Non-selective Nitric Oxide Synthase Inhibitor Research Articles

Redox imaging of dextran sodium sulfate-induced colitis mice treated with nitric oxide synthase inhibitors

Ulcerative colitis (UC) is an inflammatory bowel disease of unknown cause. Redox imbalance, as characterized by excessive production of reactive oxygen species (ROS) and nitric oxide (NO), and deficient levels of endogenous antioxidants, is associated with the pathogenesis of UC. Overhauser-enhanced magnetic resonance imaging (OMRI) combined with a spin-probe method produces high-resolution images of redox imbalance in disease states. We aimed to investigate the effect of NO synthase (NOS) inhibitors on colonic ROS generation in mice with colitis induced by dextran sodium sulfate (DSS) using the OMRI/spin-probe method. Symptoms of DSS-induced colitis were ameliorated by an inducible NOS (iNOS) inhibitor, aminoguanidine (AG), but not by a non-selective NOS inhibitor, NG-Nitro-L-arginine methyl ester (L-NAME). Intracellular ROS generation, observed as the enhancement of OMRI contrast decay rates of a 3-methoxy-2,2,5,5-tetramethylpyrrolidine-1-oxyl probe in the colons of DSS-induced colitis mice, was suppressed by AG, in agreement with the results of an in vivo electron spin resonance study. AG had an inhibitory effect on ROS production at the upper colon and rectum during the initiation stage of colitis, and at the upper and lower colon during the advanced stage. Additionally, there were partial differences in the localization of iNOS protein and nitrite/nitrate levels. L-NAME treatment showed a tendency for increased ROS generation at the upper colon during the advanced stage. The OMRI/spin-probe method provides a novel tool for screening ROS inhibitory activity in vivo in DSS-induced colitis mice.

Leonurine Reduces Oxidative Stress and Provides Neuroprotection against Ischemic Injury via Modulating Oxidative and NO/NOS Pathway.

Leonurine (Leo) has been found to have neuroprotective effects against cerebral ischemic injury. However, the exact molecular mechanism underlying its neuroprotective ability remains unclear. The aim of the present study was to investigate whether Leo could provide protection through the nitric oxide (NO)/nitric oxide synthase (NOS) pathway. We firstly explored the effects of NO/NOS signaling on oxidative stress and apoptosis in in vivo and in vitro models of cerebral ischemia. Further, we evaluated the protective effects of Leo against oxygen and glucose deprivation (OGD)-induced oxidative stress and apoptosis in PC12 cells. We found that the rats showed anxiety-like behavior, and the morphology and number of neurons were changed in a model of photochemically induced cerebral ischemia. Both in vivo and in vitro results show that the activity of superoxide dismutase (SOD) and glutathione (GSH) contents were decreased after ischemia, and reactive oxygen species (ROS) and malondialdehyde (MDA) levels were increased, indicating that cerebral ischemia induced oxidative stress and neuronal damage. Moreover, the contents of NO, total NOS, constitutive NOS (cNOS) and inducible NOS (iNOS) were increased after ischemia in rat and PC12 cells. Treatment with L-nitroarginine methyl ester (L-NAME), a nonselective NOS inhibitor, could reverse the change in NO/NOS expression and abolish these detrimental effects of ischemia. Leo treatment decreased ROS and MDA levels and increased the activity of SOD and GSH contents in PC12 cells exposed to OGD. Furthermore, Leo reduced NO/NOS production and cell apoptosis, decreased Bax expression and increased Bcl-2 levels in OGD-treated PC12 cells. All the data suggest that Leo protected against oxidative stress and neuronal apoptosis in cerebral ischemia by inhibiting the NO/NOS system. Our findings indicate that Leo could be a potential agent for the intervention of ischemic stroke and highlighted the NO/NOS-mediated oxidative stress signaling.

L-citrulline prevents heat-induced mitochondrial dysfunction and cell injury through nitric oxide-mediated Drp1 inhibition in mouse C2C12 myoblasts.

Severe heat exposure causes mitochondrial fragmentation and dysfunction, which contribute to the pathogenesis of heat-related illness. L-citrulline is a naturally occurring amino acid and has been suggested to influence heat shock responses. This study aimed to test whether L-citrulline supplementation would preserve mitochondrial integrity and attenuate heat-induced skeletal muscle injury, and elucidate the underlying mechanisms. At 37°C, L-citrulline (2 mM) increased mitochondrial elongation in mouse C2C12 myoblasts, a process associated with a reduction in mitochondrial fission protein Drp1 levels. Mechanistic studies revealed that L-citrulline increased cellular nitric oxide (NO) levels, but not S-nitrosylation of Drp1. L-citrulline caused a decrease in phosphorylation of Drp1 at Ser 616 and an increase in phosphorylation of Drp1 at Ser 637, which resulted in a reduced mitochondrial localization of Drp1. L-NAME, a non-selective NO synthase inhibitor, abolished the increase in L-citrulline-induced NO levels and inhibited Drp1 phosphorylation changes and mitochondrial elongation, which indicates involvement of a NO-dependent pathway. Under 43°C heat stress conditions, L-citrulline prevented translocation of Drp1 to mitochondria, mitochondrial fragmentation and decreased membrane potential. Finally, L-citrulline pretreatment inhibited heat-induced reactive oxygen species (ROS) overproduction, caspase 3/7 activation, apoptotic cell death, and improved cell viability. NO inhibitor L-NAME abolished all the above protective effects of L-citrulline under heat stress. Our results suggest that L-citrulline prevents heat-induced mitochondrial dysfunction and cell injury through NO-mediated Drp1 inhibition in C2C12 myoblasts. L-citrulline may be an effective treatment for heat-related illnesses and other mitochondrial diseases.

