Nitric Oxide Synthase Inhibitor L-NAME Research Articles

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.

Sensitized serotonin-mediated vasodilation in a mouse model of carcinoid disease

Background: Carcinoid tumors secrete vasoactive hormones such as serotonin that impact cardiovascular function. In consequence, patients with metastatic carcinoid disease are at risk for carcinoid syndrome, characterized by profound cutaneous flushing, hypotension, and syncope. Our recent work demonstrates that in patients with carcinoid metastases, higher serotonin levels are associated with lower vascular resistance. However, it is unknown if the vascular sensitivity to serotonin is affected by carcinoid disease. The receptor that mediates the potential vasodilatory effects of serotonin in carcinoid disease is also unknown. Therefore, we hypothesize that serotonin-mediated vasodilation is increased by carcinoid metastases. To test this, we assessed vascular reactivity to serotonin ex vivo in a mouse model of carcinoid disease. Methods: J:Nu nude mice (10 weeks) received intrasplenic injection of 2x107 BON1 neuroendocrine tumor cells (BON1, n=5) or PBS-vehicle (VEH, n=5), and were monitored for 10 weeks to allow the development of liver carcinoid metastases. Animals were then euthanized, and mesentery arteries were harvested. Isolated, pressurized mesentery arteries were pre-constricted with phenylephrine. The dose-response vasodilation to serotonin and acetylcholine was measured. These dose responses were repeated in the presence of 5-HT2B receptor antagonist LY272015 or nitric oxide synthase inhibitor L-NAME. Endothelium-independent vasodilation to sodium nitroprusside was also assessed. Data are mean ± SEM. Results: Mesentery arteries from BON1 mice had a greater vasodilation in response to serotonin compared with arteries from VEH mice (65±6% vs. 44±8%, p=0.04). Incubation with L-NAME partially inhibited the vasodilation to serotonin in the BON1 arteries (42±10%, p=0.04), but not in VEH arteries (30±11%, p=0.17). LY272015 did not affect the serotonin-induced vasodilation in the BON1 or VEH arteries (BON1: 52±10%, p=0.14, VEH: 49±17%, p=0.39). In contrast to serotonin, the vasodilation to acetylcholine did not differ between BON1 and VEH arteries (77±9% vs. 61±16%, p=0.19). There were also no differences in the vasodilation to sodium nitroprusside between groups (p=0.42). Arteries from BON1 mice had a trend for greater vasoconstriction in phenylephrine compared with VEH mice (51±7% vs. 37±9%, p=0.06). Conclusions: These findings demonstrate that serotonin-mediated vasodilation is sensitized in a mouse model of carcinoid disease. This is potentially accompanied by enhanced α-adrenergic-mediated vasoconstriction, which may be a compensatory mechanism in response to sensitized vasodilatory mechanisms. Western University of Health Sciences Intramural Grant (BHM). 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.

