Inhibitor L-NAME Research Articles

Pasteurized Akkermansia muciniphila Ameliorates Preeclampsia in Mice by Enhancing Gut Barrier Integrity, Improving Endothelial Function, and Modulating Gut Metabolic Dysregulation.

Preeclampsia (PE) is a serious complication of pregnancy linked to endothelial dysfunction and an imbalance in the gut microbiota. While Akkermansia muciniphila (AKK) has shown promise in alleviating PE symptoms, the use of live bacteria raises safety concerns. This study explored the potential of pasteurized A. muciniphila (pAKK) as a safer alternative for treating PE, focusing on its effects on endothelial function and metabolic regulation. A PE mouse model was induced via the nitric oxide synthase inhibitor L-NAME, followed by treatment with either pAKK or live AKK. Fecal metabolomic profiling was performed via liquid chromatography-tandem mass spectrometry (LC-MS/MS), and in vivo and in vitro experiments were used to assess the effects of pAKK on endothelial function and metabolic pathways. pAKK exhibited therapeutic effects comparable to those of live AKK in improving L-NAME-induced PE-like phenotypes in mice, including enhanced gut barrier function and reduced endotoxemia. pAKK also promoted placental angiogenesis by restoring endothelial nitric oxide synthase (eNOS) activity and nitric oxide (NO) production. The in vitro experiments further confirmed that pAKK alleviated L-NAME-induced NO reduction and endothelial dysfunction in human umbilical vein endothelial cells (HUVECs). Metabolomic analysis revealed that both pAKK and live AKK reversed metabolic disturbances in PE by modulating key metabolites and pathways related to unsaturated fatty acid biosynthesis, folate, and linoleic acid metabolism. As a postbiotic, pAKK may support existing treatments for preeclampsia by improving gut barrier function, restoring endothelial function, and regulating metabolic dysregulation, offering a safer alternative to live bacteria. These findings highlight the potential clinical value of pAKK as an adjunctive therapy in managing PE.

Propofol Suppresses Ferroptosis via Modulating eNOS/NO Signaling Pathway to Improve Traumatic Brain Injury.

This study aims to explore the neuroprotective effect of propofol in improving traumatic brain injury (TBI) by inhibiting ferroptosis through the modulation of the endothelial nitric oxide (NO) synthase (eNOS)/NO signaling pathway. The GSE173975 dataset was used to analyze the differentially expressed genes between TBI and sham surgery control groups in the short and long term. A TBI model was established in 2-month-old male SPF C57BL/6 mice by impact exposure of the exposed dura mater. After the establishment of the TBI model, propofol (30mg/kg) or saline was administered via intraperitoneal injection for intervention. Nissl staining and Perls staining were employed to assess neuronal function and iron deposition, respectively. Western blot technology was employed to detect the expression of proteins related to ferroptosis. Immunofluorescence staining of astrocytes and microglia was utilized to assess the neuroinflammatory response induced by TBI. The Morris water maze (MWM) and novel object recognition (NOR) tests were employed to assess cognitive dysfunction induced by TBI. Bioinformatics analysis revealed aberrant gene expression associated with iron transport, neuronal death, and inflammatory response in the initial stages of TBI. Long-term abnormalities were predominantly linked to genes involved in inflammatory response. Perls staining and protein expression analysis confirmed the occurrence of iron deposition and ferroptosis following TBI. Propofol treatment significantly reduced iron deposition and ferroptosis induced by TBI. Nissl staining demonstrated enhanced neuronal function, while TUNEL staining indicated reduced neuronal apoptosis. Immunofluorescence analysis demonstrated that propofol significantly reduced the proliferation of astrocytes and activation of microglia induced by TBI in the long term. The results of MWM and NOR tests indicated that propofol significantly improved the long-term cognitive dysfunction induced by TBI. Propofol exerts neuroprotective effects by increasing the expression of eNOS protein and the content of NO. The neuroprotective effects of propofol can be reversed by the eNOS inhibitor L-NAME. Propofol significantly improves the prognosis of TBI by inhibiting ferroptosis through the modulation of the eNOS/NO signaling pathway. The study results provide a scientific basis for the clinical use of propofol as a neuroprotective agent and offer a new direction for the development of new treatment strategies for TBI.

Exploring the anti-depressant effects and nitric oxide modulation of quercetin: A preclinical study in Socially Isolated mice

