Nitric Oxide Synthase Inhibitor Research Articles

Genistein mitigates nitroglycerine-induced migraine: modulation of nitric oxide-mediated vasodilation and oxidative stress.

Migraine is a widespread brain condition described by frequent, recurrent episodes of incapacitating, moderate-to-severe headaches with throbbing pain that are usually one-sided. It is the 2nd most debilitating state lived with disability in terms of years, with a prevalence rate of 15-20%. Significant drops in estrogen levels have been associated with triggering acute migraine attacks in certain cases. Phytoestrogens are plant-derived compounds that resemble estrogen in structure, enabling them to imitate estrogen's functions in the body by attaching to estrogen receptors. Thus, the study was aimed to explore the protective effect of genistein against migraine. Moreover, the role of nitric oxide was also studied in the observed effect of genistein. Nitric oxide (NO) is implicated in migraine pathophysiology due to its role in promoting cerebral vasodilation and modulation of pain perception. Exploring L-NAME, a nitric oxide synthase inhibitor in migraine research helps scientists better understand the role of NO in migraine. Nitroglycerine treatment significantly increased the facial-unilateral head pain and spontaneous pain, as evidenced by the increased number of head scratching and groomings. Nitroglycerine treatment also induced anxiogenic behavior in mice. A significant reduction in the number of entries in the light phase and open arm, respectively. Biochemical analysis indicated a significant increase in inflammatory and oxidative stress in the nitroglycerin group. A significant increase and decrease in brain TBARS and GSH were observed with nitroglycerine treatment, respectively. Moreover, nitroglycerine treatment has uplifted the serum TNF-α level. Genistein (20mg/kg) significantly mitigated the facial-unilateral head pain, spontaneous pain, photophobia, and anxiety-like behavior induced by nitroglycerine. Biochemical analysis showed that genistein (20mg/kg) significantly abrogated the nitroglycerine-induced lipid peroxidation and increased serum TNF-α level. Genistein treatment also upregulated the brain GSH level and downregulated the serum TNF-α level. The L-NAME-mediated alleviation of the protective effect of genistein might be attributed to the vasodilatory effect of L-NAME. Conclusively, it can be suggested that genistein might provide relief from migraine pain by inhibiting nitric oxide-mediated vasodilation and oxidative stress.

Augmentation of nitric oxide deficient hypertension by high salt diet is associated with reduced TNF-α receptor type 1 expression in the kidneys.

High salt (HS) intake induces an augmented hypertensive response to nitric oxide (NO) inhibition, though it causes minimal changes in blood pressure (BP) in NO intact condition. The cause of such augmentation is not known. HS induces tumor necrosis factor-alpha (TNFα) production that causes natriuresis via activation of its' receptor type 1 (TNFR1). We hypothesized that NO deficiency reduces renal TNFR1 activity, leading to enhanced sodium retention and hypertension. We examined the changes in renal TNFR1 protein expression (Immunohistochemistry analyses) after HS (4% NaCl) intake in wild-type mice (WT, C57BL6) treated with a NO synthase (NOS) inhibitor, nitro-L-arginine methyl ester (L-NAME; 0.05mg/min/g; osmotic mini-pump), as well as in endothelial NOS knockout mice (eNOSKO) and compared the responses in WT mice with normal salt (NS; 0.3% NaCl) intake. BP was measured with tail-cuff plethysmography and 24-hour urine collections were made using metabolic cages. HS alone did not alter mean BP in untreated mice (76±3 to 77±1 mmHg) but induced an augmented response in L-NAME treated (106±1 vs 97±2 mmHg) and in eNOSKO (107±2 vs 89±3 mmHg) mice. The percentage area of TNFR1 expression in renal tissue was higher in WT+HS (4.1+0.5%) than in WT+NS mice (2.7±0.6%). However, TNFR1 expression was significantly lower in L-NAME treated WT+NS (0.9±0.1%) and in eNOSKO+NS (1.4±0.2%) than in both WT+NS and WT+HS mice. These data indicate that TNFR1 activity is downregulated in NO deficient conditions, which facilitates salt retention leading to augmented hypertension during HS intake.

Protection by L-arginine against Epinephrine-induced Arrhythmia and Cardiotoxicity

Nitric oxide is important in control of coronary blood flow, cardiomyocyte contractility, cardiac rhythm, and thrombogenicity. We aimed to investigate the effect of the nitric oxide precursor L-arginine on cardiac arrhythmia and myocardial injury induced by epinephrine in male Sprague-Dawley rats. The possible modulation of L-arginine effects by methylene blue (MethyB), a nitric oxide synthase inhibitor was also examined. Cardiac arrhythmia was induced with 10μg/kg epinephrine given intravenously (i.v.). L-arginine (200mg/kg) or L-arginine and MethyB (100mg/kg) were intraperitoneally (i.p.) administered prior to i.v. epinephrine. Other groups received i.p. saline, only L-arginine or only MethyB (100mg/kg). Results showed that compared with the saline control, epinephrine caused a significant bradycardia (188.7±24.4 vs. 405.6±1.19beats/min), increased QRS duration (0.039±0.006 vs. 0.0185±0.0001s), decreased R wave amplitude (0.18±0.01 vs. 0.23±0.011mv), and ventricular extrasystoles. L-arginine alone showed no significant effect on heart rate (380.8±10.0 vs. 405.6±1.19beats/min) but increased QRS duration (0.029±0.0002 vs. 0.0185±0.0001s), and R wave amplitude (0.47±0.01 vs. 0.23±0.011mv). L-arginine given prior to epinephrine prevented bradycardia, shortened the duration of arrhythmia and decreased the number of extrasystoles. The administration of L-arginine and MethyB almost completely suppressed epinephrine-induced ventricular arrhythmias. Epinephrine caused severe degeneration of muscle fibres, widening of intercellular spaces, cellular infiltrations and interstitial haemorrhage. L-arginine markedly attenuated, while L-arginine/MethyB completely prevented these changes. It is concluded that L-arginine protects against cardiac arrhythmias and cardiac tissue damage caused by epinephrine.

