Blockade Of Nitric Oxide Synthesis Research Articles

Sex Dependence in Control of Renal Haemodynamics and Excretion in Streptozotocin Diabetic Rats-Role of Adenosine System and Nitric Oxide.

Recently, we compared an interplay of the adenosine system and nitric oxide (NO) in the regulation of renal function between male normoglycaemic (NG) and streptozotocin-induced diabetic rats (DM). Considering the between-sex functional differences, e.g., in the NO status, we present similar studies performed in female rats. We examined if the theophylline effects (non-selective adenosine antagonist) in NG and DM females with or without active NO synthases differed from the earlier findings. In anaesthetised female Sprague Dawley rats, both NG and DM, untreated or after NO synthesis blockade with L-NAME, theophylline effects, on blood pressure, renal hemodynamics and excretion, and renal tissue NO were investigated. Renal artery blood flow (Transonic probe), cortical, outer-, and inner-medullary flows (laser-Doppler technique), and renal tissue NO signal (selective electrode) were measured. In contrast to males, in female NG and DM rats, theophylline induced renal vasodilation. In NO-deficient females, theophylline induced comparable renal vasodilatation, confirming the vasoconstrictor influence of the renal adenosine. In NG and DM females with intact NO synthesis, adenosine inhibition diminished kidney tissue NO, contrasting with an increase reported in males. Lowered baseline renal excretion in DM females suggested stimulation of renal tubular reabsorption due to the prevalence of antinatriuretic over natriuretic tubular action of adenosine receptors. An opposite inter-receptor balance pattern emerged previously from male studies. The study exposed between-sex functional differences in the interrelation of adenosine and NO in rats with normoglycaemia and streptozotocin diabetes. The findings also suggest that in diabetes mellitus, the abundance of individual receptor types can distinctly differ between females and males.

People’s Hospital Of Huishan

To explore the mechanism of hypertension caused by sorafenib in liver cancer-bearing rats based on Vascular Endothelial-Growth Factor (VEGF) signal pathway. After the three groups (blank, liver cancer and Spontaneously Hypertensive Rat (SHR) groups, n = 30) were administered orally with sorafenib (2 mg/kg), the non-invasive tail arterial pressure measurement was employed to determine the changes in blood pressure (BP). Enzyme-linked immunosorbent assay measured the changes of VEGF and Nitric Oxide (NO) in serum. The expressions of Kinase insert Domain Receptor tyrosine kinase (KDR-CD), endothelial Nitric Oxide Synthase (eNOS), Bax and Bcl-2 were detected by Western blot. TUNEL assay detected the Apoptosis Index (AI) of cardiomyocyte. CD31 immunohistochemical staining was observed the changes of Myocardial Capillary Density (MCD). The BP and VEGF levels of the blank and SHR groups were not significantly (P > 0.05) different from those in the before treatment cohort, while the BP of the liver cancer group was significantly (P < 0.05) higher. VEGF level was significantly (P < 0.05) lower than before. Compared with the blank group, the levels of NO, KDR-CD, eNOS, Bcl-2/Bax and MCD in the liver cancer group were significantly (P < 0.05) lower than those in the after administration, while the myocardial cell AI increased significantly (P < 0.05). After antagonist intervention, the BPs and myocardial AI of the liver cancer and SHR groups were significantly lower than before the intervention. Besides, levels of KDR-CD, eNOS, Bcl-2/Bax protein, MCD, serum VEGF and NO were significantly higher than before the intervention (P<0.05). Induction mechanism of hypertension induced by sorafenib may be owing to the inhibition of the VEGF signaling pathway, and reduction of endothelial cells proliferation leading to blockade of NO synthesis. This causes vasoconstriction, while promoting myocardial cell apoptosis and decreasing capillary density, thereby inducing the occurrence of hypertension.

Exercise-Induced Modulation of Angiotensin II Responses in Femoral Veins From 2-Kidney-1-Clip Hypertensive Rats.