Objectives This study investigates the effects of quercetin, an antioxidant and nitric oxide (NO) modulator, on depressive-like behaviours triggered by social isolation stress (SIS) in mice. SIS, known to harm psychosocial functioning and increase the risk of depression, involves oxidative stress and NO in its pathophysiology. Methods 72 male mice were divided into nine groups, including the social (SC) group as the control group (stress-free with normal saline intake). The isolation (IC) groups received normal saline, quercetin at doses of 10, 20, and 40 mg/kg, the nitric oxide synthetase inhibitor L-NAME at a dose of 5 mg/kg, the NO precursor L-arginine at a dose of 100 mg/kg, an ineffective dose of quercetin combined with L-NAME and an effective dose of quercetin combined with L-arginine. Behavioural tests (open-field, forced swimming, and splash tests) were conducted, followed by measuring hippocampal nitrite levels. Results Quercetin significantly reduced immobility in the forced swimming test, increased activity in the open-field test, and enhanced grooming behaviour, particularly at 40 mg/kg. Co-administration of an ineffective dose of quercetin (10 mg/kg) with L-NAME increased immobility and grooming activity time. Interestingly, co-administration of the effective dose of quercetin (40 mg/kg) with L-arginine increased immobility time in the FST. Additionally, administration of quercetin at doses of 20 and 40 mg/kg significantly reduced the nitrite level in the hippocampus of SIS mice. Furthermore, co-administration of L-NAME and L-arginine with ineffective and effective doses of quercetin decreased and increased nitrite levels in the hippocampus and increased immobility time in the FST compared to their respective counterparts administered alone. Conclusions These results suggest quercetin’s potential in alleviating depression by modulating NO levels, pointing to its promise in treating depression associated with chronic stressors like social isolation.

Abstract 4141446: Sodium Glucose Co-transporter 2 (SGLT2) Inhibitors Promote Resiliency to High Pressure Stress in the Human Microvasculature

Emerging evidence suggests that vascular stress from cardiovascular-related co-morbidities promotes microvascular dysfunction, a key component in the development of heart failure with preserved ejection fraction. The sodium glucose co-transporter 2 (SGLT2) inhibitor empagliflozin has been shown to reduce both morbidity and mortality associated with heart failure with preserved ejection fraction, however the full scope of influence of this therapy on human microvascular function remains unknown. We hypothesized that pre-treatment of isolated human microvessels with empagliflozin will prevent stress-induced endothelial dysfunction as evidenced by preserving both the magnitude of flow-induced dilation (FID) as well as the ability to dilate to nitric oxide. Human resistance arterioles (80-250µm) from healthy adults (defined as patients with ≤1 risk factor for cardiovascular disease) were dissected from discarded surgical adipose tissue and treated with empagliflozin (1µM), or vehicle control (ethanol) for 16-20 hours prior to the flow experiment. Vessels were cannulated for videomicroscopy and subjected to high intraluminal pressure (150mmHg, 30 min), an acute stress known to induce endothelial dysfunction. Vessels were pre-constricted with endothelin-1 prior to initiation of flow. A nonlinear logistic regression was used to determine differences between curves. Compared to vehicle control, vessels pre-treated with empagliflozin (1µM ) exhibited nitric oxide-dependent FID as dilation was impaired in the presence of the nitric oxide synthase inhibitor L-NAME (EC50 Control: 10.7 vs L-NAME 83.45, p=0.0107). This data suggests that empagliflozin, an SGLT2 inhibitor, promotes microvascular resilience to stress via preservation of nitric oxide-mediated FID. The ability to elicit stress resilience may explain in part some of the cardiovascular benefits associated with SGLT2 inhibitors and may offer unique opportunities for early intervention or prevention of microvascular dysfunction associated with comorbidities that contribute to heart failure with preserved ejection fraction.

Tanacetum parthenium and parthenolide increase defensive agents and promote antisecretory activity supporting their gastroprotective actions in rodents

Tanacetum parthenium is popularly used to treat gastric disorders. However, the gastroprotective activity has not yet been tested. This study evaluated the anti-ulcer gastric effects of hydroalcoholic extract from T. parthenium (HETp) and parthenolide (PTL), its bioactive compound. Our findings revealed a significant reduction in HETp (10, 30, or 100 mg/Kg) and PTL (5 and 50 mg/Kg) in ethanol and piroxicam-induced ulcers, and this evidence was corroborated histologically. In addition, we explored the acid antisecretory activity by the pylorus ligation. Moreover, in mice pre-treated with the inhibitors indomethacin, L-NAME, and yohimbine, the antiulcerogenic effects of HETp and PTL were abolished, suggesting the involvement of nitric oxide, adrenergic receptors and prostaglandins in gastroprotective effects. Additionally, HETp and PTL prevented the depletion of SOD and CAT and decreased the activity of MPO and the levels of GSH and LPO. The results suggest that gastroprotection of HETp and PTL involves different pharmacological pathways.

Sirt5 as a mediator of endothelial and vascular dysfunction: potential therapeutic target in aging-related vascular diseases