Hypoxia-induced NOS1 as a therapeutic target in hypercholesterolemia-related colorectal cancer

BackgroundIt is well established that hypercholesterolemia increases the risk of atherosclerosis, especially because it reduces the availability of nitric oxide (NO). However, the relationship between hypercholesterolemia and NO in regulating colorectal cancer development and progression remains unknown.MethodsWe conducted bioinformatics analysis, qRT-PCR, ChIP-qPCR assays, luciferase report assays, clonogenic survival assays, and multiple mouse models to investigate the function and mechanism of hypercholesterolemia in regulating NO signaling. Additionally, NOS inhibitors were used to evaluate the potential of therapeutic strategy in anti-tumor response.ResultsHere, we show that oxidized low-density lipoprotein (oxLDL) cholesterol and its receptor LOX-1 are essential for hypercholesterolemia-induced colorectal tumorigenesis. Mechanically, the oxLDL promotes the oxidant stress-dependent induction of hypoxia signaling to transcriptionally up-regulate NO synthase (NOS) especially NOS1 expression in colorectal cancer (CRC) cells. More importantly, our results suggested that selective inhibition of NOS1 with its specific inhibitor Nω-Propyl-L-arginine is a suitable therapeutic strategy for hypercholesterolemia-related CRC with both efficacy and toxicity reduction.ConclusionsOur findings established that hypercholesterolemia induces the oxidant stress-dependent induction of hypoxia signaling to transcriptionally up-regulate NOS1 expression in CRC cells, and the clinically applicable NOS1 inhibitor Nω-Propyl-L-arginine represents an effective therapeutic strategy for hypercholesterolemia-related CRC.

Distinct metabolic responses to heme in inflammatory human and mouse macrophages – Role of nitric oxide

Activation of inflammation is tightly associated with metabolic reprogramming in macrophages. The iron-containing tetrapyrrole heme can induce pro-oxidant and pro-inflammatory effects in murine macrophages, but has been associated with polarization towards an anti-inflammatory phenotype in human macrophages. In the current study, we compared the regulatory responses to heme and the prototypical Toll-like receptor (TLR)4 ligand lipopolysaccharide (LPS) in human and mouse macrophages with a particular focus on alterations of cellular bioenergetics. In human macrophages, bulk RNA-sequencing analysis indicated that heme led to an anti-inflammatory transcriptional profile, whereas LPS induced a classical pro-inflammatory gene response. Co-stimulation of heme with LPS caused opposing regulatory patterns of inflammatory activation and cellular bioenergetics in human and mouse macrophages. Specifically, in LPS-stimulated murine, but not human macrophages, heme led to a marked suppression of oxidative phosphorylation and an up-regulation of glycolysis. The species-specific alterations in cellular bioenergetics and inflammatory responses to heme were critically dependent on the availability of nitric oxide (NO) that is generated in inflammatory mouse, but not human macrophages. Accordingly, studies with an inducible nitric oxide synthase (iNOS) inhibitor in mouse, and a pharmacological NO donor in human macrophages, reveal that NO is responsible for the opposing effects of heme in these cells. Taken together, the current findings indicate that NO is critical for the immunomodulatory role of heme in macrophages.

The Pathway-Selective Dependence of Nitric Oxide for Long-Term Potentiation in the Anterior Cingulate Cortex of Adult Mice.

Nitric oxide (NO) is a key diffusible messenger in the mammalian brain. It has been proposed that NO may diffuse in retrograde into presynaptic terminals, contributing to the induction of hippocampal long-term potentiation (LTP). Here, we present novel evidence that NO is selectively required for the synaptic potentiation of the interhemispheric projection in the anterior cingulate cortex (ACC). Unilateral low-frequency stimulation (LFS) induced a short-term synaptic potentiation on the contralateral ACC through the corpus callosum (CC). The use of the antagonists of the NMDA receptor (NMDAR), or the inhibitor of the L-type voltage-dependent Ca2+ channels (L-VDCCs), blocked the induction of this ACC-ACC potentiation. In addition, the inhibitor of NO synthase, or inhibitors for its downstream signaling pathway, also blocked this ACC-ACC potentiation. However, the application of the NOS inhibitor blocked neither the local electric stimulation-induced LTP nor the stimulation-induced recruitment of silent responses. Our results present strong evidence for the pathway-selective roles of NO in the LTP of the ACC.

The relaxation effect of endocannabinoid anandamide on isolated rat bladder and vas deferens tissues and possible mechanisms.