Asymmetric Dimethylarginine Protects Neurons from Oxygen Glucose Deprivation Insult by Modulating Connexin-36 Expression.

Background Asymmetric dimethylarginine (ADMA) is a nonselective nitric oxide synthase inhibitor. ADMA is thought to inhibit the production of nitric oxide (NO) by neurons after oxygen-glucose deprivation (OGD). The gap junction protein Connexin-36 (cx-36) is involved in the pathophysiology of stroke. We investigated whether ADMA could protect neurons from OGD insults by regulating the expression of cx-36. Methods Cultured rat cortical neuronal cells were used. Neurons were treated with OGD with or without ADMA pretreatment. The lactate dehydrogenase (LDH) release rate was used to assess neuronal injury. Intracellular NO levels were determined using 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate. Western blotting was performed to detect cx-36 expression. Results The LDH release rate increased in the supernatant of neurons after the OGD insult, whereas ADMA treatment reduced the LDH release rate. Intracellular NO levels increased following OGD treatment, and this increase was not inhibited by ADMA treatment. Expression of cx-36 was upregulated in neurons under OGD conditions, and treatment with ADMA downregulated the expression of cx-36. Conclusions ADMA protects neurons from OGD insult, and cx-36 downregulation may be a possible pathway involved in ADMA-mediated neuronal protection.

N-Nitro-L-Arginine-Methyl ester Exerts Neuroprotection byInhibiting Inflammation in Cerebral Injury Induced by Transient Ischemia/Reperfusion in Rats

Cerebral ischemia-reperfusion injury is a pivotal cause of deaths due to cerebrovascular accidents. Further research efforts are needed to reveal the mechanism underlying its aggravation or alleviation. We previously reported the antioxidant effect of N-Nitro-L-Arginine- Methyl Ester (L-NAME), a nonselective nitric oxide synthase (NOS) inhibitor, on rats subjected to transient focal cerebral ischemia-reperfusion (I/R). The aim of this work was to explore further neuroprotective anti-inflammatory effects of L-NAME. This study involved 30 adult male Wistar rats divided into three groups with ten rats in each: sham-operated (control), I/R group of rats subjected to 30 minutes of left common carotid artery (CCA) occlusion followed by 24-hour of reperfusion and test group infused with L-NAME intraperitoneally 15 minutes before the same I/R period. Neurological assessments were evaluated, Western blotting used to estimate Nuclear factor-kappa B (NF-?B), ELISA used to detect Tumor necrosis factor- α (TNF-α), and Nitric oxide metabolites were measured colorimetrically, as well as H&E staining to assess brain damage. Compared with the I/R group, the neurological score, infarction area, and the inflammatory biomarkers NF-?B, TNF-α, and NO were significantly decreased in L-NAME treated rats (P =0.001). As a conclusion from the current study, L-NAME showed potential neuroprotection through it is an anti-inflammatory effect on a rat’s model of transient focal cerebral ischemia-reperfusion.

The effect of acute intradermal administration of ascorbate on heat loss responses in older adults with uncomplicated controlled hypertension.

What is the central question of this study? Does acute intradermal administration of the antioxidant ascorbate augment local forearm cutaneous vasodilatation and sweating via nitric oxide synthase (NOS)-dependent mechanisms during exercise-heat stress in older adults with uncomplicated controlled hypertension? What is the main finding and its importance? Relative to the control site, ascorbate had no effect on forearm cutaneous vascular conductance (CVC) and sweat rate, although CVC was reduced with NOS inhibition in older adults with hypertension. Acute local administration of ascorbate to forearm skin does not modulate heat loss responses during exercise-heat stress in older adults with hypertension. Nitric oxide synthase (NOS) contributes to the heat loss responses of cutaneous vasodilatation and sweating during exercise. However, the contribution of NOS may be attenuated in individuals with uncomplicated, controlled hypertension due to elevated oxidative stress, which can reduce NO bioavailability. We evaluated the hypothesis that the acute local intradermal administration of the antioxidant ascorbate would enhance cutaneous vasodilatation and sweating via NOS-dependent mechanisms during an exercise-heat stress in adults with hypertension. Habitually active adults who were normotensive (n=14, 7 females, 62±4years) or had uncomplicated, controlled hypertension (n=13, 6 females, 62±5years) performed 30min of moderate-intensity (50% of their pre-determined peak oxygen uptake) semi-recumbent cycling in the heat (35°C, 20% relative humidity). Cutaneous vascular conductance (CVC) and sweat rate were assessed at four forearm skin sites continuously perfused with (1) lactated Ringer solution (Control), (2) 10mM antioxidant ascorbate, (3) 10mMNG -nitro-l-arginine methyl ester (l-NAME), a non-selective NOS inhibitor, or (4) a combination of ascorbate and l-NAME. Relative to Control, no effect of ascorbate was observed on CVC or sweating in either group (P=0.619). However, l-NAME reduced CVC relative to Control in both groups (P≤0.038). No effect of any treatment on sweating was observed (P≥0.306). Thus, acute local administration of ascorbate to forearm skin does not enhance the activation of heat loss responses of cutaneous vasodilatation and sweating in older adults, and those with hypertension during an exercise-heat stress.