Abstract Background Aging is one of the most dominant cardiovascular (CV) risk factors. Sirt5 belongs to the lifespan-regulating sirtuin superfamily as a protein deacylase. However, its regulatory effects on vascular function and aging are yet unclear. Purpose We therefore aimed to investigate the roles of Sirt5 on endothelial and vascular function during aging, decipher the underlying mechanisms, and to explore its potential as a therapeutic target through in vivo and in vitro studies. Methods Aortic expression of Sirt5 were studied in C5BL/6 wild type (WT) mice during aging, followed by vascular functional and structural assessment in WT mice and Sirt5-overexpressing mice (Sirt5/Tg) using the established myograph system and histological staining respectively. Furthermore, the regulatory role of Sirt5 on endothelial function was investigated in senescent primary human aortic endothelial cells (HAECs, passages 13-15) by studying its interference with endothelial nitric oxide synthase (eNOS) expression and activity in knock-down experiments using siRNA. The therapeutic potential of Sirt5 inhibition was explored at the tissue level in myograph experiments. Results Aged WT mice had increased level of Sirt5 in aorta compared to the young ones. Overexpression of Sirt5 in vivo led to significantly reduced aortic endothelial-dependent relaxation and displayed thicker collagen deposition in the adventitia compared to aged WT controls. The reduction of vascular relaxing response in Sirt5/Tg mice was prevented by an addition of eNOS inhibitor (L-name), indicating that Sirt5 regulated vascular function through the eNOS pathway. In senescent HAECs, knock-down of Sirt5 markedly induced total eNOS expression and increased its activity (phospho-eNOS Ser1177), further confirming the regulatory role of Sirt5 on eNOS pathway. In line with the above, aorta from aged WT mice displayed improved endothelial function after pharmacological inhibition of Sirt5. Conclusion Vascular Sirt5 expression increases with age and contributes to aging-related endothelial dysfunction by downregulating the eNOS pathway. Thus, Sirt5 inhibition may represent a novel therapeutic approach to maintain vascular function during aging. Ongoing studies will investigate Sirt5 silencing in-vivo using RNA interference for its therapeutic potential to prevent aging-related vascular dysfunction.

Endothelin-1- and acetylcholine-mediated effects in human and rat vessels: impact of perivascular adipose tissue, diabetes, angiotensin II, and chemerin

Objective To study the role of perivascular adipose tissue (PVAT) in the reactivity of rat and human vessels. Methods Iliac and mesenteric arteries were obtained from normotensive Sprague-Dawley rats, hypertensive transgenic (mRen2)27 rats overexpressing mouse renin, and (mRen2)27 rats made diabetic with streptozotocin. Human coronary arteries were obtained from donors. Concentration-response curves were constructed to endothelin-1 and acetylcholine with and without PVAT. The contribution of NO and endothelium-dependent hyperpolarization (EDH) were determined making use of the NO synthase inhibitor L-NAME and the EDH inhibitors apamin + TRAM-34. The endothelin type A and type B (ETA, ETB) receptor blockers BQ123 and BQ788, the chemerin inhibitors α-NETA and pravastatin, and the angiotensin receptor blocker losartan were also used. Results In rat iliac arteries, PVAT diminished endothelin-induced constriction, while the opposite was true in human coronaries. Coronary effects were unaltered by α-NETA, pravastatin, or losartan. ETB receptor-mediated relaxation in iliac arteries occurred only with PVAT, and BQ123 blocked endothelin-1-induced constriction. Diabetes upregulated the anticontractile effects of PVAT. In rat mesenteric arteries, acetylcholine-induced relaxation with PVAT relied on NO, and on NO + EDH without PVAT. Diabetes upregulated the EDH component exclusively with PVAT. Conclusion PVAT modulates ET-1-induced constriction in a vessel type-dependent manner. Its enhancing effects in coronaries involved neither chemerin nor angiotensin II. Its anticontractile effects in rat iliac arteries involved ETB receptor-mediated relaxation. Diabetes upregulated PVAT’s anticontractile effects. In mesenteric arteries, PVAT counterbalanced the EDH component of the relaxant effect of acetylcholine. Diabetes reversed this effect by upregulating the EDH component.

Differential regulation of physicochemical properties and myofibrillar protein degradation of yak meat by interactions between reactive oxygen species and reactive nitrogen species during postmortem aging.

This study aimed to explore how interactions between reactive oxygen species (ROS) and reactive nitrogen species (RNS) affect oxidative properties, nitrosative properties, and myofibrillar protein degradation during postmortem aging of yak meat. Yak longissimus dorsi was incubated with saline, ROS activator (H2O2)/inhibitor N-Acetyl-L-cysteine (NAC) and RNS activator S-Nitrosoglutathione (GSNO)/inhibitor L-NAME hydrochloride (L-NAME) combined treatments at 4 °C for 12, 24, 72, 120, and 168 h. The results indicated that regardless of whether RNS was activated or inhibited, activated ROS played a dominant role in myofibrillar protein degradation by oxidative modification to increase carbonyl content, disulfide bonds, surface hydrophobicity, and dimerized tyrosine while decreasing sulfhydryl content, thereby degrading nebulin, titin, troponin-t and desmin. Notably, the Warner-Bratzler shear force (WBSF) of the H2O2 + L-NAME group was the smallest, whereas that of the NAC + GSNO group was smaller than that of the NAC + L-NAME group. These findings provide new insights into meat tenderization patterns through the interaction between ROS and RNS. © 2024 Society of Chemical Industry.