The endocannabinoid system plays important roles in various systems, including the genitourinary system; however, its mechanism of action is not fully understood. This study aimed to investigate the direct relaxant effects of anandamide and its possible mechanisms in isolated rat bladder and vas deferens tissues. Twenty-one adult male Wistar albino rats were used. Bladder and vas deferens (prostatic and epididymal portions) tissues were mounted in 10 mL of organ baths. Relaxation responses to anandamide were recorded at 3 and 10 μM concentrations. After the rest period, the procedures were repeated in the presence of cannabinoid (CB) and vanilloid receptor antagonists, various potassium channel blockers, cyclo-oxygenase, and nitric oxide synthase inhibitors. In different tissues to investigate the Ca2+-channel antagonistic effect of anandamide, concentration-response curves to CaCl2 were obtained in the absence and presence of anandamide. Anandamide caused a significant relaxation response in the bladder and epididymal vas deferens tissues, but not in the prostatic portion. The effect of anandamide was antagonized in the presence of the CB1 antagonist AM251 or the non-selective potassium channel blocker tetraethylammonium in bladder tissue. In the epididymal vas deferens, anandamide significantly inhibited the calcium contraction responses, especially at high concentrations. The CB2 antagonist AM630 reversed this inhibition. The results show that anandamide has a direct relaxant effect on the isolated rat bladder and epididymal vas deferens. Anandamide triggers different mechanisms in different types of tissues, and further studies are needed to elucidate the mechanism of action of anandamide.

Ethnomedicinal Study and Evaluation of the Anxiolytic-like and Diuretic Effects of the Orchid Stanhopea tigrina Bateman ex Lindl-(Orchidaceae).

Stanhopea tigrina Bateman ex Lindl. (Orchidaceae) is an orchid endemic to Mexico, known as "Calavera" or "calaverita", in the Huasteca Potosina (central region of Mexico). This plant species is used for the folk treatment of mental disorders and urological kidney disorders, according to the ethnomedicinal information obtained in this study. Ethanolic extracts of leaves (HE) and pseudobulb (PE) were obtained by microwave-assisted extraction (MAE). Gas Chromatography coupled with Mass Spectrometry (GC-MS) was used to carry out the chemical characterization of HE and PE. The pharmacological effects (antioxidant, diuretic, anxiolytic, locomotor, hypnotic, and sedative) of HE and PE were evaluated. The possible mechanism of action of the anxiolytic-like activity induced by HE was assessed using inhibitors of the GABAergic, adrenergic, and serotonergic systems. The possible mechanism of the diuretic action of HE was assessed using prostaglandin inhibitory antagonists and nitric oxide synthase (NOS) blockers. HE at 50 and 100 mg/kg exerted anxiolytic-like activity without inducing hypnosis or sedation. Flumazenil, prazosin, and ketanserin inhibited the anxiolytic-like activity shown by HE, which suggests the participation of GABA, α1-adrenergic receptors, and 5-HT2 receptors, respectively. The diuretic effect was reversed by the non-selective NOS inhibitor L-NAME, which caused the reduction in nitric oxide (NO). These results demonstrate that the ethanolic extract of S. tigrina leaves exhibited anxiolytic-like activity and diuretic effects without inducing hypnosis or sedation. This work validates the medicinal uses of this orchid species.

Inhibition of activin receptor 2 signalling ameliorates metabolic dysfunction–associated steatotic liver disease in western diet/L-NAME induced cardiometabolic disease

ObjectiveBlockade of activin 2 receptor (ACVR2) signaling has been shown to improve insulin sensitivity and aid in weight loss. Inhibition of ACVR2 signaling restores cardiac function in multiple heart failure models. However, its potential in the treatment of obesity-related cardiometabolic disease remains unknown. Here, we investigated targeting ACVR2 signaling in cardiometabolic disease manifested with metabolic dysfunction–associated steatotic liver disease (MASLD). MethodsMice were fed a high-fat, high-sugar diet combined with the administration of nitric oxide synthase inhibitor L-NAME in drinking water, which causes hypertensive stress. For the last eight weeks, the mice were treated with the soluble ACVR2B decoy receptor (sACVR2B-Fc). ResultssACVR2B-Fc protected against the development of comorbidities associated with cardiometabolic disease. This was most pronounced in the liver where ACVR2 blockade attenuated the development of MASLD including cessation of pro-fibrotic activation. It also significantly reduced total plasma cholesterol levels, impeded brown adipose tissue whitening, and improved cardiac diastolic function. In vitro, ACVR2 ligands activin A, activin B and GDF11 induced profibrotic signaling and the proliferation of human cardiac fibroblasts. ConclusionsBlockade of ACVR2B exerts broad beneficial effects for therapy of cardiometabolic disease. By reducing obesity, ameliorating cardiovascular deterioration and restraining MASLD, blockade of ACVR2B signaling proves a potential target in MASLD and its comorbidities.

Loss of nitric oxide bioavailability promotes arterial stiffening in a tissue transglutaminase-dependent manner