The present study investigated the angiotensin II (Ang II) responses in rat femoral veins taken from 2-kidney-1clip (2K1C) hypertensive rats at 4 weeks after clipping, as well as the effects of exercise on these responses. In this manner, femoral veins taken from 2K1C rats kept at rest or exposed to acute exercise or to exercise training were challenged with Ang II or endothelin-1 (ET-1) in organ bath. Simultaneously, the presence of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) were determined in these preparations by western blotting. In these experiments, femoral veins exhibited subdued Ang II responses. However, after nitric oxide (NO) synthesis blockade, the responses were higher in the femoral veins taken from animals kept at rest [0.137(0.049–0.245); n = 10] than those obtained in trained animals kept at rest [0.008(0.001–0.041); n = 10] or studied after a single bout of exercise [0.001(0.001–0.054); n = 11]. In preparations in which, in addition to NO synthesis, both the local production of prostanoids and the action of ET-1 on type A (ETA) or B (ETB) receptors were inhibited, the differences induced by exercise were no longer observed. In addition, neither ET-1 responses nor the presence of COX-1 and COX-2 in these preparations were modified by the employed exercise protocols. In conclusion, NO maintains Ang II responses reduced in femoral veins of 2K1C animals at rest. However, vasodilator prostanoids as well as other relaxing mechanisms, activated by ETB stimulation, are mobilized by exercise to cooperate with NO in order to maintain controlled Ang II responses in femoral veins.

Synthesis of the Mechanisms of Opioid Tolerance: Do We Still Say NO?

The use of morphine as a first-line agent for moderate-to-severe pain is limited by the development of analgesic tolerance. Initially opioid receptor desensitization in response to repeated stimulation, thought to underpin the establishment of tolerance, was linked to a compensatory increase in adenylate cyclase responsiveness. The subsequent demonstration of cross-talk between N-methyl-D-aspartate (NMDA) glutamate receptors and opioid receptors led to the recognition of a role for nitric oxide (NO), wherein blockade of NO synthesis could prevent tolerance developing. Investigations of the link between NO levels and opioid receptor desensitization implicated a number of events including kinase recruitment and peroxynitrite-mediated protein regulation. Recent experimental advances and the identification of new cellular constituents have expanded the potential signaling candidates to include unexpected, intermediary compounds not previously linked to this process such as zinc, histidine triad nucleotide-binding protein 1 (HINT1), micro-ribonucleic acid (mi-RNA) and regulator of G protein signaling Z (RGSZ). A further complication is a lack of consistency in the protocols used to create tolerance, with some using acute methods measured in minutes to hours and others using days. There is also an emphasis on the cellular changes that are extant only after tolerance has been established. Although a review of the literature demonstrates a lack of spatio-temporal detail, there still appears to be a pivotal role for nitric oxide, as well as both intracellular and intercellular cross-talk. The use of more consistent approaches to verify these underlying mechanism(s) could provide an avenue for targeted drug development to rescue opioid efficacy.

Enhancing Plasticity Mechanisms in the Mouse Motor Cortex by Anodal Transcranial Direct-Current Stimulation: The Contribution of Nitric Oxide Signaling.

Consistent body of evidence shows that transcranial direct-current stimulation (tDCS) over the primary motor cortex (M1) facilitates motor learning and promotes recovery after stroke. However, the knowledge of molecular mechanisms behind tDCS effects needs to be deepened for a more rational use of this technique in clinical settings. Here we characterized the effects of anodal tDCS of M1, focusing on its impact on glutamatergic synaptic transmission and plasticity. Mice subjected to tDCS displayed increased long-term potentiation (LTP) and enhanced basal synaptic transmission at layer II/III horizontal connections. They performed better than sham-stimulated mice in the single-pellet reaching task and exhibited increased forelimb strength. Dendritic spine density of layer II/III pyramidal neurons was also increased by tDCS. At molecular level, tDCS enhanced: 1) BDNF expression, 2) phosphorylation of CREB, CaMKII, and GluA1, and 3) S-nitrosylation of GluA1 and HDAC2. Blockade of nitric oxide synthesis by L-NAME prevented the tDCS-induced enhancement of GluA1 phosphorylation at Ser831 and BDNF levels, as well as of miniature excitatory postsynaptic current (mEPSC) frequency, LTP and reaching performance. Collectively, these findings demonstrate that anodal tDCS engages plasticity mechanisms in the M1 and highlight a role for nitric oxide (NO) as a novel mediator of tDCS effects.

Neural motor complexes propagate continuously along the full length of mouse small intestine and colon.