Estradiol Augments Sensory Nerve‐Mediated Vasodilation in Young Women

Local heating of the skin elicits biphasic vasodilation: a sensory nerve‐mediated initial peak (axon reflex) and an endothelial‐dependent plateau. We have previously shown that sex hormones, particularly estradiol (E2), improves cutaneous endothelial‐dependent vasodilation, but less is known about the effects of E2 on cutaneous sensory nerve‐mediated vasodilation.PURPOSEWe tested the hypothesis that E2 enhances sensory‐nerve mediated vasodilation in response to local heating of the skin in young women.METHODSWe retrospectively analyzed data in 15 healthy young women (age 24 ± 4 yr; MAP 84 ± 6 mmHg; BMI 24 ± 3 kg/m2). Endogenous sex hormone production was suppressed with daily administration of a gonadotropin‐releasing hormone antagonist (GnRHant; Ganirelix) for 10 days. E2 (0.1 mg/day, Vivelle‐Dot) was added back via transdermal patch on days 4–10. Cutaneous vasodilatory responses to local heating were measured on days 4 and 10 using laser Doppler flowmetry during microdialysis perfusions of lactated Ringer’s (control), and in a subset of women (n=6), L‐NAME (non‐selective nitric oxide synthase inhibition). Cutaneous vascular conductance (CVC) was calculated during the initial peak of the axon reflex during local heating (42°C) and normalized to maximal vasodilation achieved by perfusion of sodium nitroprusside (28mM) and heating to 43°C.RESULTSSensory nerve‐mediated vasodilation increased during E2 administration compared to GnRHant (E2: 71 ± 15, GnRHant: 57 ± 18 %CVCmax; p=0.046). Blockade of nitric oxide with L‐NAME did not blunt the axon reflex during GnRHant (control: 57 ± 18 vs. L‐NAME: 50 ± 17 %CVCmax; p=0.401) or E2 administration (control: 71 ± 15 vs. L‐NAME: 52 ± 19 %CVCmax; p=0.152).CONCLUSIONThese preliminary findings suggest that E2 enhances sensory nerve‐mediated vasodilation in young women, and that this may occur through mechanisms independent of nitric oxide. Future mechanistic studies isolating the nitric oxide component and other pathways could help elucidate the role of E2 in sensory nerve‐mediated vasodilation.

Aortic Relaxation in Castrated Male Rats and Responses to Estrogen Treatment

Previous studies suggest that risk factors for cardiovascular diseases (CVD) have increased in the transgender population after they undergo cross‐sex hormone therapy (CSHT). Several studies, including ours in animal models as well as in humans, have shown that estrogen exerts direct beneficial effects on the vessel wall in healthy females. A growing body of evidence also suggests that androgen exhibits protective actions on the cardiovascular system. However, the effects of CSHT in cardiovascular physiology in males or females are less understood.The current study was, therefore, undertaken to evaluate the effects of estrogen treatment on aortic reactivity in castrated (CAS) male rats. Specifically, we investigated the effects of in vivo treatment with 17β‐estradiol (E2) on the aortic responses of CAS male as well as ovariectomized (OVX) female rats.Age‐matched CAS male and OVX female rats were implanted subcutaneously with either 17β‐estradiol (E2,1.5 mg) or a placebo‐containing pellet for 30 days. Endothelium‐dependent vasorelaxation (EDV) in response to acetylcholine (ACh, 10‐8 to 10‐5 M) in aorta precontracted with phenylephrine (PE, 2 µM) was measured before and after pretreatment with indomethacin (10 µM, cyclooxygenase inhibitor), followed by N‐nitro‐ L‐arginine methyl ester [L‐NAME, 200 µM, a nonselective nitric oxide synthase (NOS) inhibitor]. Endothelium‐independent vasorelaxation was assessed by measuring sodium nitroprusside (SNP)‐induced relaxation response in endothelium‐denuded rings in addition to the contractile response curves to PE before and after pretreatment with L‐NAME in aortic rings.EDV to ACh was significantly impaired in aortic rings from E2‐treated CAS rats compared to their placebo‐treated counterparts and E2‐treated OVX rats. The impairment of EDV to ACh in E2‐treated CAS rats was accompanied by a significant decrease in nitric oxide (NO) contribution to ACh‐induced relaxation in this group. The smooth muscle sensitivity to NO as measured by SNP relaxation response curve was not altered, whereas the responsiveness to PE was slightly enhanced in the aortas of E2‐treated CAS group compared to those in other experimental groups. Endothelium‐derived NO release during smooth muscle contraction, as assessed by the potentiation of the response to PE after NOS inhibition, was not different in the aortas of experimental groups.Overall, our data demonstrate that aortic function was altered in E2‐treated CAS male rats. Here, we provide the first evidence of impairment in aortic relaxation in E2‐treated CAS male rats, possibly via a significant decrease in the relative contribution of NO to the regulation of vascular relaxation response. Furthermore, the comparison of current data in the CAS male rats with our recently published data on the aortic reactivity in the intact males appears to rule out any effect of testosterone deprivation on the stimulated release of NO in male rat aortas. Additional studies will be needed to document the direction and magnitude of CSHT interaction with vasculatures.