Abstract P178: Sex Differences in the Role of the Immune ET-1/ET A Axis in Controlling Blood Pressure during a Hypertension Challenge

Immune cells like dendritic cells (DCs) and T cells are essential for the development of hypertension. Upregulation of the vasoactive peptide endothelin-1 (ET-1) is key in the progression of hypertension. ET-1 acting via its ET A receptor raises blood pressure and promotes end-organ damage and inflammation. DCs and T cells express ET-1 receptors and respond to changing levels of ET-1. However, it is unknown whether activation of the ET-1/ET A receptor axis on DCs or T cells is critical in driving hypertension, or if a differential activation of this axis in immune cells leads to the sex differences that exist in the pathophysiology of this disease. We hypothesized that activation of the ET-1/ ET A axis on immune cells is crucial in the development of hypertension, with a more prominent role in males than females. Male and female mice lacking ET A receptor on DCs (DC ET A KO) or on T cells (T cell ET A KO) and floxed ET A control mice were given the nitric oxide synthase inhibitor L-NAME (0.5 mg/mL) in drinking water for 3 wks to induce hypertension. Systolic blood pressure (SBP) was measured 3 times/wk via tail cuff plethysmography. Glomerular filtration rate (GFR) was evaluated at baseline and after L-NAME treatment via the transcutaneous FITC-sinistrin clearance method. Absence of ET A receptor on DCs resulted in a ~10 mmHg decrease in SBP at baseline in both sexes, and this difference increased throughout the protocol in males (Floxed ET A vs. DC ET A KO: L-NAME: 150.8±6.7 vs. 130.8±2.6 mmHg, n=4/group, p=0.002), but not in females (137.4±5.7 vs. 137.2±2.4 mmHg, n=3/group). Lack of the ET A receptor on T cells led to a similar decrease of ~10 mmHg in SBP at baseline. However, the difference in SBP widened when males were challenged with L-NAME (Floxed ET A vs. T cell ET A KO: 135.2±2.0 vs. 118.0±4.1, n=11/group, p=0.003), whereas the group difference in SBP disappeared in females (131.5±4.0 vs. 132.4±4.7, n=8-9/group). Following L-NAME, GFR trended to decline in control male mice, while it increased in T cell ET A KO males. Our data suggest that activation of ET A receptors on DCs and T cells modulates SBP in response to a hypertensive insult in males, but not in females. Additionally, absence of ET-1/ ET A axis activation on T cells suggests improved renal function during hypertension. Our results suggest that the use of ET A antagonism in the clinic may be more protective during hypertension in males than females.

Role of L-type Ca2+-channels in the vasorelaxing response to finerenone in arteries of human visceral adipose tissue.

Inadequate blood supply to the expanding adipose tissue (AT) is involved in the unhealthy AT remodeling and cardiometabolic consequences of obesity. Because of the pathophysiological role of upregulated mineralocorticoid receptor (MR) signaling in the complications of obesity, this study tested the vasoactive properties of finerenone, a nonsteroidal MR antagonist, in arteries of human AT. Arteries isolated from the visceral AT of obese subjects were studied in a wire myograph. Finerenone resulted in a concentration-dependent relaxation of arteries precontracted with either the thromboxane-A2 analog U46619, ET-1, or high-K+ solution; the steroidal MR antagonist potassium canrenoate, by contrast, did not relax arteries contracted with either U46619 or high-K+ solution. Finerenone-induced relaxation after precontraction with U46619 was greater in the arteries of obese versus nonobese subjects. Mechanistically, the vasorelaxing response to finerenone was not influenced by preincubation with the nitric oxide synthase inhibitor L-NAME or by endothelium removal. Interestingly, finerenone, like the dihydropyridine Ca2+-channel blocker nifedipine, relaxed arteries contracted with the L-type Ca2+-channel agonist Bay K8644. In conclusion, finerenone relaxes arteries of human visceral AT, likely through antagonism of L-type Ca2+ channels. This finding identifies a novel mechanism by which finerenone may improve AT perfusion, hence protecting against the cardiometabolic complications of obesity.

New morpholine-containing pyrimidinones act on α-adrenoceptors

Drugs that act on α-adrenoceptors may contain morpholine and pyrimidinone heterocycles. The aim of this study was to synthesize a series of pyrimidinones (S6a-e and S8) and characterize their α-adrenoceptor activity. Cytotoxicity assays (MTT and LDH) were performed in A7r5 and HUVECs. Concentration-effect curves to phenylephrine (Phe) were performed in rat aortic rings in the presence of compounds S6a-e and S8 or vehicle. Nitric oxide (NO) production and NO stable metabolic products, nitrite and nitrate, expressed as total nitrogen oxides (NOx) were assessed in HUVECs by confocal microscopy with the DAF-2DA probe and by the Griess reaction, respectively. Molecular docking simulations were performed using the 6a compound and α2A-adrenoceptor. In the evaluated conditions, the percentage of viable cells and the release of LDH were similar between control cells and cells exposed to the tested pyrimidinones. S6d, S6e, S8, and the positive control prazosin (but not S6a, S6b, and S6c) decreased Phe-induced contractions in endothelium-denuded aortic rings. S6a, S6b, and S6c decreased Phe-induced contractions in endothelium-intact aortic rings. The effect of S6a was abolished by L-NAME. NO production and NOx levels were inhibited in the presence of the α2 receptor antagonist yohimbine and the NOS inhibitor L-NAME. The 6a docking simulation estimated that the mean binding free energy of the compound was lower than the estimated value for yohimbine. These data suggest that S6d, S6e, and S8 may be α1-adrenoceptor antagonists while S6a acts as an agonist of α2-adrenoceptors.