Age and APOE4 genotype alter cerebrovascular function and stiffness

Two of the greatest risk factors for late-onset Alzheimer’s disease (LOAD) are age and the APOE4 genotype. The gene encoding for apolipoprotein E ( APOE) has three isoforms in humans ( E2, E3, E4). E3 has the highest prevalence, while E4 exponentially increases the risk for AD. Additionally, it is well known that the E4 genotype is accompanied by an altered cerebral blood flow. However, the mechanisms underlying these blood flow changes in E4 individuals remain unclear and may be mediated by changes to cerebral artery structure and function. We aimed to determine if APOE genotype alters the impact of age on cerebral artery endothelial function and stiffness. We studied male and female, young E3 (n=20; ~6 months), aged E3 (n=9; ~24 months young E4 (n=22; ~6 months), and aged E4 (n=18; ~24 months) mice. We assessed endothelium-dependent and -independent vasodilation and vasoconstriction in isolated, pressurized posterior cerebral arteries (PCAs). Passive stiffness was measured in isolated PCAs after incubation in a calcium-free solution. Data are mean ± SD. Both old E3 and old E4 mice had impaired PCA endothelium-dependent vasodilation to acetylcholine compared with young mice ( E3: 29±10.6% vs 46±15.2%, p=0.02; E4: 30±10.8 vs 49±14.3%, p=0.0004). Sex comparisons found that the PCA maximal dilation to acetylcholine was 21% greater in young E3 females compared with young E3 males (52±12.5% vs 41±16%, p=0.04), and 13% greater in young E4 females compared with young E4 males (52±10% vs 45±16%, p=0.07), but there were no sex differences among the old groups. Additionally, the PCA responses to acetylcholine in the presence of nitric oxide synthase inhibitor L-NAME did not differ between groups (p>0.05). Endothelium-independent dilation, measured as the PCA response to sodium nitroprusside, also did not differ between groups (p>0.05). Aged E3 mice, compared with young E3, had a lower maximal constriction to potassium chloride (20±11% vs 59±16%, p<0.0001) and endothelin-1 (23±16% vs 53±14%, p=0.0005). In contrast, young E4 and old E4 mice had a similar maximal constriction to potassium chloride (35±13% vs 43±18%, p>0.05) and endothelin-1 (48±12% vs 47±20%, p>0.05). Lastly, we found that old mice had a greater PCA β stiffness index compared with young mice for both the E3 (15.5±4.5 vs 10.6±1.4 AU, p=0.0008) and E4 (13.4±2.9 vs 10.2±2.5 AU, p>0.0001) groups. In summary, we found that age impairs cerebral artery endothelial function in both APOE3 and APOE4 mice, and young female mice have better endothelial function than their male counterparts regardless of genotype. In addition, vasoconstrictor responsiveness appears to decline in old age in APOE3 mice, whereas vasoconstrictor responsiveness is maintained in old APOE4 mice. This maintained sensitivity to vasoconstrictors in old age may explain why APOE4 individuals have reduced cerebral blood flow and greater LOAD risk. Luvaas Family Fund, Alzheimer’s Association ALZDISCOVERY-1049110. 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.

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.

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.

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.

Metabolic dysfunction induced by HFD + L-NAME preferentially affects hippocampal mitochondria, impacting spatial memory in rats.

High-fat diet-induced metabolic changes are not restricted to the onset of cardiovascular diseases, but also include effects on brain functions related to learning and memory. This study aimed to evaluate mitochondrial markers and function, as well as cognitive function, in a rat model of metabolic dysfunction. Eight-week-old male Wistar rats were subjected to either a control diet or a two-hit protocol combining a high fat diet (HFD) with the nitric oxide synthase inhibitor L-NAME in the drinking water. HFD plus L-NAME induced obesity, hypertension, and increased serum cholesterol. These rats exhibited bioenergetic dysfunction in the hippocampus, characterized by decreased oxygen (O2) consumption related to ATP production, with no changes in H2O2 production. Furthermore, OPA1 protein expression was upregulated in the hippocampus of HFD + L-NAME rats, with no alterations in other morphology-related proteins. Consistently, HFD + L-NAME rats showed disruption of performance in the Morris Water Maze Reference Memory test. The neocortex did not exhibit either bioenergetic changes or alterations in H2O2 production. Calcium uptake rate and retention capacity in the neocortex of HFD + L-NAME rats were not altered. Our results indicate that hippocampal mitochondrial bioenergetic function is disturbed in rats exposed to a HFD plus L-NAME, thus disrupting spatial learning, whereas neocortical function remains unaffected.

Endothelial Hemoglobin Alpha Mediates Iron Regulation of Endothelial Nitric Oxide