Cyclical propagating waves of muscle contraction have been recorded in isolated small intestine or colon, referred to here as motor complexes (MCs). Small intestinal and colonic MCs are neurogenic, occur at similar frequencies, and propagate orally or aborally. Whether they can be coordinated between the different gut regions is unclear. Motor behavior of whole length mouse intestines, from duodenum to terminal rectum, was recorded by intraluminal multisensor catheter. Small intestinal MCs were recorded in 27/30 preparations, and colonic MCs were recorded in all preparations (n = 30) with similar frequencies (0.54 ± 0.03 and 0.58 ± 0.02 counts/min, respectively). MCs propagated across the ileo-colonic junction in 10/30 preparations, forming "full intestine" MCs. The cholinesterase inhibitor physostigmine increased the probability of a full intestine MC but had no significant effect on frequency, speed, or direction. Nitric oxide synthesis blockade by Nω-nitro-l-arginine, after physostigmine, increased MC frequency in small intestine only. Hyoscine-resistant MCs were recorded in the colon but not small intestine (n = 5). All MCs were abolished by hexamethonium (n = 18) or tetrodotoxin (n = 2). The enteric neural mechanism required for motor complexes is present along the full length of both the small and large intestine. In some cases, colonic MCs can be initiated in the distal colon and propagate through the ileo-colonic junction, all the way to duodenum. In conclusion, the ileo-colonic junction provides functional neural continuity for propagating motor activity that originates in the small or large intestine.NEW & NOTEWORTHY Intraluminal manometric recordings revealed motor complexes can propagate antegradely or retrogradely across the ileo-colonic junction, spanning the entire small and large intestines. The fundamental enteric neural mechanism(s) underlying cyclic motor complexes exists throughout the length of the small and large intestine.

The role of nitric oxide signaling in pulmonary circulation of high- and low-altitude newborn sheep under basal and acute hypoxic conditions

Nitric oxide (NO) is the main vasodilator agent that drives the rapid decrease of pulmonary vascular resistance for the respiratory onset during the fetal to neonatal transition. Nevertheless, the enhanced NO generation by the neonatal pulmonary arterial endothelium does not prevent development of hypoxic pulmonary hypertension in species without an evolutionary story at high altitude. Therefore, this study aims to describe the limits of the NO function at high-altitude during neonatal life in the sheep as an animal model without tolerance to perinatal hypoxia. We studied the effect of blockade of NO synthesis with l-NAME in the cardiopulmonary response of lowland (580 m) and highland (3600 m) newborn lambs basally and under an episode of acute hypoxia. We also determined the pulmonary expression of proteins that mediate the actions of the NO vasodilator pathway in the pulmonary vasoactive tone and remodeling. We observed an enhanced nitrergic function in highland lambs under basal conditions, evidenced as a markedly greater increase in basal mean pulmonary arterial pressure (mPAP) and resistance (PVR) under blockade of NO synthesis. Further, acute hypoxic challenge in lowland lambs infused with l-NAME markedly increased their mPAP and PVR to values greater than baseline, whilst in highland animals under NO synthesis blockade, these variables did not show additional increase in response to low PO2. Highland animals showed increased pulmonary RhoA expression, decreased PSer188-RhoA fraction, increased PSer311-p65-NFқβ fraction and up-regulated smooth muscle α-actin, relative to lowland controls. Taken together our data suggest that NO-mediated vasodilation is important to keep a low pulmonary vascular resistance under basal conditions and acute hypoxia at low-altitude. At high-altitude, the enhanced nitrergic signaling partially prevents excessive pulmonary hypertension but does not protect against acute hypoxia. The decreased vasodilator efficacy of nitrergic tone in high altitude lambs could be in part due to increased RhoA signaling that opposes to NO action in the hypoxic pulmonary circulation.

Blockade of NMDA Receptors and Nitric Oxide Synthesis Potentiated Morphine-Induced Anti-Allodynia via Attenuating Pain-Related Amygdala pCREB/CREB Signaling Pathway