Although arterial stiffening undeniably contributes to cardiovascular disease, the cellular and molecular mechanisms leading to increased arterial stiffness are not well understood. Increased arterial stiffness in disease states such as obesity and type 2 diabetes typically coexists with indices of endothelial dysfunction including diminished nitric oxide (NO) bioavailability. In fact, it is commonly assumed that endothelial dysfunction precedes, and causally contributes to, arterial stiffening; yet the molecular underpinnings for this causal relationship remain elusive. We and others previously showed that activation of tissue transglutaminase (TG2), a crosslinking enzyme that also works as a G-protein and cellular scaffold, promotes cytoskeletal actin polymerization and stiffening of vascular smooth muscle cells (VSMC), thus contributing to whole-artery stiffening. It is also reported that NO is an endogenous inhibitor of TG2. Accordingly, herein, we tested the hypothesis that increased exposure of VSMC to NO suppresses TG2 activity and actin polymerization. Conversely, we hypothesized that loss of NO bioavailability promotes arterial stiffening, and that this effect is mediated by TG2 activity and downstream activation of LIM kinase (LIMK), an enzyme that modulates fibrillar (F)-actin severing via cofilin phosphorylation. All differences reported herein are significant at P<0.05. In support of our hypotheses, we show that treatment of cultured human aortic VSMC with the NO donor S-nitrosoglutathione (200μM for 2 hours) reduces TG2 activity (N-epsilon gamma glutamyl Lysine presence) and F-actin formation (phalloidin staining) induced by TG2 activation (dithiothreitol, 200μM). Furthermore, we show that exposure of mouse isolated mesenteric arteries to the NO synthase inhibitor L-NAME (300μM for 18 hours) causes an increase in arterial stiffness ( i.e., increased incremental modulus of elasticity) as determined using pressure myography and videomicroscopy. Of note, L-NAME-induced arterial stiffening is prevented when arteries are co-treated with a TG2 inhibitor (cystamine, 1mM) or LIMK inhibitor (LIMKi, 1μM). Taken together, these findings support the notion that NO is a key downregulator of TG2 activity and demonstrate that reduced NO bioavailability promotes arterial stiffening in a VSMC TG2-LIMK-F-actin dependent manner. As such, this work provides insight into plausible molecular mechanisms by which endothelial dysfunction leads to arterial stiffening. R01HL153264 (to LAML and JP), and MU Research Excellence Program (REP) postdoctoral fellowship (to FIRP). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Divergent Vasodilator Mechanisms to Endothelial-Dependent Agonists with Advancing Age

Introduction: Microvascular function is maintained by a number of vasodilating factors, with nitric oxide (NO) and endothelial hyperpolarizing factors (EDHF) predominating. Advancing age is an independent risk factor in the development of cardiovascular disease, and NO-mediated vasodilation declines, or is unchanged, depending on the vascular bed examined, number of cardiovascular risk factors, or specific endothelial-dependent agonist used. Mechanisms contributing to cutaneous microvascular dysfunction with age have been attributed largely to reductions in NO signaling, but the role of EDHF-mediated dilation, particularly to acetylcholine (ACh), remains unclear. The purpose of this study was to determine the endothelial mechanisms contributing to age-induced cutaneous microvascular function and compare cutaneous microvascular function responses to those from isolated adipose arteriole preparations. We hypothesized that cutaneous microvascular vasodilation to ACh in older adults will be mediated primarily by EDHFs relative to young adults and that isolated adipose arteriole preparations will corroborate these findings. Methods: Four intradermal microdialysis fibers were placed in the skin of the ventral forearm of 9 young (3F/6M; 29±7 yrs) and 3 older adults (3F/0M; 59±11 yrs) for infusion of Ringer’s solution; L-NAME (10 mM), an NO synthase inhibitor; tetraethylammonium chloride (TEA; 50 mM), a non-specific K+ channel inhibitor; and a combination of L-NAME and TEA. Laser Doppler flowmetry and brachial mean arterial pressure were measured during infusion of ACh (10 mM and 100 mM) and in response to local heating to 39°C. Maximal vasodilation was assessed with heating to 43°C and infusion of sodium nitroprusside (28 mM). Adipose resistance arterioles for pressure myography were obtained from a gluteal biopsy or from surgical discard tissue, which were pre-constricted (ET-1), and ACh responses (10−9–10−4 M) were assessed in the presence of the above inhibitors. Cutaneous vascular conductance (CVC) and isolated arteriole responses are presented as % maximum vasodilation. Results: In response to local heating in young adults, L-NAME reduced vasodilator magnitude (Ringers 69.0±18.1% vs. L-NAME 30.3±12.1%; p< 0.05) while TEA did not have any effect (69±18% vs. 49±28%; p=0.20). Combined L-NAME and TEA reduced vasodilation (69±18% vs. 19±6%; p<0.05). Conversely, vasodilator magnitude to ACh was not different across the Ringers, L-NAME, or TEA sites (p=0.93-0.94), while combined inhibition of NOS and K+ channels with L-NAME and TEA, respectively, reduced vasodilator responsiveness (p<0.05). In isolated resistance arterioles, there were no differences in %max dilation to ACh across inhibitor sites (p=0.19) in young adults. In older adults, neither independent nor combined inhibition of NOS or K+ channels impacted vasodilator responses to local heating, while vasodilator magnitude to ACh was most impacted by combined inhibition with L-NAME and TEA (83±14% vs. 32±15%; p<0.05). Conclusion: Our data demonstrate that vasodilator plasticity in response to local heating is divergent with advancing age, relying on non-NO or K+ mechanisms relative to young adults, while NO and K+ mechanisms are suffcient to maintain ACh-mediated vasodilation. These findings further support the concept of diversity in vasodilator mechanisms within the microvasculature. NHLBI K99-HL161491 (WEH), K08-HL150340 (JJM), R01-HL160752 (JKF), R01-HL133029 (AB), and NICHD R01-HD099340 (MJD). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Inhibition of NF-κB Activation Improves Non-Nitric Oxide-Mediated Cutaneous Microvascular Function in Reproductive-Aged Healthy Women