Modafinil exerts anticonvulsive effects against lithium-pilocarpine-induced status epilepticus in rats: A role for tumor necrosis factor-α and nitric oxide signaling

BackgroundStatus epilepticus (SE) is a continuous episode of seizures which leads to hippocampal neurodegeneration, severe systemic inflammation, and extreme damage to the brain. Modafinil, a psychostimulant and wake-promoting agent, has exerted neuroprotective and anti-inflammatory effects in previous preclinical studies. The aim of this study was to assess effects of modafinil on the lithium-pilocarpine-induced SE rat model and to explore possible involvement of tumor necrosis factor-α (TNF-α) and nitric oxide (NO) pathways in this regard. MethodsStatus epilepticus was provoked by injection of lithium chloride (127 mg/kg, intraperitoneally [i.p]) and pilocarpine (60 mg/kg, i.p.) in rats. Animals received different modafinil doses (50, 75, 100, and 150 mg/kg, i.p.) and SE scores were documented over 3 hours of duration. Moreover, the role of the nitrergic pathway in the effects of modafinil was evaluated by injection of the non-selective NO synthase (NOS) inhibitor L-NG-Nitro arginine methyl ester (L-NAME, 10 mg/kg, i.p.), the selective neuronal NOS inhibitor 7-nitroindazole (30 mg/kg, i.p.), and the selective inducible NOS inhibitor aminoguanidine (100 mg/kg, i.p.) 15 min before saline/vehicle or modafinil. The ELISA method was used to quantify TNF-α and NO metabolite levels in the isolated hippocampus. ResultsModafinil at 100 mg/kg significantly decreased SE scores (P < 0.01). Pre-treatment with L-NAME, 7-nitroindazole, and aminoguanidine significantly reversed the anticonvulsive effects of modafinil. Status epilepticus-induced animals showed significantly higher NO metabolite and TNF-α levels in their hippocampal tissues, an effect that was reversed by modafinil (100 mg/kg, i.p.) treatment. Administration of NOS inhibitors resulted in excessive NO level reduction but an escalation of TNF-α level in modafinil-treated SE-animals. ConclusionOur study revealed anticonvulsive effects of modafinil in the lithium-pilocarpine-induced SE rat model via possible involvement of TNF-α and nitrergic pathways.

Boldine, an Alkaloid from Peumus boldus Molina, Induces Endothelium-Dependent Vasodilation in the Perfused Rat Kidney: Involvement of Nitric Oxide and Small-Conductance Ca2+-Activated K+ Channel

Boldine, 2,9-dihydroxy-1,10-dimethoxyaporphine, is the main alkaloid found in the leaves and bark of Peumus boldus Molina. In recent years, boldine has demonstrated several pharmacological properties that benefit endothelial function, blood pressure control, and reduce damage in kidney diseases. However, the renal vasodilator effects and mechanisms remain unknown. Herein, perfused rat kidneys were used to study the ability of boldine to induce vasodilation of renal arteries. For that, left kidney preparations with and without functional endothelium were contracted with phenylephrine and received 10–300 nmol boldine injections. The preparations were then perfused for 15 min with phenylephrine plus L-NAME, indomethacin, KCl, tetraethylammonium, glibenclamide, apamin, charybdotoxin, or iberiotoxin. In 30, 100, and 300 nmol doses, boldine induced a dose-and endothelium-dependent relaxing effect on the renal vascular bed. No vasodilator effects were observed in preparations lacking functional endothelium. While the inhibition of the cyclooxygenase enzyme through the addition of indomethacin did not cause any change in the vasodilating action of boldine, the nonselective nitric oxide synthase inhibitor L-NAME fully precluded the vasodilatory action of boldine at all doses tested. The perfusion with KCl or tetraethylammonium (nonselective K+ channels blocker) also abolished the vasodilatory effect of boldine, indicating the participation of K+ channels in the renal action of boldine. The perfusion with glibenclamide (selective ATP-sensitive K+ channels blocker), iberiotoxin (selective high-conductance Ca2+-activated K+ channel blocker), and charybdotoxin (selective high- and intermediate-conductance Ca2+-activated K+ channel blocker) did not modify the vasodilatory action of boldine. On the other hand, the perfusion with apamin (selective small-conductance Ca2+-activated K+ channel blocker) completely prevented the vasodilatory action of boldine at all doses tested. Together, the present study showed the renal vasodilatory properties of boldine, an effect dependent on the generation of nitric oxide and the opening of a small-conductance Ca2+-activated K+ channel.