Iron deficiency is associated with heart failure, chronic kidney disease, and chronic inflammation. All of these pathologies are associated with endothelial dysfunction and impaired nitric oxide (NO) signaling. Previous clinical studies have found iron deficiency anemia (IDA) increases endothelial NO signaling and attributed this change to the reduction in circulating hemoglobin. However, while free hemoglobin is a potent scavenger of NO, 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. For these reasons we focused on endothelial iron regulation. Using single cell RNA sequencing data of isolated mesenteric and adipose endothelial cells generated by our lab, we have observed arterial endothelial cells have higher transferrin receptor expression compared to capillary, veinous, and lymphatic endothelial cells. This suggests that the arterial endothelium, which controls blood flow and is especially susceptible to endothelial dysfunction, may be particularly sensitive to changes in iron status. Additionally, our lab has previously shown the alpha chain of hemoglobin (Hbα) is expressed in the endothelium of small arteries and scavenges NO. We therefore 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. 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 aid in the progression of anemia. 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. One week later complete blood counts were assessed, tissues were collected, and blood flow studies were performed. As expected, IDA mice had low blood hemoglobin (Con: 15.54 ± 0.43 vs. IDA: 6.04 ± 0.35 g/dL; p < 0.0001). FeDex did not rescue the anemia (8.55 ± 0.74 g/dL; p < 0.0001 vs. Con), 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. Rescuing vascular iron stores with FeDex restored the L-NAME response back to control levels suggesting 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 (Hba1 fl/fl-Cdh5-CreER T2+) developed by us. Lack of endothelial Hbα did not prevent the increased response to L-NAME in IDA mice, but did prevent the rescue by FeDex. These data suggest a model in which endothelial Hbα participates in regulating NO in response to iron, but not the increased NO in IDA.

Age-induced nitrative stress decreases retrograde transport of proNGF via TrkA and increases proNGF retrograde transport and neurodegeneration via p75NTR.

Axonal transport of pro nerve growth factor (proNGF) is impaired in aged basal forebrain cholinergic neurons (BFCNs), which is associated with their degeneration. ProNGF is neurotrophic in the presence of its receptor tropomyosin-related kinase A (TrkA) but induces apoptosis via the pan-neurotrophin receptor (p75NTR) when TrkA is absent. It is well established that TrkA is lost while p75NTR is maintained in aged BFCNs, but whether aging differentially affects transport of proNGF via each receptor is unknown. Nitrative stress increases during aging, but whether age-induced nitrative stress differentially affects proNGF transport via TrkA versus p75NTR has not yet been studied. Answering these questions is essential for developing an accurate understanding of the mechanisms contributing to age-induced loss of proNGF transport and BFCN degeneration. In this study, fluorescence microscopy was used to analyze axonal transport of quantum dot labeled proNGF in rat BFCNs in vitro. Receptor specific effects were studied with proNGF mutants that selectively bind to either TrkA (proNGF-KKE) or p75NTR (proNGF-Δ9-13). Signaling factor activity was quantified via immunostaining. Young BFCNs transported proNGF-KKE but not proNGF-Δ9-13, and proNGF transport was not different in p75NTR knockout BFCNs compared to wildtype BFCNs. These results indicate that young BFCNs transport proNGF via TrkA. In vitro aging increased transport of proNGF-Δ9-13 but decreased transport of proNGF-KKE. Treatment with the nitric oxide synthase inhibitor L-NAME reduced retrograde transport of proNGF-Δ9-13 in aged BFCNs while increasing retrograde transport of proNGF-KKE but did not affect TrkA or p75NTR levels. ProNGF-Δ9-13 induced greater pro-apoptotic signaling and neurodegeneration and less pro-survival signaling relative to proNGF-KKE. Together, these results indicate that age-induced nitrative stress decreases proNGF transport via TrkA while increasing proNGF transport via p75NTR. These transport deficits are associated with decreased survival signaling, increased apoptotic signaling, and neurodegeneration. Our findings elucidate the receptor specificity of age-and nitrative stress-induced proNGF transport deficits. These results may help to rescue the neurotrophic signaling of proNGF in aging to reduce age-induced loss of BFCN function and cognitive decline.