P-321 Uterine fibroids secrete vasodilatory factors that induce their response via the opioid pathway

Abstract Study question Do uterine fibroids produce vasoactive factors that could affect vasculature and induce fibroid associated symptoms? Summary answer Ex vivo secretions from uterine fibroids induce a vasodilatory response in rat mesenteric arteries that is mediated via the opioid receptor pathway. What is known already Uterine fibroids (leiomyoma) are a common but clinically neglected gynaecological condition, where benign smooth muscle tumours arise from the myometrium and cause heavy menstrual bleeding (HMB), pain and infertility. Fibroids affect up to one in two women of reproductive age and are the main reason for failure of both medical and surgical treatments in women with HMB. Fibroids are proposed to affect the systemic vasculature system, with angiogenic growth factors and abnormal vasculature implicated in fibroid pathophysiology. However, the precise mechanism of fibroid induced HMB has yet to be fully elucidated. Study design, size, duration Fibroid and myometrium biopsies were prospectively collected from 13 patients undergoing MyoSure, myomectomy or hysterectomy for the treatment of HMB associated with fibroids at Liverpool Women’s Hospital, a tertiary referral centre. The collection and use of human tissue was ethically approved, and all participants gave informed written consent. Tissue biopsies were incubated in a basal medium for 24 hours. Conditioned medium was collected and used for wire myography assays to examine vascular reactivity. Participants/materials, setting, methods Second order mesenteric arteries from female Wistar rats were mounted on a wire myograph system and toned with uridine triphosphate (UTP) before being exposed to control (non-conditioned), normal myometrium (n = 4) or fibroid (n = 9) conditioned medium. Vascular activity was measured relative to UTP induced vasoconstriction. To determine the mediatory factors of observed vasoreactivity, inhibitors of well characterised pathways were systematically tested in the presence of conditioned medium. Main results and the role of chance Fibroid conditioned medium caused a marked dilation of UTP induced vasoconstriction in rat mesenteric arteries, which was significantly greater than that induced by non-conditioned, or control myometrium conditioned medium. Blockage of ATP-sensitive potassium (KATP) channels with PNU37883A completely inhibited fibroid induced vasodilation. However, blockage of other potassium channels did not inhibit dilation, suggesting that a KATP-mediated PKA-dependent pathway is being stimulated. To test the potential involvement of endothelial-derived nitric oxide synthase in vasodilation, vessels were treated with the inhibitor L-NAME, which partially blocked fibroid induced vasodilation. The specificity of nitric oxide synthase inhibition was confirmed by treating vessels with acetylcholine or carbachol, which induced vasodilation via activation of nitric oxide synthase. In the presence of L-NAME, the vasodilatory effects of acetylcholine and carbachol were blocked, thus confirming inhibitor specificity. An adenosine receptor inhibitor, theophylline, had no effect on fibroid induced vasodilation, thus ruling out the adenosine pathway. Blockage of bradykinin and adrenergic receptors also failed to prevent fibroid induced dilation in mesenteric arteries. However, blocking opioid receptors with naloxone completely inhibited vasodilation in the presence of fibroid conditioned medium. These findings suggest that fibroid induced vasodilation is mediated via endothelial nitric oxide synthase and an unknown opioid compound. Limitations, reasons for caution This study was performed using excised tissue in an ex vivo environment, which may not fully simulate the fibroid secretome in vivo. Although rat mesenteric arteries are a well-established model, human uterine arteries may be more appropriate to study fibroid induced vasoreactivity. Wider implications of the findings Fibroids are responsible for up to half of all hysterectomies and healthcare costs run into the billions globally. This study demonstrates that fibroid secreted factors can impact vasoreactivity, which has important connotations for disease pathophysiology regarding HMB. Targeting vasoactive compounds produced by fibroids may be a future novel treatment option. Trial registration number NA

Activation of Piezo1 or TRPV2 channels inhibits human ureteral contractions via NO release from the mucosa.

We aimed to investigate the expression and motor modulatory roles of several mechano-sensitive channels (MSCs) in human ureter. Human proximal ureters were obtained from eighty patients subjected to nephrectomy. Expression of MSCs at mRNA, protein and functional levels were examined. Contractions of longitudinal ureter strips were recorded in organ bath. A fluorescent probe Diaminofluoresceins was used to measure nitric oxide (NO). RT-PCR analyses revealed predominant expression of Piezo1 and TRPV2 mRNA in intact ureter and mucosa. Immunofluorescence assays indicate proteins of MSCs (Piezo1/Piezo2, TRPV2 and TRPV4) were mainly distributed in the urothelium. Ca2+ imaging confirmed functional expression of TRPV2, TRPV4 and Piezo1 in cultured urothelial cells. Specific agonists of Piezo1 (Yoda1, 3-300μM) and TRPV2 (cannabidiol, 3-300μM) attenuated the frequency of ureteral contractions in a dose-dependent manner while the TRPV4 agonist GSK1016790A (100nM-1μM) exerted no effect. The inhibitory effects of Piezo1 and TRPV2 agonists were significantly blocked by the selective antagonists (Dooku 1 for Piezo1, Tranilast for TRPV2), removal of the mucosa, and pretreatment with NO synthase inhibitor L-NAME (10μM). Yoda1 (30μM) and cannabidiol (50μM) increased production of NO in cultured urothelial cells. Our results suggest that activation of Piezo1 or TRPV2 evokes NO production and release from mucosa that may mediate mechanical stimulus-induced reduction of ureter contractions. Our findings support the idea that targeting Piezo1 and TRPV2 channels may be a promising pharmacological strategy for ureter stone passage or colic pain relief.