PURPOSE: The human cutaneous circulation is used as an in vivo bioassay to examine mechanisms of systemic vascular function and dysfunction in health and disease. In cohorts of patients with accelerated cardiovascular disease risk, systemic interventions to investigate inflammatory mechanisms [e.g., oral salsalate; nuclear factor kappa-B (NF-κB) inhibitor] have been used to elucidate mechanisms underlying microvascular dysfunction. In healthy control subjects, inhibition of inflammatory mechanisms modestly reduces microvascular function. We hypothesize that inhibition of NF-κB activation may impair nitric oxide (NO)-mediated cutaneous microvascular function in reproductive-aged healthy women. METHODS: In a randomized, single-blind, placebo-controlled design, oral salsalate (1500mg/b.i.d./5 days) was used to assess the impact of systemic inflammation on cutaneous microvascular function. Two intradermal microdialysis fibers were placed into the ventral forearm skin of eleven healthy women [Mean (SEM): 34 (2) yrs]. Laser-Doppler flowmetry was used to measure cutaneous blood flow while increasing concentrations of acetylcholine (10−10 to 10−1 M; endothelium-dependent vasodilator) were perfused with a control (lactated Ringer’s) or a NO synthase inhibitor (15 mM NG-nitro-L-arginine methyl ester). Following the dose-response protocol, maximal cutaneous vasodilation was induced by increasing local skin temperature to 43°C and perfusing 28 mM sodium nitroprusside. Cutaneous vascular conductance (CVC) was calculated as the quotient of red blood cell flux over mean arterial pressure and normalized as a percentage of the site-specific maximum (%CVCmax). The NO-dependent contribution was calculated as the difference between the area under the dose-response curve (AUC) of the control and the NO synthase-inhibited sites. RESULTS: There was an overall main effect of salsalate treatment ( p < 0.001). Both the control and NO synthase-inhibited sites shifted upwards following salsalate treatment (Placebo - Control: 45.78 (8.81)% vs NO synthase inhibited: 33.62 (8.10)%; Salsalate - Control: 56.30 (8.48)% vs NO synthase inhibited: 41.57 (7.85)%; both p < 0.05). There was no change in the NO-dependent contribution between treatments (Placebo: 164.30 (35.40) AU vs Salsalate: 180.30 (51.17) AU; p=0.7028).CONCLUSION: Inhibition of NF-κB activation improved cutaneous microvascular function in reproductive-aged healthy women. This improvement was mediated via non-nitric oxide-dependent mechanisms. Supported by NIH Grant R01 HL161000 (LMA) and Diversity Supplement R01 HL161000-02S2 (VGC). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

CD4+ T Cell Effector Status Is Dependent on Aryl Hydrocarbon Receptor Activation during High Salt Diet

Recent studies suggest that patients with hypertension and individuals that consume a HSD are depleted of numerous tryptophan-derived indoles and other AHR-binding metabolites that are key in intestinal CD4+ T cell homeostasis. Thus, the inappropriate immunologic response by CD4+ T cells, and their role in hypertension, may be due in part to loss of AHR activation. Therefore, we hypothesized that AHR activation contributes to maintaining effector T cell status in salt-sensitive mice. To examine the influence of a representative homeostatic or proinflammatory environment and the subsequent response to salt, we cultured naïve CD4+ T cells under homeostatic cytokine or pathogenic cytokine conditions and exposed the cultures to increased NaCl conditions with or without the AHR-activating metabolite, FICZ (6-Formylindolo(3,2-b)-carbazole). TH17 cells cultured under homeostatic high salt conditions were depressed with a concomitant increase in response to FICZ. In contrast, TH17 cells cultured under pathogenic high salt conditions had augmented IL-17A-expressing cells without a response to AHR activation. These data suggest that excess salt is disrupting the ability to induce IL-17A and increasing AHR activity overcomes this disruption to promote IL-17A, without expansion of the pathogenic TH17 pool, and this observation was specific to non-pathogenic microenvironments (homeostatic TH17: media: 11±0.6%, +NaCl: 4.9±0.3%*, +NaCl/FICZ: 9.9±0.8%, of CD4; pathogenic TH17: media: 1.0±0.2%, +NaCl: 2.2±0.2%*, +NaCl/FICZ: 2.2±0.2%*, *vs respective media, n=8/group). Given these data, we then fed salt-sensitive mice (nitric oxide synthase inhibition) a HSD or a HSD supplemented with the AHR agonist indole-3-carbinol (I3C) and examined Peyer’s patches, a lymphoid tissue in close proximity to intestinal lumen contents enriched with salt and the I3C-metabolites and noted expansion of non-pathogenic TH17 cells. In conclusion, increasing AHR ligand availability through supplementing I3C may provide a gut-centric experimental approach in targeting experimental salt-sensitivity as it relates to immunologic disturbances. F31HL151264. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Estrogen, Exercise Training, and Nitric-oxide Mediated Sympatholysis