PDE2 is cardioprotective in chronic heart failure

Abstract Background Patients with chronic heart failure (HF) often develop life-threatening arrhythmia due to structural and electrophysiological cardiac remodelling. The chronic activation of the sympathetic nervous system is initially able to compensate the reduced myocardial contraction. Finally, long-term activation of the β-adrenergic pathway leads to deleterious activation of cAMP-dependent kinases and dysregulation of intracellular Ca2+ handling promoting HF progression and development of arrhythmia. Interestingly, phosphodiesterase 2 (PDE2), an enzyme to hydrolyze cAMP, is upregulated in human HF. Thus, PDE2 might reduce enhanced cAMP levels and pro-arrhythmic Ca2+ releases in HF. Purpose We investigated the effects of genetic PDE2 deletion on cardiac function, cellular contraction and arrhythmia occurrence in murine HF model. Methods HF was induced via high fat (60 %) diet (HFD) and the treatment with NO-synthase inhibitor L-NAME (0.5 g/l) for 5 weeks in cardiomyocyte-specific PDE2-knockout mice (KO) and control mice (WT). We characterized cardiac function by echocardiography, arrhythmia development by ECG-telemetry, cellular contraction by video-based analysis of sarcomere-shortening and cellular Ca2+ releases using Ca2+ imaging techniques. Results After 5 weeks HFD and L-NAME, mice gained weight, displayed increased blood pressure and reduced systolic and diastolic cardiac function. Interestingly, PDE2 KO showed reduced contractility and ejection fraction compared to WT mice after 5 weeks HF. On cellular level, the contraction of isolated cardiomyocytes slightly increased in HF indicating cardiac hypertrophy. However, the β-adrenergic stimulation with isoprenaline (ISO) enhanced cellular contraction only in non-failing WT mice, but was blunted in cells from failing WT hearts reflecting a desensitization of the β-adrenergic pathway. In contrast, the ISO-induced increase in cellular contraction was preserved in failing hearts from KO mice. Interestingly, PDE2 inhibition with BAY 60-7550 increased the ISO-induced cellular contraction in HF from WT, which was not observed in cells from non-failing mice. In HF, the expression of Ca2+ cycling proteins like phospholamban (PLB) were affected. Failing PDE2 KO mice showed higher phosphorylation levels of PLB compared to WT mice with HF. In diseased hearts, pro-arrhythmic triggers like spontaneous Ca2+ waves and Ca2+ sparks were increased. Importantly, diseased PDE2 KO mice developed more arrhythmia in-vivo upon arrhythmia provocation than WT animals. Conclusion Cardiac PDE2 deletion was detrimental in HF showing reduced cardiac function and higher number of arrhythmia. PDE2 stimulation might serve as potential therapeutical target in HF limiting the consequences of chronic β-adrenergic hyperactivation.

Abstract 11591: Mechanism of Finerenone-Induced Vasorelaxation in Arteries From Human Visceral Adipose Tissue

Introduction: Inadequate perfusion has emerged as a main determinant of adipose tissue (AT) dysfunction in obesity, sparking interest about possible ways to improve it. Among several mechanisms, mineralcorticoid receptor (MR) signaling has been shown to play a key role in the cross-talk between AT and the vasculature. Hypothesis: This study, therefore, tested the vasoactive properties of finerenone, a nonsteroidal MR antagonist, in arteries harvested from human AT. Methods: Small arteries (<1 mm in diameter) were isolated from visceral AT and studied ex vivo in a wire myograph. After vessels had been contracted, changes in vascular tone were determined under different experimental conditions. Results: Finerenone elicited a concentration-dependent relaxation in arteries of obese individuals contracted with the thromboxane A2 analogue U46619 (n=9; P<0001 vs. baseline); the steroidal MR antagonist potassium canrenoate, by contrast, did not induce any vasorelaxation (n=3; P>0.05). The relaxing effect of finerenone on U46619-induced constriction was not different in arteries from lean (n=3) and obese subjects (P=0.155). Mechanistically, the vasorelaxing response to finerenone was not influenced by pre-incubation with the nitric oxide (NO) synthase inhibitor L-NAME (n=4; P=0.19 vs. saline), hence ruling out an involvement of NO. Interestingly, finerenone was also able to relax arteries contracted with endothelin-1 (n=7; P<0.001) or with high-K + solution (n=4; P<0001), as well as those contracted with the L-type Ca 2+ -channel agonist BayK8644 (n=5; P=0.007); similarly, the calcium-channel blocker nifedipine induced relaxation of arteries contracted with BayK8644 (n=3; P<0.001). Conclusions: Finerenone induces NO-independent vasorelaxation in arterioles from human visceral AT, likely through antagonism of L-type Ca2 + -channels. This finding identifies a novel action of finerenone to improve AT perfusion, thereby potentially expanding the emerging observations about the cardiovascular benefits of this agent.