The present study investigated the role of the amygdala N-methyl-d-aspartate (NMDA) receptors/nitric oxide synthase pathway in morphine-induced anti-allodynia. Concurrently with the bilateral cannulation of the central amygdala, chronic constriction of the sciatic nerve was performed on male Wistar rats. Morphine (3–5 mg/kg) was administered intraperitoneally to induce anti-allodynia. When D-AP5, a selective NMDA receptor antagonist, (.05–.1 µg/rat) or NG-Nitro-L-arginine methyl ester hydrochloride (L-NAME), the nitric oxide synthase inhibitor (.1–.5 µg/rat), were microinjected into the central amygdala, the higher doses potentiated an ineffective dose of morphine (3 mg/kg). Microinjection of the same doses of D-AP5 and L-NAME without morphine had no effect. Comicroinjection of the ineffective doses of L-NAME (.1 µg/rat) and D-AP5 (.05 µg/rat) with a 5-minute interval, enhanced the anti-allodynic effect of morphine (3 mg/kg). Western blot analysis was employed to evaluate the levels of cyclic adenosine monophosphate-response element-binding protein (CREB) and phosphorylated CREB (pCREB) in the amygdala tissues. Our results showed that neuropathic pain increased the pCREB/CREB ratio in the amygdala, and this ratio was decreased after morphine-induced anti-allodynia. The potentiative effect of the coadministration of D-AP5/L-NAME on an ineffective dose of morphine also decreased the amygdala pCREB/CREB levels. Therefore, it seems that the amygdala pCREB/CREB signaling pathway plays a critical role in processing neuropathic pain. Moreover, the glutamate NMDA receptors and nitric oxide system in the amygdala may be involved in morphine-induced anti-allodynia. PerspectiveNeuropathic pain is difficult to treat and the exact mechanisms remain unknown. This article suggests the importance of the amygdala glutamatergic and nitric oxide systems in morphine-induced anti-allodynia. These findings might be used in clinical studies to reach a better understanding of neuropathic pain mechanisms and treatment.

Abstract P222: Oxidative Stress Causes Salt Sensitive Hypertension via Activation of Na + -Cl – Cotransporter (ncc)

Background: We have reported that blockade of nitric oxide synthesis by L-NAME causes salt-sensitive hypertension via activating NCC. which was confirmed by the finding that systemic administration of L-NAME failed to cause salt-sensitive hypertension in NCC deficient mice. In vitro study showed when mDCT cells are treated with L-NAME, NCC phosphorylation is increased and NO donner reduced its phosphorylation. However, the precise direct effect of NO on NCC phosphorylation is not clear. In this study we clarified the role of oxidative stress but not angiotensin signaling in NCC phosphorylation Method: The passages 3 - 15 of mDCT cells were used in this study. The cells were starved 1 hour before the experiment. L-NAME (10-40 μM), SOD mimetic 4-Hydroxy-TEMPO (10 μM), NO donor sodium nitroprusside, (SNP 100 μM - 2 mM), pSPAK inhibitor STOCK2S-26016, and Losartan (10 μM) were used to treat the mDCT cells for 10 min and 30 min. Oxidative stress was measured by ROS production using the lucigenin method. To confirm the oxidative stress function, we performed vivo experiment with SOD mimetics--4-hydroxy-TEMPO. The C57BL/6J Kwl mice received implantation of subcutaneous osmotic mini-pump containing TEMPO (20 mg/kg/day) with 8 % high salt diet and L-NAME (3.7 μM/day in drinking water) for 4 weeks. Result: L-NA ME increased oxidative stress by ROS production (2.0 ± 0.15 times higher than control) and p-SPAK signaling, which was normalized by TEMPO, an SOD mimetic and p-SPAK inhibitor. In contrast, AT1R inhibitor Losartan did not affect L-NAME-induced activation of NCC in mDCT cells. Moreover, the function of TEMPO was also confirmed in vivo study, which attenuated the L-NAME- induced increases in superoxide, mean BP (HS 108.7 ± 3.7 mmHg; HSL 130.5 ± 2.9 mmHg; HSL + TEMPO 116.0 ± 1.7 mmHg) and p-NCC expression in the C57BL/6J mice. Conclusion: Oxidative stress - pSPAK pathway plays a pivotal role in the NO-induced inhibition of NCC.

Opioidergic conditioning of the human heart muscle in nitric oxide-dependent mechanism.

Opioidergic conditioning is well documented to trigger cardioprotection against ischemia/ reperfusion (I/R) injury. Previous studies on animal models have suggested that nitric oxide (NO) mediates the beneficial effect of opioids, but the role of NO in humans seems to be controversial. The aim of the study was to assess the influence of NO modulators on opioid-induced cardioprotection in the human myocardium. Trabeculae of the human right atria were electrically driven in an organ bath and subjected to simulated I/R injury. The non-selective inhibitor of nitric oxide synthase (NOS) - N-methyl-l-arginine (LNMMA), the donor of NO - S-Nitroso-N-acetylpenicillamine (SNAP) or morphine (in the amount of 10-4 M) were used at the time of re-oxygenation. The additional trabecula was subjected to the hypoxia protocol only (control). The contractility of the myocardium was assessed as the maximal force of a contraction (Amax), the rate of rise of the force of a contraction (Slope L) and the cardiac muscle relaxation - as the rate of decay of the force of a contraction (Slope T). The application of 100 μM LNMMA resulted in the decrease of Amax, Slope L and Slope T during the re-oxygenation period as compared to control. The application of 10-4 M morphine and/or 100 μM SNAP resulted in a partial reversal of the detrimental influence of LNMMA. At the re-oxygenation period, the blockade of NO synthesis has a deleterious effect on the systolic and diastolic function of the human myocardium as well as attenuates the beneficial effect of morphine conditioning.

