogenic and endothelium-dependent relaxations were evaluated in the corpus cavernosum of rabbits exposed to UCMS. Eighteen male New Zealand white rabbits were assigned into three groups (n = 6 in each group): controls; UCMS; and UCMS rabbits treated with resveratrol (20 mg/kg/day, i.p.) for 12-week period of stress induction. UCMS was induced by a couple of defined adverse conditions applied in a shuffled order for 12 weeks. Neurogenic and endothelium-dependent relaxations of corpus cavernosum were assessed by using organ bath studies. Both the electrical field stimulation (EFS)-induced neurogenic and carbachol-induced endothelium-dependent relaxant responses significantly decreased in physiological stress and resveratrol treatment exhibited a marked improvement in these relaxation responses in vitro. Our results indicated that chronic psychological stress could lead to ED by reducing neurogenic and endothelium-dependent relaxations and resveratrol prevents impairment of the functional responses, suggesting a potential new treatment approach for treatment of ED during psychological stress.

Expression of a Nitric Oxide Synthesizing Protein in Arterial Endothelial Cells in Response to Different Anti-Anginal Agents Used in Acute Coronary Syndromes.

Organic "nitro" compounds such as nitroglycerine, isosorbide dinitrate are useful in the control of chest pain in acute coronary syndrome. But the mechanism of it in pain regulation remains speculative. Here, increase of NO production was investigated by the possible regulation of constitutive nitric oxide synthase (cNOS) function from goat arterial endothelial cells. This protein was purified and sequence wise characterized as protein disulfide isomerase (PDI) in response to different nitro compounds. The NO generating protein was isolated from arterial endothelial cells and prepared to homogeneity. NO was determined by methemoglobin method. Protein sequence was analyzed by (µLC/MS/MS). A protein of Mr. ~57 kDa was isolated and found to be activated by not only "nitro" compounds but also by acetyl salicylic acid, insulin and glucose. The global BLAST of the protein sequence showed a significant alignment of the protein sequence with PDI. This protein trivially called pluri activator stimulated endothelial NOS (PLASENOS). The enzyme was stimulated by the above-mentioned activators in the presence of Ca2+. Lineweaver-Burk plot of this NOS like activities were demonstrated with its specific substrate l-arginine as Vmax = 5(nmol NO/mg of protein/hr) and Km≈ 0.5µM by the above activators. The enzyme activity was inhibited by the l-NAME, the specific inhibitor of NOS. The organic nitro compounds, acetyl salicylic acid, insulin and glucose were found to activate PLASENOS in the arterial endothelial cells for a continuous supply of NO to control the chest pain in acute coronary syndrome.