Regulation of the Endogenous Opiate Signaling Pathway against Oxidative Stress and Inflammation: A Considerable Approach for Exploring Preclinical Treatment of Parkinson’s Disease

Introduction: Oxidative stress and inflammation are major factors contributing to the progressive death of dopaminergic neurons in Parkinson’s disease (PD). Recent studies have demonstrated that morphine’s biosynthetic pathway, coupled with nitric oxide (NO) release, is evolutionarily conserved throughout animals and humans. Moreover, dopamine is a key precursor for morphine biosynthesis. Method: The present study evaluated a series of preclinical experiments to evaluate the effects of low-level morphine treatment upon neuro-immune tissues exposed to rotenone and 6-OHDA as models of PD, followed by an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell proliferation assay and cell/tissue computer-assisted imaging analyses to assess cell/neuronal viability. Results: Morphine at normal physiological concentrations (i.e., 10−6 M and 10−7 M) provided neuroprotection, as it significantly inhibited rotenone and 6-OHDA dopaminergic insults; thereby, reducing and/or forestalling cell death in invertebrate ganglia and human nerve cells. To ensure that morphine caused this neuroprotective effect, naloxone, a potent opiate receptor antagonist, was employed and the results showed that it blocked morphine’s neuroprotective effects. Additionally, co-incubation of NO synthase inhibitor L-NAME also blocked morphine’s neuroprotective effects against rotenone and 6-OHDA insults. Conclusions: Taken together, the present preclinical study showed that while morphine can attenuate lipopolysaccharide-induced inflammation and cell death, both naloxone and L-NAME can abolish this effect. Preincubation of morphine precursors (i.e., L-3,4-dihydroxyphenylalanine, reticuline, and trihexyphenidyl [THP] at physiological concentrations) mimics the observed morphine effect. However, high concentrations of THP, a precursor of the morphine biosynthetic pathway, induced cell death, indicating the physiological importance of morphine biosynthesis in neural tissues. Thus, understanding the morphine biosynthetic pathway coupled with a NO signaling mechanism as a molecular target for neuroprotection against oxidative stress and inflammation in other preclinical models of PD is warranted.

Cilostazol induces vasorelaxation through the activation of the eNOS/NO/cGMP pathway, prostanoids, AMPK, PKC, potassium channels, and calcium channels

ObjectiveThis study aimed to investigate vasoactive effect mechanisms of cilostazol in rat thoracic aorta. Materials and methodsThe vessel rings prepared from the thoracic aortas of the male rats were placed in the chambers of the isolated tissue bath system. The resting tone was adjusted to 1 g. Following the equilibration phase, potassium chloride or phenylephrine was used to contract the vessel rings. When achieving a steady contraction, cilostazol was applied cumulatively (10−8–10−4 M). In the presence of potassium channel blockers or signaling pathway inhibitors, the same experimental procedure was performed. ResultsCilostazol exhibited a significant vasorelaxant effect in a concentration-dependent manner (pD2: 5.94 ± 0.94) (p < .001). The vasorelaxant effect level of cilostazol was significantly reduced by the endothelial nitric oxide synthase inhibitor L-NAME (10−4 M), soluble guanylate cyclase inhibitor methylene blue (10 µM), cyclooxygenase 1/2 inhibitor indomethacin (5 µM), adenosine monophosphate-activated protein kinase inhibitor compound C (10 µM), non-selective potassium channel blocker tetraethylammonium chloride (10 mM), large-conductance calcium-activated potassium channel blocker iberiotoxin (20 nM), voltage-gated potassium channel blocker 4-Aminopyridine (1 mM), and inward-rectifier potassium channel blocker BaCl2 (30 µM) (p < .001). Moreover, incubation of cilostazol (10−4 M) significantly reduced caffeine (10 mM), cyclopiazonic acid (10 µM), and phorbol 12-myristate 13-acetate-induced (100 µM) vascular contractions (p < .001). ConclusionsIn the rat thoracic aorta, the vasodilator action level of cilostazol is quite noticeable. The vasorelaxant effects of cilostazol are mediated by the eNOS/NO/cGMP pathway, prostanoids, AMPK pathway, PKC, potassium channels, and calcium channels.