Abstract P282: Low Dose LNAME Causes Salt Sensitive Hypertension via Activation of NCC

Background: Blood pressure is regulated both by vascular resistance and blood volume. Our group recently clarified norepinephrine activates sodium channel (sodium chloride cotransporter: NCC) via epigenetic modulation (Nat Med 2011). The accumulating data strongly suggest that vasoactive substances are not only act on vasculature but also effect on sodium homeostasis. We hypothesized that blockade of nitric oxide synthesis by L-NAME has a direct effect on sodium channels in the kidney. Method: C57BL/6J Mice were treated with 8%NaCl (high salt; HS) or 8%NaCl +L-NAME (0.7mg/10ml). Blood pressure was measured by telemetry. After 4 weeks’ treatment, the function of sodium channel was monitored by pharmacologically and molecular biologically. mDCT cell was used to observe the direct effect of L-NAME on NCC. Result: Low dose L-NAME or high salt loading alone did not change blood pressure in the C57BL/6J mouse, but L-NAME shifted the pressure natriuresis curve toward the right and induced salt-sensitive hypertension in HS group. No significant changes were observed in morphological changes in the kidney. However, significant changes of sodium and chloride excretion were observed after hydrochlorothiazide challenge test but not in amiloride test, which was consistent with increases in membrane NCC. This highly suggested NCC, not ENaC was activated by L-NAME to cause salt-sensitive hypertension. To determine whether L-NAME active NCC directly or not, we treated mDCT cells with L-NAME. The membrane NCC expression was increased in a time and dose depended manner. Conclusion: Low dose L-NAME inappropriately and directly activate NCC in both vivo and vitro, and finally induces salt-sensitive hypertension.

Role of endothelial nitric oxide in control of peripheral vascular conductance during muscle metaboreflex activation.

The muscle metaboreflex is a powerful pressor reflex induced by the activation of chemically sensitive muscle afferents as a result of metabolite accumulation. During submaximal dynamic exercise, the rise in arterial pressure is primarily due to increases in cardiac output, since there is little systemic vasoconstriction. Indeed, in normal animals, we have often shown a small, but significant, peripheral vasodilation during metaboreflex activation, which is mediated, at least in part, by release of epinephrine and activation of vascular β2-receptors. We tested whether this vasodilation is in part due to increased release of nitric oxide caused by the rise in cardiac output eliciting endothelium-dependent flow-mediated vasodilation. The muscle metaboreflex was activated via graded reductions in hindlimb blood flow during mild exercise with and without nitric oxide synthesis blockade [NG-nitro-l-arginine methyl ester (l-NAME); 5 mg/kg]. We assessed the role of increased cardiac output in mediating peripheral vasodilation via the slope of the relationship between the rise in nonischemic vascular conductance (conductance of all vascular beds excluding hindlimbs) vs. the rise in cardiac output. l-NAME increased mean arterial pressure at rest and during exercise. The metaboreflex-induced increases in mean arterial pressure were unaltered by l-NAME, whereas the increases in cardiac output and nonischemic vascular conductance were attenuated. However, the slope of the relationship between nonischemic vascular conductance and cardiac output was not affected by l-NAME, indicating that the rise in cardiac output did not elicit vasodilation via increased release of nitric oxide. Thus, although nitric oxide is intrinsic to the vascular tonus, endothelial-dependent flow-mediated vasodilation plays little role in the small peripheral vasodilation observed during muscle metaboreflex activation.

Hyperbaric oxygen therapy may overcome nitric oxide blockage during cyanide intoxication.