Our laboratory has previously reported that nitric oxide (NO)-mediated inhibition of sympathetic vasoconstriction in contracting muscle (sympatholysis) was enhanced in female, compared with male rats. Estrogen has been shown to modulate NO synthase (NOS) expression and NO-mediated sympatholysis. Therefore, this study investigated the hypothesis that NO-mediated inhibition of sympathetic vasoconstriction in resting and contracting skeletal muscle of female rats would be a function of estrogen bioavailability. Further, exercise training has been shown to enhance sympatholysis. Thus, we also investigated the hypothesis that exercise training would restore sympatholysis in estrogen-deficient rats. Female Sprague-Dawley rats were randomized to sedentary ovary-intact (SOI, n=10), sedentary ovariectomized (SOVX, n=9), exercise-trained (10 weeks, 5 days/week, 600m, 40m·min−1, 5% grade) ovary-intact (TOI, n=13), or exercise-trained ovariectomized (TOVX, n=10) groups. OVX rats had both ovaries surgically removed. Following sedentary behavior or exercise training, rats were anesthetized and instrumented for measurement of blood pressure, femoral vascular conductance (FVC), stimulation of the lumbar sympathetic chain, and contraction of the triceps surae muscle group. The percentage change of FVC (%FVC) in response to sympathetic chain stimulation delivered at 2 and 5 Hz was determined at rest and during muscle contraction (60% maximal contractile force) in control, selective neuronal NOS (nNOS) inhibition (SMTC; 0.6 mg·kg−1 IV), and non-selective NOS inhibition (L-NAME; 10 mg·kg−1 IV) conditions. In the Control condition, sympathetic vasoconstrictor responsiveness (%FVC) in resting and contracting skeletal muscle was not different between groups. Under resting conditions, sympathetic vasoconstrictor responsiveness was not different following selective nNOS inhibition (p>0.05), but was increased (p<0.05, main effect of drug) following non-selective NOS inhibition compared with control and selective nNOS inhibition conditions at both 2 Hz and 5 Hz. During muscle contraction, sympathetic vasoconstrictor responsiveness to 2 Hz sympathetic stimulation was unchanged (p>0.05) in the presence of selective nNOS inhibition, but was increased (p<0.05, main effect of drug) following non-selective NOS inhibition compared with control and selective nNOS inhibition conditions. At 5 Hz, sympathetic vasoconstrictor responsiveness was increased (p<0.05, main effect of drug) by selective nNOS inhibition, and increased further (p<0.05, main effect of drug) by non-selective NOS inhibition. The magnitude of sympatholysis at 2 Hz was not different (p>0.05) between groups or drug conditions. At 5 Hz, sympatholysis was blunted (p<0.05, main effect of drug) in the selective nNOS inhibition condition, and further reduced (p<0.05, main effect of drug condition) following non-selective NOS inhibition in all groups. Sympatholysis was not different (p>0.05) between sedentary and exercise trained groups. In conclusion: 1) sympathetic vasoconstrictor responsiveness in resting and contracting skeletal muscle was not dependent on estrogen bioavailability; 2) NO blunted vasoconstriction in all groups, however, NO-mediated sympatholysis was not altered by estrogen status, and; 3) exercise training did not alter sympathetic vasoconstrictor responsiveness or sympatholysis regardless of estrogen bioavailability. NSERC, Canada, Canadian Foundation for Innovation. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Effects of Cross-Sex Hormone Therapy on Metabolic Parameters and Aortic Relaxation in Castrated Male Rats

With the rising awareness, the number of individuals identifying themselves as transgender in the US is increasing over the years. Cross-sex hormone therapy (CSHT) may alter numerous cardiometabolic parameters leading to the increased risk of cardiovascular diseases (CVD) in transgender population. Epidemiological studies suggest that transgender females (male to female, MtF) on estrogen therapy develop higher risk of CVD compared to transgender males (female to male, FtM) taking testosterone. Underlying mechanisms of impaired cardiovascular functions in MtF are not well documented. This study was undertaken to examine the effects of 17β-estradiol (E2) on certain cardiometabolic parameters and aortic reactivity in castrated (CAS) male Sprague Dawley (SD) rats. The 8-10-weeks old CAS rats were subcutaneously implanted with either 1.5 mg of E2 (CAS+E2) or placebo (CAS+PL) containing pellets for ~35 days. The age-matched SD intact male (IM) rats were also included in the experimental groups. After performing the glucose tolerance test, animals were sacrificed, and the blood was collected for later measurements of the HbA1C and triglyceride (TG) levels. Endothelium-dependent vasorelaxation (EDV) to acetylcholine (ACh) in aortic rings pre-contracted with phenylephrine (PE) was measured before and after treatment with indomethacin (a cyclooxygenase inhibitor), followed by L-NAME [a nonselective nitric oxide (NO) synthase (NOS) inhibitor]. Contractile responses to PE before and after L-NAME in aortic rings and sodium nitroprusside (SNP)-induced relaxation responses in endothelium-denuded rings were also measured. Here, we report the decreased body weight and visceral white adipose tissue, and improved HbA1C and glucose tolerance in E2-treated CAS rats. However, the E2-treated CAS rats exhibited elevated plasma level of TG. Furthermore, the vascular relaxation to ACh was significantly impaired in aortas from this group compared to those in CAS+PL and IM rats. The impairment of ACh responses in CAS+E2 rats was accompanied by a significant decrease in NO contribution to ACh vasorelaxation. The basal NO levels and the smooth muscle sensitivity to NO were not altered, whereas the maximum contractile responses to PE were enhanced in aortas of CAS+E2 group compared to IM. In conclusion, our data suggests that the glucose homeostasis was improved in CAS+E2 rats. Regardless, this group exhibited increased level of TG in plasma and decreased aortic vasorelaxation to ACh. The impairment of EDV in E2-treated CAS rats might be due to reduced NO-mediated vasorelaxation or increased contractile responses observed in this group. Additional studies are needed to document the underlying mechanisms for improvement of glucose homeostasis and impairment of EDV in CAS+E2 rats. NIDA/NIGMS, 1SC1DA052120-01 to MF and Bridge Grant from University of the Pacific to RR. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The differential effects of acute and prolonged L-NAME exposure on A23187-induced NO-dependent dilation in human arterioles