Effect of the nitric oxide synthesis inhibitor L-NAME on the isolated rat heart after hypokinesia

The effects of a non-selective nitric oxide synthase inhibitor L-NAME on the functional parameters of the isolated rat heart after a 30-day period of hypokinesia were studied. Electron paramagnetic resonance spectroscopy was employed in the analysis of a role for L-NAME in the intensity of nitric oxide production in rat heart tissues. The intensity of nitric oxide synthesis was assessed by the intensity of the signal belonging to the (DETC)2-Fe2+-NO complex. It was found that L-NAME decreased nitric oxide production on average by 69%. The Langendorff isolated perfused heart was used to evaluate cardiac activity, and the following parameters were measured: pressure generated by the left ventricle, heart rate, and coronary flow. Addition of the nitric oxide synthesis inhibitor L-NAME induced an increase in inotropic function and normalization of heart rate.

DIABETES IMPAIRS PERIVASCULAR ADIPOSE TISSUE (PVAT)-MEDIATED VASCULAR RESPONSES IN RAT MESENTERIC AND ILIAC ARTERIES

Objective: To study the role of PVAT in vascular function of blood vessels from hypertensive and diabetic rats. Design and method: Mesenteric and iliac artery rings were isolated ± PVAT from Sprague Dawley (SD), and diabetic and non-diabetic TGR (mREN2)27 (REN2) rats. Vascular activity was measured in organ baths by constructing concentration-response curves to endothelin (ET-1) or acetylcholine (ACh). To assess the involvement of NO and endothelium-dependent hyperpolarization (EDH) pathways, ACh effects were studied with the NO synthase inhibitor L-NAME and/or the EDH inhibitors apamine+TRAM-34. ET-1 effects were additionally studied in the presence of the endothelin type A and type B receptor (ETAR, ETBR) blockers BQ123 and BQ788. Constrictor data are expressed as a % of the response to 100 mmol/L K+, and dilator effects as a % of the preconstriction to the thromboxane A2 agonist U46619. Results: With PVAT, ET-1 constricted SD and REN2 iliac arteries identically (Emax 126±28 and 119±48, pEC50 7.68±0.29 and 7.59±0.35). BQ788 potentiated constriction, while BQ123 prevented it. Without PVAT, BQ788 had potentiating effect, while BQ123 exerted blockade. Indicating that acute ETBR stimulation results in PVAT-dependent vasodilation. DM reduced the effect of ET-1 in PVAT+ vessels (Emax 78±56, pEC50 6.36±2.59). Yet, PVAT removal restored the ET-1 effect to normal, while BQ788 was still without effect, and BQ123 exerted its usual blockade. This indicates that in DM, PVAT continuously releases relaxant factors that counteract ET-1-mediated effects, even without ETBR stimulation. ACh relaxed PVAT+ SD and REN2 mesenteric arteries identically (Emax 85±15 and 81±16, pEC50 5.70±0.79 and 6.43±0.61), additionally L-NAME blocked was comparable, while EDH blockade had no effect. PVAT removal did not alter ACh responses, and L-NAME and EDH inhibition yielded an identical degree of blockade, with additive effects. This indicates that PVAT counteracts EDH-mediated responses. DM did not alter the response to ACh, but under DM conditions, EDH inhibition yielded blockade in the presence of PVAT, this effect was additive to that of L-NAME. Thus, DM diminishes the anti-EDH effects of PVAT. Conclusions: PVAT releases both relaxant and contractile factors that affect vascular reactivity, and DM impairs PVAT function.