To determine the effects of a blockade of nitric oxide (NO) synthesis on hyperbaric oxygen (HBO₂) therapy during cyanide (CN) intoxication. 39 anesthetized female Sprague-Dawley rats were exposed to CN intoxication (5.4 mg/kg intra-arterially) with or without previous nitric oxide synthase (NOS) inhibition by L-NG-nitroarginine methyl ester (L-NAME) injection (40 mg/kg intraperitoneally). Subsequently, either HBO₂ therapy (284 kPa/90 minutes), normobaric oxygen therapy (100% oxygen/90 minutes) or nothing was administered. Intracerebral microdialysis was used to measure the interstitial brain concentration of lactate, glucose, glycerol and lactate/pyruvate ratios. L-NAME potentiated CN intoxication by higher maximum and prolonged lactate (in mM: 0. 5 ± 0.3 vs. 0.7 ± 0.4, P ⟨ 0.005) concentrations compared with solely CN-intoxicated rats. The same trend was found for mean glucose, glycerol and lactate/pyruvate ratio levels. During HBO₂ treatment a sustained reduction occurred in mean lactate levels (in mM: 0.5 ± 0.5 vs. 0.7 ± 0.4, P ⟨ 0.01) regardless of NOS blockade by L-NAME. The same trend was found for mean glucose and glycerol levels. The results suggest that blocking NOS using L-NAME can worsen acute CN intoxication. HBO₂ treatment can partially overcome this block and continue to ameliorate CN intoxication.

Glucagon-Like Peptide-1 Excites Firing and Increases GABAergic Miniature Postsynaptic Currents (mPSCs) in Gonadotropin-Releasing Hormone (GnRH) Neurons of the Male Mice via Activation of Nitric Oxide (NO) and Suppression of Endocannabinoid Signaling Pathways.

Glucagon-like peptide-1 (GLP-1), a metabolic signal molecule, regulates reproduction, although, the involved molecular mechanisms have not been elucidated, yet. Therefore, responsiveness of gonadotropin-releasing hormone (GnRH) neurons to the GLP-1 analog Exendin-4 and elucidation of molecular pathways acting downstream to the GLP-1 receptor (GLP-1R) have been challenged. Loose patch-clamp recordings revealed that Exendin-4 (100 nM–5 μM) elevated firing rate in hypothalamic GnRH-GFP neurons of male mice via activation of GLP-1R. Whole-cell patch-clamp measurements demonstrated increased excitatory GABAergic miniature postsynaptic currents (mPSCs) frequency after Exendin-4 administration, which was eliminated by the GLP-1R antagonist Exendin-3(9–39) (1 μM). Intracellular application of the G-protein inhibitor GDP-β-S (2 mM) impeded action of Exendin-4 on mPSCs, suggesting direct excitatory action of GLP-1 on GnRH neurons. Blockade of nitric-oxide (NO) synthesis by Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME; 100 μM) or N5-[Imino(propylamino)methyl]-L-ornithine hydrochloride (NPLA; 1 μM) or intracellular scavenging of NO by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO; 1 mM) partially attenuated the excitatory effect of Exendin-4. Similar partial inhibition was achieved by hindering endocannabinoid pathway using cannabinoid receptor type-1 (CB1) inverse-agonist 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-(1-piperidyl) pyrazole-3-carboxamide (AM251; 1 μM). Simultaneous blockade of NO and endocannabinoid signaling mechanisms eliminated action of Exendin-4 suggesting involvement of both retrograde machineries. Intracellular application of the transient receptor potential vanilloid 1 (TRPV1)-antagonist 2E-N-(2, 3-Dihydro-1,4-benzodioxin-6-yl)-3-[4-(1, 1-dimethylethyl)phenyl]-2-Propenamide (AMG9810; 10 μM) or the fatty acid amide hydrolase (FAAH)-inhibitor PF3845 (5 μM) impeded the GLP-1-triggered endocannabinoid pathway indicating an anandamide-TRPV1-sensitive control of 2-arachidonoylglycerol (2-AG) production. Furthermore, GLP-1 immunoreactive (IR) axons innervated GnRH neurons in the hypothalamus suggesting that GLP-1 of both peripheral and neuronal sources can modulate GnRH neurons. RT-qPCR study confirmed the expression of GLP-1R and neuronal NO synthase (nNOS) mRNAs in GnRH-GFP neurons. Immuno-electron microscopic analysis revealed the presence of nNOS protein in GnRH neurons. These results indicate that GLP-1 exerts direct facilitatory actions via GLP-1R on GnRH neurons and modulates NO and 2-AG retrograde signaling mechanisms that control the presynaptic excitatory GABAergic inputs to GnRH neurons.