The Ca2+ ionophore A23187 induces endothelium-dependent and non-receptor-mediated vasodilation in human adipose arterioles (HAA). Preliminary data from our group suggest that while A23187 dilation mainly depends on NO as a vasodilator factor in healthy HAAs, there is a component of non-NO and likely hydrogen peroxide (H2O2)-mediated dilation, especially in arterioles from patients with coronary artery disease (CAD). The mechanisms underlying the release of two different vasodilator factors remain largely unclear. There is evidence that NO-mediated S-nitrosylation inhibits the catalytic activity of mitochondrial electron transport chain (ETC) complexes or other reactive oxygen species (ROS)-producing enzymes such as NADPH oxidases (NOX). The objective of this study was to compare the effects of acute (30 minutes) and prolonged (90 minutes) nitric oxide (NO) synthase inhibitor nitro-L-arginine methyl ester (L-NAME) exposure on A23187 dilation to elucidate whether prolonged incubation with L-NAME results in transition from NO-dependent to H2O2-dependent vasodilation in HAAs. HAAs (~150 μm ID), isolated from fat tissues obtained from subjects with and without CAD, were cannulated and pressurized at 60 mmHg for videomicroscopic measurement of vessel diameters. In non-CAD HAAs constricted with endothelin-1, A23187 (10−11 to 10−7 M) elicited concentration-dependent dilation (maximal dilation at 10−7 M: 68±5%, n=5), and this dilation was largely abolished after acute exposure to L-NAME (maximal dilation at 10−7 M: 21±7%, n=5). Prolonged L-NAME incubation restored A23187-dependent dilation (maximal dilation at 10−7 M: 74±2%, n=5), which was subsequently inhibited by hydrogen peroxide scavenger catalase (500 U/ml; maximal dilation at 10−7 M: 14±4%, n=5). HAAs obtained from CAD patients, responded to A23187 in a similar dose-dependent fashion (maximal dilation at 10−7 M: 68±3%, n=5), and this dilation was not affected by acute L-NAME exposure (maximal dilation at 10−7 M: 65±3%, n=5), but was reduced by catalase (maximal dilation at 10−7 M: 31±6%, n=6). To further determine the role of mitochondria-derived ROS in A23187-induced dilation, we treated arterioles with ETC inhibitor rotenone (1 mM). In non-CAD HAAs not pretreated with L-NAME, rotenone had no effect on A23187-induced dilation (maximal dilation at 10−7 M: 68±6%, n=5). In the presence of rotenone, however, prolonged L-NAME incubation no longer restored A23187-dependent dilation (maximal dilation at 10−7 M: 20±3%, n=5). Together, these data provide the first evidence that in healthy HAAs, prolonged L-NAME exposure changes the mechanism of vasodilation from NO to H2O2-mediated CAD-like vasodilation in response to A23187. In addition, the signaling process likely involves mitochondrial ROS. To elucidate the precise mechanisms underlying this process, future studies will be required to evaluate whether in healthy vasculature, basal NO inhibits mitochondrial ETC or other proteins such as NOXs through NO-mediated protein modification, which can be reversed by prolonged L-NAME incubation thereby promoting a switch from NO to H2O2 -mediated dilation. This work was supported by the National Heart, Lung, and Blood Institute [R01HL096647]. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Neuronal nitric oxide synthase is the main isoform responsible for 6-nitrodopamine release from mouse isolated vas deferens

Rat and human vas deferens (VD) present basal release 6-nitrodopamine (6-ND), which is significantly reduced when the VD is pre-incubated with the nitric oxide (NO) synthase inhibitor L-NAME, or in the case of the rat, when the VD was obtained from rats chronically treated with L-NAME. 6-Nitrodopamine is considered a major mediator of both rat and human VD contractility. Since the origin of 6-ND is not clear in the VD, here it was investigated the basal release of 6-ND from control, eNOS−/−, nNOS−/− and iNOS−/− mice VD to ascertain which NOS isoform(s) play(s) an essential role in 6-ND biosynthesis in the mouse VD. Euthanasia was performed by isoflurane overdose and both VD were removed and immediately placed in Krebs-Henseleit solution (KHS). The release of 6-ND was measured by LC-MS/MS. To evaluate contractility, the mice VD strips were submitted to EFS (60 V for 20 sec, at 2-16 Hz in square-wave pulses, 0.3 ms pulse width, and 0.1 ms delay), using a Grass S88 stimulator (Astro-Medical, Industrial Park, RI, USA). The tissues were allowed to equilibrate under a resting tension of 10 mN, and the isometric tension was registered using a PowerLab system. Isolated VD from control, nNOS−/−), eNOS−/−, and iNOS−/− mice present basal release of both 6-ND and dopamine, as detected by LC-MS/MS. The basal release of 6-ND from isolated VD of nNOS−/− mice was significantly reduced when compared to that obtained from control mice VD, whereas that of dopamine was significantly increased. The basal release of both 6-ND and dopamine from eNOS−/− mice VD was not different compared to that obtained from control mice VD. The basal release of 6-ND from iNOS−/− mice was significantly increased compared to that observed in control mice VD. Electric-field stimulation (EFS) caused frequency-dependent (2Hz - 32Hz) contractions of the mouse isolated VD. The contractions induced by EFS were significantly decreased in nNOS−/− mouse isolated VD, but unaltered in isolated VD obtained from either eNOS−/− or iNOS−/− mice. The results clearly demonstrate that neuronal nitric oxide synthase is the main isoform responsible for 6-ND synthesis/release from mouse isolated VD. The results obtained with the contractions induced by EFS in the mouse VD further support the concept that 6-ND is a major modulator of VD contractility, since the contractions of the VD was only reduced in tissue obtained from nNOS−/− mice. The reduction in the EFS-induced contractions observed in the vas deferens obtained from nNOS−/− mice could be attributed to the reduction observed in the basal release of 6-ND. This research is supported by the São Paulo Research Foundation (FAPESP). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Antidepressant Treatment Preserves Microvascular Endothelium-Dependent Dilation in Women Who Have Had Preeclampsia