Release of 6-nitrodopamine from human popliteal artery and vein

6-Nitrodopamine (6-ND) is a novel catecholamine that is released from human umbilical cord vessels, and it causes vascular relaxation by acting as a dopamine D2-receptor antagonist. Here it was investigated whether human peripheral vessels obtained from patients who have undergone surgery for leg amputation release 6-ND, and its action in these tissues. Popliteal artery and vein strips present basal release of 6-ND, as measured by liquid chromatography coupled to tandem mass spectrometry. The release was significantly reduced when the tissues were pre-treated with the nitric oxide synthase inhibitor L-NAME (100 μM), or when the endothelium was mechanically removed. In U-46619 (3 nM) pre-contracted rings, 6-ND induced concentration-dependent relaxations (pEC50 8.18 ± 0.05 and 8.40 ± 0.08, in artery and vein rings, respectively). The concentration-dependent relaxations induced by 6-ND were unaffected in tissues pre-treated with L-NAME, but significantly reduced in tissues where the endothelium has been mechanically removed. In U-46619 (3 nM) pre-contracted rings, the selective dopamine D2 receptor antagonist L-741,626 also caused concentration-dependent relaxations (pEC50 8.92 ± 0.22 and 8.79 ± 0.19, in artery and vein rings, respectively). The concentration-dependent relaxations induced by L-741,626 were unaffected in tissues pre-treated with L-NAME, but significantly reduced in tissues where the endothelium has been mechanically removed. This is the first demonstration that 6-nitrodopamine is released from human peripheral artery and vein rings. The results also indicate that endothelium-derived dopamine is a major contractile agent in the popliteal artery and vein, and that selective dopamine D2-receptor antagonists such as 6-ND, may have therapeutic potential in the treatment of human peripheral vascular diseases.

(082) The Effect of Sex Steroids on RhoA/ROCK Pathway in Rat Distal Vagina

Abstract Introduction The RhoA/ROCK calcium-sensitizing pathway plays a role in clitoral contractility, and therefore we deemed interesting to investigate its involvement in the vagina. Objective The aim of this study was to investigate the sex steroid regulation of the vagina contractility through the RhoA/ROCK pathway, using a validated animal model. Methods Ovariectomized Sprague-Dawley rats (OVX) were treated with 17β-estradiol (E2), testosterone (T), with or without letrozole (T+L), and compared with intact animals. Contractility studies were performed on vaginal strips to test the effect of ROCK inhibitor Y-27632 and NO synthase inhibitor L-NAME. In vaginal tissues, ROCK1 immunolocalization was investigated, mRNA expression was analyzed by semi-quantitative RT-PCR, and RhoA membrane translocation was evaluated by Western blot (Wb) analysis. Finally, rat smooth muscle cells (rvSMCs) were isolated from distal vagina of intact animals and the quantification of RhoA inhibitory protein RhoGDI was performed by Wb after stimulation with NO-donor SNP, with or without the soluble guanylate cyclase inhibitor ODQ or the PKGR1 inhibitor KT5823 administration. Results The results show that ROCK1 was immunolocalized in the smooth muscle bundles and in the blood vessel wall of vagina, while a weak positivity was detected in the epithelium. Y-27632 induced a dose-dependent relaxation of noradrenaline pre-contracted vaginal strips, decreased by OVX and completely restored by E2, while T and T+L further decreased it even below OVX level increased it. Accordingly, in Wb analysis, compared to controls, OVX significantly induced RhoA activation, as reveled by its membrane translocation, with T reverting it at a level significantly lower than in controls. This effect was not exerted by E2. Abolishing the NO formation via L-NAME increased Y-27632 responsiveness in the OVX+T group; L-NAME had only a partial effect in controls whilst it did not modulate Y-27632 responsiveness in OVX. Finally, stimulation of rvSMCs with SNP significantly increased the RhoGDI protein expression, an effect counteracted by ODQ and, partially, by the protein kinase inhibitor KT5823 incubation. Conclusions In conclusion, in vagina androgen administration in OVX functionally decreases RhoA/ROCK activity by hampering RhoA membrane translocation and plays a critical role in the inhibitory mechanism of the smooth muscle distal vagina contractility. Accordingly, androgens, by inhibiting the RhoA/ROCK pathway, could positively contribute to vaginal smooth muscle relaxation, favoring the sexual intercourse. Disclosure No

Enhancement of Sphingomyelinase-Induced Endothelial Nitric Oxide Synthase-Mediated Vasorelaxation in a Murine Model of Type 2 Diabetes.