Interplay between nitric oxide and gonadotrophin-releasing hormone in the neuromodulation of the corpus luteum during late pregnancy in the rat

BackgroundNitric oxide and GnRH are biological factors that participate in the regulation of reproductive functions. To our knowledge, there are no studies that link NO and GnRH in the sympathetic ganglia. Thus, the aim of the present work was to investigate the influence of NO on GnRH release from the coeliac ganglion and its effect on luteal regression at the end of pregnancy in the rat.MethodsThe ex vivo system composed by the coeliac ganglion, the superior ovarian nerve, and the ovary of rats on day 21 of pregnancy was incubated for 180 min with the addition, into the ganglionic compartment, of L-NG-nitro arginine methyl ester (L-NAME), a non-selective NO synthase inhibitor. The control group consisted in untreated organ systems.ResultsThe addition of L-NAME in the coeliac ganglion compartment decreased NO as well as GnRH release from the coeliac ganglion. In the ovarian compartment, and with respect to the control group, we observed a reduced release of GnRH, NO, and noradrenaline, but an increased production of progesterone, estradiol, and expression of their limiting biosynthetic enzymes, 3β-HSD and P450 aromatase, respectively. The inhibition of NO production by L-NAME in the coeliac ganglion compartment also reduced luteal apoptosis, lipid peroxidation, and nitrotyrosine, whereas it increased the total antioxidant capacity within the corpora lutea.ConclusionCollectively, the results indicate that NO production by the coeliac ganglion modulates the physiology of the ovary and luteal regression during late pregnancy in rats.

Effect of soya phosphatidylcholine and possible underlying mechanism on ischemia/reperfusion injury in isolated perfused rat heart: an experimental and computational study.

This study was designed to assess the effect of soya phosphatidylcholine (SPC) against ischemia/reperfusion (I/R) injury and the possible underlying mechanism using experimental and computational studies. I/R injury was induced by global ischemia for 30min followed by reperfusion for 120min. The perfusion of the SPC was performed for 10min before inducing global ischemia. In the mechanistic study, the involvement of specific cellular pathways was identified using various inhibitors such as ATP-dependent potassium channel (KATP) inhibitor (glibenclamide), protein kinase C (PKC) inhibitor (chelerythrine), non-selective nitric oxide synthase inhibitor (L-NAME), and endothelium remover (Triton X-100). The computational study of various ligands was performed on toll-like receptor 4 (TLR4) protein using AutoDock version 4.0. SPC (100 μM) significantly decreased the levels of cardiac damage markers and %infarction compared with the vehicle control (VC). Furthermore, cardiodynamics (indices of left ventricular contraction (dp/dtmax), indices of left ventricular relaxation (dp/dtmin), coronary flow, and antioxidant enzyme levels were significantly improved as compared with VC. This protective effect was attenuated by glibenclamide, chelerythrine, and Triton X-100, but it was not attenuated by L-NAME. The computational study showed a significant bonding affinity of SPC to the TLR4-MD2 complex. Thus, SPC reduced myocardial I/R injury in isolated perfused rat hearts, which might be governed by the KATP channel, PKC, endothelium response, and TLR4-MyD88 signaling pathway.

Особенности механизмов NO-опосредованной дилатации пиальных артерий на воздействие ацетилхолина у стареющих крыс

The article reports the results of the study focusing on age-related changes in the contribution of NO, synthesized by various forms of nitric oxide synthases (NOS) and exogenous L-arginine in acetylcholine-induced dilation of cerebral vessels in rats. A comparative assessment of the responses of pial arteries of various diameters to acetylcholine chloride (ACh, 10–7 M, 8 min) was carried in the absence and in the presence of NOS blockade and exogenous L-arginine (0.25 mM, 30 min) in Sprague-Dawley rats aged 4 and 18 months. NOS blockade was performed by using a non-selective NOS inhibitor (L-NAME, 10–3 M, 12 min) and an inducible NOS inhibitor (aminoguanidine, 1 mM, 10 min). The change in the number and degree of arterial dilation was assessed by measuring the width of the erythrocyte flow in three separate groups of arteries: small (&lt;20 μm in diameter), medium (20–40 μm), and large (&gt;40 μm). It was found that in 18-month-old rats, the contribution of NO, synthesized by constitutive forms of NOS, decreases in the implementation of ACh-mediated endothelium-dependent dilatatory reactions in small and medium pial arteries, while an increase was observed for pial. At the same time, the contribution of inducible NOS to dilation of all types of arteries is on the increase. These processes are accompanied by a decrease in the bioavailability of exogenous L-arginine for NOS.

Nitric oxide contributes to cerebrovascular shear-mediated dilatation but not steady-state cerebrovascular reactivity to carbon dioxide.