PS 04–03] LONG-TERM TREATMENT WITH N-NITRO-L-ARGININE METHYL ESTER (L-NAME) INCREASES ACTIVITY AND EXPRESSION OF NA+−CL−COTRANSPORTER TO CAUSE SALT-SENSITIVE HYPERTENSION

Objective: Blood pressure is regulated both by vascular resistance and blood volume. Vascular resistance is controlled by vasoactive hormones such as angiotensin II, norepinephrine and Nitric oxide. Blood volume is mainly regulated by the kidney, how much of salt is excreted. Angiotensin II is known to regulate sodium channel to increase sodium uptake. Our group recently clarified norepinephrine also activate sodium channel (sodium chloride cotransporter: NCC) via epigenetic modulation (Nat Med 2011). The accumulating data strongly suggest that vasoactive substances are not only act on vasculature but also affect on sodium homeostasis. We hypothesized that blockade of Nitric oxide synthesis by L-NAME has a direct effect on sodium channels in the kidney. Design and Method: C57BL/6J Mice were treated with 8%NaCl (high salt; HS) or 8%NaCl +L-NAME (0.7 mg/10 ml). Blood pressure was measured by telemetry. The function of sodium channel was monitored by pharmacologically and molecular biologically. NCC was blocked by hydrochlorothiazide (HCTZ) and other sodium channel (epithelial sodium channels; ENaC) was blocked by amiloride. Their protein expression was evaluated by Western blot. Results: Low dose L-NAME or high salt loading alone did not induce blood pressure changes in the C57BL/6J mouse.However, the mice developed hypertension during co-treatment with low dose L-NAME and high salt loading. To determine which type of sodium channel is involved, we injected both amiloride and HCTZ into the mice to inhibit the function of ENaC and NCC, respectively. The sodium excretion profile in urine showed that the mouse responded to HCTZ than amiloride suggesting that NCC is activated by L-NAME and high salt diet. It was confirmed by western blot. Conclusions: These results highly suggest that NO activate NCC to lead to salt-sensitive hypertension. Further we will use NCC-/- mice to confirm the blood pressure changes by HS+L-NAME. And check out the possible signal pathway between NCC and Nitric Oxide.

Nitric Oxide Upregulates Proteasomal Protein Degradation in Neurons.

Nitric oxide (NO) is involved in many neuronal functions such as neuromodulation and intracellular signaling. Recent studies have demonstrated that nitric oxide is involved in regulation of proteasomal protein degradation. However, its role in neuronal protein degradation still remains unclear. In our study, we investigated the influence of endogenous nitric oxide production in this process. We have shown that nitric oxide synthase blockade prevents decline of the UbG76V-GFP fluorescence (GFP-based proteasomal protein degradation reporter) in neuronal processes of the cultured hippocampal neurons. It suggests that nitric oxide may regulate ubiquitin-dependent proteasomal protein degradation in neurons. Also, we have confirmed that the NO synthesis blockade alone significantly impairs long-term potentiation, and demonstrated for the first time that simultaneous blockade of the NO and proteins synthesis leads to the long-term potentiation amplitude rescue to the control values. Obtained results suggest that nitric oxide is involved in the protein degradation in proteasomes in physiological conditions.

Role of endothelial nitric oxide in control of peripheral vascular conductance during muscle metaboreflex activation

The muscle metaboreflex is a powerful pressor reflex induced by the activation of chemically sensitive muscle afferents as a result of metabolite accumulation. During submaximal dynamic exercise, the rise in arterial pressure is primarily due to increases in cardiac output as there is little systemic vasoconstriction. Indeed, in normal animals we have shown a small, but significant, peripheral vasodilation during metaboreflex activation which is mediated at least in part by release of epinephrine and activation of vascular β2 receptors (Kaur et al., Am. J. Physiol. Heart Circ., 2015). We tested whether this vasodilation is in part due to increased release of nitric oxide caused by the rise in cardiac output eliciting endothelium‐dependent flow‐mediated vasodilation. The muscle metaboreflex was activated via graded reductions in hindlimb blood flow during mild exercise with and without nitric oxide synthesis blockade (L‐nitroarginine methyl ester (L‐NAME); 5mg/kg). We accessed the role of increased cardiac output in mediating peripheral vasodilation via the slope of the relationship between the rise in non‐ischemic vascular conductance (conductance of all vascular beds excluding hindlimbs) vs. the rise in cardiac output. L‐NAME increased mean arterial pressure at rest and during exercise. The metaboreflex‐induced increases in mean arterial pressure were unaltered by L‐NAME; whereas, the increases in cardiac output and non‐ischemic vascular conductance were attenuated. However, the slope of the relationship between non‐ischemic vascular conductance and cardiac output was not affected by L‐NAME indicating that the rise in cardiac output did not elicit vasodilation via increased release of nitric oxide. Thus, although nitric oxide is intrinsic to the vascular tonus, endothelial‐dependent flow‐mediated vasodilation plays little role in the small peripheral vasodilation observed during muscle metaboreflex activation.Support or Funding Information(NIH HL55473)