(007) TESTOSTERONE POSITIVELY REGULATES ADENOSINE-INDUCED RELAXATION IN THE VAGINA: AN EXPERIMENTAL STUDY IN RATS

Abstract Introduction It has been previously demonstrated that testosterone (T) has a pivotal role in controlling vaginal relaxation through the related molecular machinery, i.e., improving the nitric oxide (NO)/protein kinase G (PKG)/cyclic guanosine monophosphate (cGMP) vaginal signaling and maintaining the integrity of the muscular wall. Adenosine (ADO) is a purinergic neuromodulator that, in the male, shows a positive effect on penile erection by controlling smooth muscle relaxation and local blood flow increase. However, the role, if any, of T on the ADO-induced relaxation signaling is yet to be investigated. Objective The aim of this study was to evaluate the effect of sex steroids on ADO-mediated vaginal relaxation using a validated animal model of ovariectomized (OVX) and sex steroid supplemented Sprague-Dawley rats. Methods Subgroups of OVX rats were treated with 17β-estradiol (E2; 10 mg/kg/die), testosterone (T; 30 mg/kg/week), or testosterone and letrozole (T+L, 2.5 mg/kg/die) for 6 weeks. The experimental groups were compared with a group of intact rats. In vitro contractility studies were performed on noradrenaline (NA) pre-contracted distal vaginal strips, isolated from each experimental group, stimulated with increasing doses of ADO (1 μM - 1 mM), with or without NO-synthase inhibitor L-NAME (100 μM) pretreatment, or treated with ADORA2A selective agonist CGS21680 (10 Pm-100 μM). Messenger ribonucleic acid (mRNA) isolated from vaginal tissue was analyzed by semi-quantitative Reverse Transcriptase-Polymerase Chain Reaction. Results The results showed that OVX resulted in a decreased responsiveness to ADO, completely restored by T supplementation, with or without letrozole. In contrast, E2 did not affect the hyporesponsiveness to ADO in OVX rats. Likewise, ADO induced dose-dependent relaxation was significantly reduced by in vitro L-NAME (100 μM) only in the OVX animals treated with T or T+L. Accordingly, vagina expressed all ADO receptors (ADORA1, ADORA2A, ADORA2B, ADORA3) mRNAs, with pro-relaxant ADORA2A (ADO receptor coupled to adenylyl cyclase activation) being significantly modulated by hormonal treatments: OVX significantly reduced ADORA2A mRNA expression, an effect counteracted by either T or T+L administration, but not by E2. These data are supported by the observed T-induced upregulation of cyclic adenosine monophosphate (cAMP)-dependent genes, where T, but not E2, treatment upregulated the mRNA expression of the ubiquitous isoform of ADCY (ADCY4), protein kinase A (PKA) catalytic subunit b (PKAcb), PKA regulatory subunit 2b (PKAr2b) and cAMP responsive element modulator (CREM). Finally, as further demonstration of the functional activity of ADO signaling in distal vagina, stimulation by increasing doses of CGS21680, a selective agonist of ADORA2A receptor, enhanced the relaxant effect induced by T and T+L treatments but not by E2. Conclusions Our data demonstrated for the first time a novel ADO-mediated relaxant mechanism in vagina and suggested that T positively modulates the responsiveness to ADO stimulation, thus confirming this process as a major player in the relaxant mechanism regulation of muscle contractility in vagina. Disclosure No.