Cerebrovascular CO2 reactivity (CVR) is often considered a bioassay of cerebrovascular endothelial function. We recently introduced a test of cerebral shear-mediated dilatation (cSMD) that may better reflect endothelial function. We aimed to determine the nitric oxide (NO)-dependency of CVR and cSMD. Eleven volunteers underwent a steady-state CVR test and transient CO2 test of cSMD during intravenous infusion of the NO synthase inhibitor NG -monomethyl-l-arginine (l-NMMA) or volume-matched saline (placebo; single-blinded and counter-balanced). We measured cerebral blood flow (CBF; duplex ultrasound), intra-arterial blood pressure and . Paired arterial and jugular venous blood sampling allowed for the determination of trans-cerebral NO2- exchange (ozone-based chemiluminescence). l-NMMA reduced arterial NO2- by ∼25% versus saline (74.3±39.9 vs. 98.1±34.2nM; P=0.03). The steady-state CVR (20.1±11.6nM/min at baseline vs. 3.2±16.7nM/min at +9 mmHg ; P=0.017) and transient cSMD tests (3.4±5.9nM/min at baseline vs. -1.8±8.2nM/min at 120 s post-CO2 ; P=0.044) shifted trans-cerebral NO2- exchange towards a greater net release (a negative value indicates release). Although this trans-cerebral NO2- release was abolished by l-NMMA, CVR did not differ between the saline and l-NMMA trials (57.2±14.6 vs. 54.1±12.1ml/min/mmHg; P=0.49), nor did l-NMMA impact peak internal carotid artery dilatation during the steady-state CVR test (6.2±4.5 vs. 6.2±5.0% dilatation; P=0.960). However, l-NMMA reduced cSMD by ∼37% compared to saline (2.91±1.38 vs. 4.65±2.50%; P=0.009). Our findings indicate that NO is not an obligatory regulator of steady-state CVR. Further, our novel transient CO2 test of cSMD is largely NO-dependent and provides an in vivo bioassay of NO-mediated cerebrovascular function in humans. KEY POINTS: Emerging evidence indicates that a transient CO2 stimulus elicits shear-mediated dilatation of the internal carotid artery, termed cerebral shear-mediated dilatation. Whether or not cerebrovascular reactivity to a steady-state CO2 stimulus is NO-dependent remains unclear in humans. During both a steady-state cerebrovascular reactivity test and a transient CO2 test of cerebral shear-mediated dilatation, trans-cerebral nitrite exchange shifted towards a net release indicating cerebrovascular NO production; this response was not evident following intravenous infusion of the non-selective NO synthase inhibitor NG -monomethyl-l-arginine. NO synthase blockade did not alter cerebrovascular reactivity in the steady-state CO2 test; however, cerebral shear-mediated dilatation following a transient CO2 stimulus was reduced by ∼37% following intravenous infusion of NG -monomethyl-l-arginine. NO is not obligatory for cerebrovascular reactivity to CO2 , but is a key contributor to cerebral shear-mediated dilatation.

TRPA1 Channel Activation With Cinnamaldehyde Induces Cutaneous Vasodilation Through NOS, but Not COX and KCa Channel, Mechanisms in Humans.

Transient receptor potential ankyrin 1 (TRPA1) channel activation induces cutaneous vasodilation in humans in vivo. However, the mechanisms underlying this response remains equivocal. We hypothesized that nitric oxide synthase (NOS) and Ca2+ activated K+ (KCa) channels contribute to the TRPA1 channel-induced cutaneous vasodilation with no involvement of cyclooxygenase (COX). Cutaneous vascular conductance (CVC) in 9 healthy young adults was assessed at 4 dorsal forearm skin sites treated by intradermal microdialysis with (1) 1.985% dimethyl sulfoxide + 0.015% lactated Ringer solution with propylene glycol (vehicle control), (2) 10 mM l-NAME, a nonselective NOS inhibitor, (3) 10 mM ketorolac, a nonselective COX inhibitor, or (4) 50 mM tetraethylammonium, a nonselective KCa channel blocker. Cinnamaldehyde, a TRPA1 channel activator, was administered to each skin site in a dose-dependent manner (2.9%, 8.8%, 26%, and 80%, each lasting ≥30 minutes). Administration of ≥8.8% cinnamaldehyde increased CVC from baseline at the vehicle control site by as much as 27.4% (95% confidence interval of 5.3; P < 0.001). NOS inhibitor attenuated the cinnamaldehyde-induced increases in CVC at the 8.8%, 26%, and 80% concentrations relative to the vehicle control site (all P ≤ 0.05). In contrast, both the COX inhibitor and KCa channel blockers did not attenuate the cinnamaldehyde induced-increases in CVC relative to the vehicle control site for all concentrations (all P ≥ 0.130). We conclude that in human skin in vivo, NOS plays a role in modulating the regulation of cutaneous vasodilation in response to TRPA1 channel activation with no detectable contributions of COX and KCa channels.

Solitary Nitric Oxide Signaling Mediates Mild Stress-Induced Anxiety and Norepinephrine Release in the Bed Nucleus of the Stria Terminalis during Protracted Ethanol Withdrawal.

Ethanol withdrawal (EtOHW) alters the pattern of neurohormonal and behavioral response toward internal and external stimuli, which mediates relapse to alcohol use even after a long period of abstinence. Increased noradrenergic signaling from the nucleus tractus solitarius (NTS) to the bed nucleus of the stria terminalis (BNST) during EtOHW underlies withdrawal-induced anxiety, while nitric oxide synthase (NOS) inhibitors injected into the periaqueductal area attenuate EtOHW-induced anxiety. Therefore, this study investigated the involvement of NOS within the NTS in anxiety and increased norepinephrine (NE) release in the BNST during protracted EtOHW in rats exposed to a mild stress. Rats were intraperitoneally administered 3 g/kg/day EtOH for 21 days followed by 28 days of withdrawal, and on the 28th day of withdrawal, the rats were subjected to restraint stress for 7 minutes. The elevated plus maze test was employed to evaluate anxiety-like behavior in rats, and in vivo microdialysis was used to measure the extracellular NE level in the BNST. In elevated plus maze tests, EtOHW rats but not EtOH-naive rats exhibited anxiety-like behavior when challenged with 7-minute mild restraint stress, which was, respectively, mitigated by prior intra-NTS infusion of the nitric oxide scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO), nonselective NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME), or selective neuronal NOS (nNOS) inhibitor 7-nitroindazole (7-NI). Each of these agents also decreased the plasma corticosterone levels in EtOHW rats. In in vivo microdialysis, prior intra-NTS infusion of carboxy-PTIO, L-NAME, or 7-NI attenuated the mild stress-induced NE release in the BNST of EtOHW rats. Additionally, EtOHW rats showed increased solitary nNOS gene and protein expression. Moreover, the anxiolytic effect of intra-NTS administration of 7-NI was abolished by subsequent intra-NTS administration of sodium nitroprusside. These results suggest that elevation of solitary nitric oxide signaling derived from nNOS mediates stress-precipitated anxiety and norepinephrine release in the BNST during protracted EtOHW.