Nitric oxide signaling in the ischemic postconditioning of human heart muscle (RCD code: III)

Background: Ischemic heart conditioning is well documented to trigger the intrinsic protective mechanisms of resistance against ischemia/reperfusion (I/R) injury. Previous studies on animal model have suggested that the nitric oxide (NO) mediates the beneficial effect of ischemic postconditioning (POC). We tested the hypothesis that POC provide cardioprotection in the NO-dependent mechanism in the human myocardium. Methods: Human atrial trabeculae were subjected to simulated I/R injury. To achieve POC triple brief hypoxia periods were followed by the lethal hypoxia. Non-selective inhibitor of NO synthase: NG-monomethyl-L-arginine (L-NMMA) was used at the time of re-oxygenation in the POC protocol. Contractility of the myocardium was assessed as the maximal force of a contraction (Amax), the rate of rise of the force of a contraction (Slope L) and cardiac muscle relaxation – as the rate of decay of the force of a contraction (Slope T). Results: Co-application of L-NMMA with POC resulted in the decrease of Amax, Slope L and Slope T during re-oxygenation period as compared to POC only. Conclusions: At re-oxygenation period, the blockade of NO synthesis has deleterious effect on systolic and diastolic function of human myocardium, as well as attenuates the beneficial effect of ischemic.

Effects of a New Glutamic Acid Derivative on Myocardial Contractility of Stressed Animals under Conditions of Nitric Oxide Synthesis Blockade.

Glufimet (glutamic acid derivative) in a dose of 28.7 mg/kg limited the reduction of the cardiac functional reserve in animals subjected to 24-h stress under conditions of nonselective NO synthase blockade with L-NAME (10 mg/kg). Adrenoreactivity and increased afterload tests showed that the increment of myocardial contraction/relaxation rates, left-ventricular pressure, and HR were significantly higher in glufimet-treated stressed animals with NO synthesis blockade than in animals which received no glufimet. The efficiency of glufimet was higher than that of phenibut (the reference drug).

L-ARGININE, BUT NOT L-NAME PROTECTS AGAINST LIVER INJURY INDUCED BY EXPERIMENTAL ISCHEMIA-REPERFUSION

<p><strong>Background</strong>. Hepatic ischemia-reperfusion (I/R) injury occurs upon restoration of hepatic blood flow after a period of ischemia.<br /><strong>Objective.</strong> The study establishes that stimulation or blockade of nitric oxide synthesis has a protective effect during ischemia-reperfusion.<br /><strong>Methods.</strong> Male albino rats which were divided into four equal groups: sham-operated control, ischemia and reperfusion group (0.9 % saline i.p.) for 3 days, group pre-treated with L-arginine (25 mg/kg i.p.), group pre-treated with L-NAME (10 mg/kg i.p.) for 3 days before ischemia-reperfusion maneuver. Complete ischemia of the median and left hepatic lobes was induced by clamping the left branches of the portal vein and the hepatic artery for 45 min. Rats were sacrificed after 3-h reperfusion. Nitric oxide synthase 3 (endothelial) and nitric oxide synthase 2 (inducible) expression, nitric oxide stabile metabolites (NO2, NO3) content, AST and ALT activities were determined. Histological examination of liver tissue was performed.<br /><strong>Conclusions.</strong> Relative NO deficiency, due to eNOS inhibition, is central in the pathogenesis of hepatic ischemia reperfusion injury. Replacing NO content with either precursors or via donor drugs represents novel methods in ameliorating ischemia-reperfusion injury.<br /><strong></strong></p><p><strong>KEY WORDS:</strong> hepatic ischemia-reperfusion, nitric oxide, NOS isoforms, L-arginine, L-NAME<strong></strong></p>