(046) THE ROLE OF ADENOSINE IN THE RELAXANT MECHANISM OF RAT DISTAL VAGINA

Abstract Introduction Adenosine (ADO) is a purinergic neuromodulator that in the male exerts a positive effect on penile erection by controlling smooth muscle (SM) relaxation. However, its role in the relaxant/contractile pathways in the vagina is yet to be investigated. Objective The aim of this study was to characterize the effect of ADO stimulation on SM activity in distal vagina, evaluating its possible effect on the regulation of nitric oxide (NO) pathway using a validated animal model of female Sprague-Dawley rats. Methods The immunolocalization of ADORA2A and ADORA2B in distal vagina tissues was detected by immunohistological staining. In vitro contractility studies on noradrenaline (NA)-precontracted vaginal strips from intact Sprague-Dawley rats were performed; rat smooth muscle cells (rvSMCs) from distal vagina of intact animals were stimulated with ADO for cyclic adenosine monophosphate (cAMP) quantification. mRNA (messenger ribonucleic acid) isolated from vaginal tissue was analyzed by semi-quantitative Reverse Transcriptase-Polymerase Chain Reaction. Results All ADORAs mRNA were expressed in the vaginal tissue, with a significant prevalence of ADORA2B; furthermore, ADORA2A and ADORA2B immunolocalization highlighted that both receptors are highly expressed in the epithelium, in blood capillaries of the vascular bed and in the smooth muscle bundles of vagina wall. In vitro ADO stimulation induced a dose-dependent relaxation (IC50=31.9±2.38 μM), which was significantly reduced by the administration of NO-synthase inhibitor L-NAME; similar effects were observed after the mechanical ablation of endothelium and in vitro administration of the soluble guanylate cyclase inhibitor ODQ, highlighting the pivotal contribution of NO signaling to the ADO relaxant activity. In vitro stimulation with increasing doses of the selective ADO receptor ADORA2A agonist CGS2-1680 induced a dose-dependent relaxation (IC50=132.6±3.47 nM); a similar but less potent relaxant effect was observed with ADORA2B selective agonist BAY60-6583. The relaxing effect induced by ADO on NA-precontracted vaginal strips was completely counteracted by the stimulation with the re-spectively selective ADORA2A and ADORA2B antagonists SHC-442416 and PSB603; a similar effect was induced by ADORA1 selective antagonist SLV320, but not by ADORA3 selective antagonists VUF5574. The mRNA relative expression of enzymes regulating the ADO turnover [adenosine deami-nase (ADA), 5′-nucleotidase (5NTD), AMP deaminase type 1 and type 2 (AMPD1, AMPD2) and adenosine kinase (ADK)] showed a higher expression of genes regulating the de novo ADO/cAMP syn-thesis (ADK, 5NTD), compared to those acting on ADO degradation (ADA, AMPD1, AMPD2). Finally, stimulation of rvSMCs with increasing doses of ADO induced a significant production of intracellular cAMP after 3 minutes (1 and 10 μM). Conclusions These data show that ADO acts as an important relaxant modulator of the vagina through the activation of its receptors, specifically ADORA2A, and the consequent cAMP-dependent adenylate cyclase re-sponse. However, in vitro studies show that ADO also acts trough the NO-dependent pathway. These results therefore demonstrate for the first time a new relaxant pathway in distal vagina, ADO signaling, that results deeply intertwined with the well-known NO pathway. This finding unravels new biological mechanisms underlying vagina physiology that need to be further studied in future research. Disclosure No.

Stimulation of the calcium-sensing receptor induces relaxations through CGRP and NK1 receptor-mediated pathways in male rat mesenteric arteries.

Stimulation of the calcium-sensing receptor (CaSR) regulates vascular contractility, but cellular mechanisms involved remain unclear. This study investigated the role of perivascular sensory nerves in CaSR-induced relaxations of male rat mesenteric arteries. In fluorescence studies, colocalisation between synaptophysin, a synaptic vesicle marker, and the CaSR was present in the adventitial layer of arterial segments. Using wire myography, increasing external Ca2+ concentration ([Ca2+]o) from 1 to 10 mM induced vasorelaxations, previously shown to involve the CaSR, which were inhibited by pretreatment with capsaicin. [Ca2+]o-induced vasorelaxations were partially reduced by the calcitonin gene-related peptide (CGRP) receptor blockers, CGRP 8-37 and BIBN 4096, and the neurokinin 1 (NK1) receptor blocker L733,060. The inhibitory effect of CGRP 8-37 required a functional endothelium whereas the inhibitory action of L733,060 did not. Complete inhibition of [Ca2+]o-induced vasorelaxations occurred when CGRP 8-37 and L733,060 were applied together. [Ca2+]o-induced vasorelaxations in the presence of capsaicin were abolished by the ATP-dependent K+ channel (KATP) blocker PNU 37883, but unaffected by the endothelium nitric oxide synthase (eNOS) inhibitor L-NAME. We suggest that the CaSR on perivascular sensory nerves mediate relaxations in rat mesenteric arteries via endothelium-dependent and -independent mechanisms involving CGRP and NK1 receptor-activated NO production and KATP channels, respectively.