Ethyl Acetate Fraction from Leandra dasytricha (A. Gray) Cong. Leaves Promotes Vasodilatation and Reduces Blood Pressure in Normotensive and Hypertensive Rats.

Leandra dasytricha (A. Gray) Cong. is widely distributed in the south of Brazil and is commonly used for cardiovascular and kidney ailments. For this study, we used male Wistar normotensive rats (NTRs) and spontaneously hypertensive rats (SHRs) to verify the effects of the ethyl acetate fraction (EAF) obtained from L. dasytricha leaves on isolated aorta relaxation and in the arterial blood pressure. The EAF was analyzed by LC-DAD-MS, and several components were annotated, including hydrolysable tannins, triterpenes, and O- and C-glycosylated dihydrochalcones, such as the most intense ion peak relative to C-hexosyl phloretin (nothofagin; compound number 13). The EAF caused a concentration and endothelium-dependent relaxation of the aorta in both NTRs and SHRs. This effect was abolished in the endothelium-denuded aorta. L-NAME, a nonselective nitric oxide synthase inhibitor, and ODQ, a soluble guanylate cyclase inhibitor, entirely blocked the EAF-induced relaxation. The presence of a muscarinic receptor antagonist or a cyclooxygenase inhibitor did not alter the EAF's effectiveness in relaxing the aorta. The preincubation with tetraethylammonium, a Ca2+-activated K+ channel blocker, and with 4-aminopyridine, a voltage-dependent K+ channel blocker, significantly interfered with the EAF's relaxation. However, the incubation with glibenclamide, an ATP-sensitive K+ channel blocker, and barium chloride, an inward-rectifier K+ channel blocker, did not interfere with the EAF-induced relaxation. The EAF treatment also caused a dose-dependent decrease in the mean arterial pressure, systolic arterial pressure, and diastolic arterial pressure of both NTRs and SHRs, without significantly interfering with heart rate values. In conclusion, this study demonstrated the EAF-induced vasorelaxant and hypotensive actions, primarily dependent on the endothelium function and mainly with the participation of the nitric oxide and Ca2+-activated and voltage-dependent K+ channels.

Analgesic and anti-inflammatory effects of modafinil in a mouse model of neuropathic pain: A role for nitrergic and serotonergic pathways

ABSTRACT Objectives To evaluate the effects of modafinil on neuropathic pain induced by sciatic nerve cuffing in mice, and possible contribution of nitrergic/inflammatory and serotonergic systems. Methods Neuropathic pain was induced by applying a polyethylene cuff around the left sciatic nerve. Seven days later, mice received modafinil (50, 100, and 200 mg/kg; intraperitoneal [i.p.]) and morphine (10 mg/kg, i.p.) as control. Mice also received pretreatments of the nonselective nitric oxide (NO) synthase (NOS) inhibitor L-NAME, the selective neuronal NOS inhibitor 7-nitroindazole, the selective inducible NOS inhibitor aminoguanidine, and the selective serotonin reuptake inhibitor citalopram before modafinil (100 mg/kg). von Frey test was used to evaluate mechanical allodynia. Additionally, sciatic nerves were collected for histopathological analysis. Tissue levels of NO metabolites, tumor necrosis factor (TNF)-α, and interleukin (IL)-6 were assessed. Results Animals whose sciatic nerves were cuffed had a significantly (P<0.001) decreased paw withdrawal threshold (PWT) compared with the sham-operated group. Modafinil (100 mg/kg) and morphine significantly reversed PWT (P<0.001). Pretreatments with L-NAME, 7-nitroindazole, aminoguanidine, and citalopram in different groups markedly reversed analgesic effects of modafinil. Tissue homogenates of Cuffed sciatic nerves showed significantly higher levels of NO metabolites, TNF-α and IL-6 (P<0.001). Modafinil lowered NO metabolites, TNF-α, and IL-6 levels (P<0.001). Histopathology illustrated marked axonal degeneration and shrinkage in the cuffed sciatic nerve, which were improved in the modafinil-treated group. Conclusions Modafinil exerts analgesic and neuroprotective effects in cuff-induced neuropathic mice via possible involvement of the nitrergic/inflammatory and serotonergic systems.

Effect of Lenalidomide on Pentylenetetrazole-Induced Clonic Seizure Threshold in Mice: A Role for N-Methyl-D-Aspartic Acid Receptor/Nitric Oxide Pathway.