Women with a history of preeclampsia (hxPE) demonstrate persistent microvascular endothelial dysfunction which may contribute to the progression of cardiovascular disease. Women with hxPE also have higher rates of depression compared to women with a history of healthy pregnancy. Interestingly, accumulating evidence indicates that antidepressant treatment may have vasculoprotective effects and attenuate preeclampsia risk. However, whether antidepressant treatment impacts microvascular function in these women remains unknown. Therefore, we examined the effect of chronic antidepressant use on microvascular function in women with hxPE. We hypothesized that antidepressant-treated women with hxPE would have greater endothelium-mediated microvascular vasodilation than unmedicated women with hxPE. Five women with hxPE who were currently treated with an antidepressant (hxPE+AD; 36±3 years; 26±7 months postpartum), six who were not (hxPE-AD; 34±2 years; 26±5 months postpartum), and six unmedicated women with a history of uncomplicated pregnancy (HC; 37±1 years; 35±7 months postpartum) participated in one study visit. We measured cutaneous vascular conductance (CVC=laser doppler flowmetry flux/MAP) responses to graded perfusion of acetylcholine (10−10-10−1 mM) via microdialysis in a control (lactated Ringer's) site and a site treated with 15 mM NG-nitro-L-arginine methyl ester [L-NAME; nitric oxide (NO) synthase inhibitor]. hxPE-AD had reduced endothelium-dependent dilation compared to HC (acetylcholine ³10−3 mM; P<0.03). hxPE+AD had greater endothelium-dependent dilation compared to hxPE-AD (acetylcholine 10−2 and 10−3 mM; P<0.02) and there were no differences between hxPE+AD and HC (all P>0.05). L-NAME reduced endothelium-dependent dilation in all groups (all P<0.001), and there was no difference between groups in NO-dependent dilation [area under the curve (AUC) at a control site − AUC at a L-NAME treated site; P=0.522]. These data suggest that microvascular endothelium-dependent dilation is preserved in women with a history of preeclampsia treated with an antidepressant, potentially mediated through non-NO-dependent mechanisms. Funding: American Heart Association CDA 937990. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

N-Methyl-D-aspartic acid (NMDA) mediated vasodilation in aorta

Although N-Methyl-D-aspartic acid (NMDA) mediated vasodilation has been extensively studied in cerebral circulation, there are limited studies that address the NMDA-mediated vascular responses at the peripheral vascular system. In this study our objective is to address the peripheral vascular responses mediated by NMDA. Vascular responses were obtained via in vitro myography on thoracic aorta segments isolated from C57BL/6 wild type male mice. NMDA at the concentration range of 100 micromolar to 1 millimolar in aortic segments precontracted by 1 micromolar phenylephrine. Maximal vasodilation of 75% in reference to baseline prior to drug application was observed at 3mM NMDA. NMDA receptor blockade by MK-801 did not change NMDA-induced vasodilation (55% vs 58%). Neither vehicle of NMDA, nor corresponding osmolar solutions of mannitol to NMDA concentrations induced vasodilation. Nitric oxide synthase (NOS) inhibition by 100 micromolar m Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) reduced 3mM NMDA induced vasodilation from 75% to 16%. Removal of endothelial cell layer did not change the vasodilation mediated by NMDA. NMDA at millimolar concentrations relaxes vascular smooth muscle via non-endothelium dependent and NMDA-receptor independent mechanisms. Vasodilatory response to NMDA appears partially mediated by NOS. It is possible that NMDA is exerting its vasodilatory effects via non-NMDA receptors on the vascular smooth muscle. Whether NMDA-induced vasorelaxation is observed in resistance arteries remains warranted. PSC CUNY 66608-00 54. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The gut-brain and gut-macrophage contribution to gastrointestinal dysfunction with systemic inflammation

The gastrointestinal tract is one of the main organs affected during systemic inflammation and disrupted gastrointestinal motility is a major clinical manifestation. Many studies have investigated the involvement of neuroimmune interactions in regulating colonic motility during localized colonic inflammation, i.e., colitis. However, little is known about how the enteric nervous system and intestinal macrophages contribute to dysregulated motility during systemic inflammation. Given that systemic inflammation commonly results from the innate immune response against bacterial infection, we mimicked bacterial infection by administering lipopolysaccharide (LPS) to rats and assessed colonic motility using ex vivo video imaging techniques. We utilized the Cx3cr1-Dtr rat model of transient depletion of macrophages to investigate the role of intestinal macrophages in regulating colonic motility during LPS infection. To investigate the role of inhibitory enteric neurotransmission on colonic motility following LPS, we applied the nitric oxide synthase inhibitor, Nω-nitro-L-arginine (NOLA). Our results confirmed an increase in colonic contraction frequency during LPS-induced systemic inflammation. However, neither the depletion of intestinal macrophages, nor the suppression of inhibitory enteric nervous system activity impacted colonic motility disruption during inflammation. This implies that the interplay between the enteric nervous system and intestinal macrophages is nuanced, and complex, and further investigation is needed to clarify their joint roles in colonic motility.