#1918 L-NAME accelerates the progression of diabetic kidney disease in mice

Abstract Background and Aims Diabetic kidney disease (DKD) represents one of the major causes of end stage kidney disease worldwide. Despite DN having such a high incidence, mouse models of DN rarely mimic all aspects of the human disease, besides showing very slow progression and sometimes maintenance issues. For this reason, in this project we aimed to develop an accelerated, reproducible and accessible mouse model of DN. Method For our experiments we chose 9-week-old female and male of the leptin receptor knock-out (BKS.Cg-Dock7m +/+ Leprdb/J) mice as type 2 diabetes model. As these mice are still not prone to develop severe kidney injury despite their diabetic condition, we administered the eNOS inhibitor L-NAME for 6 weeks, in order to suppress nitric oxide production and therefore to aggravate kidney damage. Mice were divided in three groups: one vehicle control group, one receiving 40/mg/kg/day of L-NAME and one receiving 80/mg/kg/day of L-NAME dissolved in drinking water. Blood pressure and glomerular filtration rate (GFR) were measured at different time points, together with glycemia and albuminuria. Weight and water intake were also monitored throughout the study. At the end of the experiment, animals were killed and blood and organs collected for further analysis. Results Mice treated with 80 mg/kg/day of L-NAME showed an average increase of 10 mmHg in systolic blood pressure, with a peak of 30 mmHg increase at week 4 of treatment. On the other hand, 40 mg/kg/day of L-NAME did show any effect on blood pressure. Despite the different effect on blood pressure, three weeks of L-NAME administration caused a significant reduction in GFR at both doses, with 5% reduction for the 40 mg-group and 23% for the 80 mg-group. For the 80 mg-group we observed an even further GFR decline after 5 weeks of treatment, with a 28% reduction. No change in GFR was observed in the controls. This decrease in GFR was accompanied by a 10-fold increase in urinary albumin for both experimental groups already after 3 weeks of treatment, which after 4 weeks slightly declined but remained significantly higher than the controls (p < 0.0001). At a histological level, PAS staining of kidney sections revealed that in particular mice treated with 80 mg of L-NAME have enlarged Bowman capsule and glomerular tuft area, besides a reduced glomerular capillary density, coinciding with mesangial expansion. We also stained for overall collagen deposition with picrosirius red staining in order to detect possible fibrosis formation. We then observed that 80 mg of L-NAME accelerates interstitial fibrosis in the cortical area of the kidney, which also overlapped with an increased deposition of collagen I. This increase in collage I was also detected in kidney cortex of mice treated with 40 mg of L-NAME, although their overall collagen deposition and therefore fibrotic tissue did not differ from the control group. The medullar region of kidneys was also analyzed, but none of the two experimental groups revealed significant differences in terms overall collagen or collagen I deposition. Unlike mesangial expansion and fibrosis, L-NAME did not influence microphages infiltration, as revealed by CD68 staining in kidney sections. This suggests that L-NAME treatment does not accelerate kidney damage by triggering or accelerating inflammatory processes. This was also confirmed by qPCR analysis of kidney tissue, where the expression pro-inflammatory markers, such as IL1β, TNFα and MCP-1, did not change in the experimental groups. Conclusion Our results show that L-NAME considerably accelerates the progression of DKD in diabetic mice, with GFR declining and progressive albuminuria already after 3 weeks. Such disfunction is confirmed at 6 weeks by histological analysis, where mesangial expansion and fibrosis were detected. This model can be used to assess the therapeutic potential of novel interventions aimed to slow down DKD.

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.

Endothelial Hemoglobin Alpha Mediates Iron Regulation of Endothelial Nitric Oxide

Iron deficiency anemia (IDA) is a common comorbidity in cardiovascular disease. Previous clinical studies have found iron deficiency anemia (IDA) increases endothelial NO signaling and attributed this change to the reduction in circulating hemoglobin. However, it has been shown in ex vivo preparations that endothelial NO signaling is unaffected by the presence of erythrocytes suggesting changes in circulating hemoglobin do not alter NO signaling. Our lab has previously shown the hemoglobin-α (Hbα) is expressed in the endothelium of small arteries and scavenges NO. We hypothesized endothelial Hbα, rather than blood hemoglobin is responsible for the increased NO signaling in IDA. We first tested this hypothesis in a mouse model of IDA in which we were able to replete vascular iron stores. Male and female C57BL/6 mice were fed an iron deficient (2-6 ppm Fe) or nutrient matched control diet (48 ppm Fe) beginning at weaning. IDA mice underwent phlebotomies (10% blood volume) at 9 and 11 weeks old. To restore vascular iron stores, a subset of IDA mice received a single i.p. injection of iron dextran (FeDex; 20 mg/kg) at 12 weeks. All measurements were taken one week later. As expected, IDA mice had low blood hemoglobin, and FeDex did not rescue the anemia but did rescue vascular iron stores as measured by ferritin light chain protein. To investigate NO signaling, we used laser speckle contrast imaging to measure changes in in vivo blood flow in response to the NO synthase inhibitor L-NAME. The magnitude of the response to L-NAME is an indicator of NO signaling. IDA mice exhibited an exaggerated response to L-NAME indicating increased NO signaling. FeDex treatment restored the L-NAME response back to control levels suggesting reduced vascular iron stores, rather than the anemia itself increased NO signaling. In order to investigate the role of endothelial Hbα, we repeated these studies in endothelial specific Hbα knockout mice (Hba1fl/fl-Cdh5-CreERT2+). Lack of endothelial Hbα did not prevent the increased response to L-NAME in IDA mice but did prevent the rescue by FeDex. This suggests a model in which endothelial Hba participates in regulating NO in response to iron but not the increased NO in IDA. Because Hbα does not explain the initial increase in NO, we have begun investigating other anemia-associated NO stimulators. Erythropoetin (EPO) is known to stimulate NO production in endothelial cells and is elevated in our anemia model regardless of FeDex treatment. Future work will investigate the role of EPO in regulating NO in IDA. University of Virginia Basic and Translational Research Training Grant T32 007284 (LSD, MAL, SAL); NIH HL088554; LaunchPad; Lipedema Foundation; AHA Predoctoral Fellowship (MAL). 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.