Background and PurposeAccumulating evidence suggest that lenalidomide, a structural analog of thalidomide, has neuro-modulatory and neuroprotective properties. In the present study, we investigated effects of acute administration of lenalidomide on clonic seizure threshold in mice induced by pentylenetetrazole (PTZ) and possible role of N-methyl-D-aspartic acid receptor (NMDAR) and nitric oxide (NO) pathway.MethodsWe have utilized a clonic model of seizure in NMRI mice induced by PTZ to evaluate the potential effect of lenalidomide on seizure threshold. Different doses of lenalidomide (5, 10, 20, and 50 mg/kg, intraperitoneal [i.p.]) were administered 1 hour before PTZ. To evaluate probable role of NMDAR/NO signaling, the non-selective NO synthase inhibitor L-NG-nitroarginine methyl ester (L-NAME; 10 mg/kg, i.p.), neuronal NOS (nNOS) inhibitor 7-nitroindazole (7-NI; 30 mg/kg, i.p.), selective inducible NOS inhibitor aminoguanidine (AG; 100 mg/kg, i.p.), selective NMDAR antagonist MK-801 (0.01 mg/kg, i.p.), and selective NMDAR agonist D-serine (30 mg/kg, i.p.) were injected 15 minutes before lenalidomide.ResultsLenalidomide at 10 and 20 mg/kg significantly elevated the PTZ-induced seizure thresholds. Interestingly, L-NAME (10 mg/kg, i.p), 7-NI (30 mg/kg, i.p), and AG (100 mg/kg, i.p) reversed the anticonvulsive effect of lenalidomide (10 mg/kg). Moreover, treatment with the NMDAR agonist D-serine (30 mg/kg, i.p.) did not alter the anticonvulsive properties of lenalidomide (10 mg/kg, i.p). However, the NMDAR antagonist MK-801 (0.01 mg/kg, i.p) significantly reversed the anticonvulsive effects of lenalidomide (10 mg/kg).ConclusionsOur study demonstrated a role for the NMDAR/NO pathway in the anticonvulsive effects of lenalidomide on the PTZ-induced clonic seizures in mice.

TRPA1 channel activation with cinnamaldehyde induces cutaneous vasodilation through NOS, but not COX and KCa channel, mechanisms in human

Transient receptor potential (TRP) channels exist in various cells and tissues including nerves and vascular endothelium. TRP channels are implicated in the regulation of several sensory organs associated with pain, pressure, and temperature. While there exists a number of TRP channels, transient receptor potential vanilloid 1 (TRPV1) and 4 (TRPV4) channels in human skin have been shown to modulate cutaneous vasodilation (Fujii et al., 2019; Wong et. al., 2012). Transient receptor potential ankyrin 1 (TRPA1) subtype, which belongs to the TRP channel, is thought to be activated by cold and possibly warm/hot temperatures (Wang and Siemens, 2015). Recently, we showed that TRPA1 channel activation with cinnamaldehyde induced cutaneous vasodilation in humans (Fujii et al., 2020). However, the mechanisms underlying this response remains to be determined. A previous study using mouse ear showed that nitric oxide (NO) synthase (NOS), which is a meditator of endothelium-dependent vasodilation, can modulate the TRPA1 channel induced cutaneous vasodilation (Aubdool et al., 2016). However, it is unclear if this relationship exists in humans. Moreover, whether cyclooxygenase (COX) and Ca2+ activated K+ (KCa) channels, which are also established modulators of endothelium-dependent vasodilation, contribute to TRPA1 channel induced cutaneous vasodilation remains to be directly assessed in humans. Based on the above, we evaluated the hypothesis that NOS, COX, and KCa channels mediate the TRPA1 channel induced cutaneous vasodilation in humans. To assess our hypothesis, cutaneous vascular conductance (CVC) was assessed in 9 healthy young adults (29 ± 6 years, 4 women) at four dorsal forearm skin sites treated by intradermal microdialysis with either: 1) vehicle control (98 % propylene glycol + 0.015% lactated Ringer solution + 1.985 % dimethyl sulfoxide), 2)10 mM N(G)-Nitro-L-arginine methylester, a non-selective NOS inhibitor, 3)10 mM ketorolac, a non-selective COX inhibitor, or 4) 50 mM tetraethylammonium, a non-selective KCa channel blocker. Cinnamaldehyde, a TRPA1 channel activator, was administered to each skin site in a dose-dependent manner (2.9, 8.8, 26 and 80% of cinnamaldehyde concentrations, each lasting at least 30min). Administration of ≥8.8 % cinnamaldehyde increased CVC from baseline at the vehicle control site (e.g., 27.4± 2.7 % increase in CVC, P<0.05). NOS inhibition attenuated the cinnamaldehyde induced-cutaneous vasodilation at 2.9, 8.8, 26 and 80% concentrations relative to the vehicle control site (all P<0.05). COX inhibition and KCa channel blockade did not attenuate the cinnamaldehyde induced-cutaneous vasodilation relative to the vehicle control site at all concentrations (all P>0.05). We conclude that in human skin in vivo, NOS plays a role in modulating the regulation of cutaneous vasodilation in response to TRPA1 channel activation with negligible contributions of COX and KCa channels.