Sphingolipids are important biological mediators both in health and disease. We investigated the vascular effects of enhanced sphingomyelinase (SMase) activity in a mouse model of type 2 diabetes mellitus (T2DM) to gain an understanding of the signaling pathways involved. Myography was used to measure changes in the tone of the thoracic aorta after administration of 0.2 U/mL neutral SMase in the presence or absence of the thromboxane prostanoid (TP) receptor antagonist SQ 29,548 and the nitric oxide synthase (NOS) inhibitor L-NAME. In precontracted aortic segments of non-diabetic mice, SMase induced transient contraction and subsequent weak relaxation, whereas vessels of diabetic (Leprdb/Leprdb, referred to as db/db) mice showed marked relaxation. In the presence of the TP receptor antagonist, SMase induced enhanced relaxation in both groups, which was 3-fold stronger in the vessels of db/db mice as compared to controls and could not be abolished by ceramidase or sphingosine-kinase inhibitors. Co-administration of the NOS inhibitor L-NAME abolished vasorelaxation in both groups. Our results indicate dual vasoactive effects of SMase: TP-mediated vasoconstriction and NO-mediated vasorelaxation. Surprisingly, in spite of the general endothelial dysfunction in T2DM, the endothelial NOS-mediated vasorelaxant effect of SMase was markedly enhanced.

The effects of standard and soy-free chow on cerebral artery function in ovariectomized female mice

As females age, they transition through menopause, experiencing a decrease in estrogen and an increase in cardiovascular and neurodegenerative disease risk. When studying the effects of menopause in mice, modeled by ovariectomy, it is important to consider the effects of diet. Most standard rodent chows contain phytoestrogen-rich soybean meal, which can mimic the effects of estrogen. Understanding the impact of this soybean meal on outcomes is crucial to proper experimental design. Thus, the aim of this study was to compare the effects of standard and soy-free chows on cerebral artery endothelial function and cognitive function. We studied young female C57Bl/6J mice (n=43; ~6 months) divided into three groups: sham-controls on a soy-free diet (sham-SF), ovariectomized on a soy-free diet (OVX-SF), or OVX on a standard chow diet (OVX-SC). We assessed endothelium-dependent and -independent vasodilation in isolated pressurized posterior cerebral arteries (PCAs). We measured gene expression in cerebral arteries by real-time PCR. To assess behavioral function, we measured instinctual behavior via nest building. Data are presented as mean±SD. Compared with sham-SF, OVX-SF mice had impaired PCA endothelium-dependent vasodilation as evidenced by a maximal response that was 21% lower to acetylcholine (ACh, 60±14% vs. 47±14%, p=0.002) and 27% lower to insulin (52±17% vs. 38±8%, p=0.005). OVX-SC mice had a 27% greater PCA maximal vasodilation to insulin compared with OVX-SF mice (52±11% vs. 38±8%, p=0.001). The PCA responses to ACh and insulin did not differ between sham-SF and OVX-SC (p&gt;0.05). Additionally, the PCA response to ACh and insulin in the presence of nitric oxide synthase inhibitor L-NAME did not differ between groups (p&gt;0.05). Endothelium-independent vasodilation, measured as the PCA response to sodium nitroprusside, also did not differ between groups (p&gt;0.05). There was a trend for lower gene expression of superoxide dismutase 2 ( Sod2) in cerebral arteries from OVX-SF mice (0.7±0.2 AU) compared with OVX-SC (1.0±0.4 AU, p=0.14) but this difference did not reach statistical significance. Cerebral artery gene expression was not different between groups for Esr1 (estrogen receptor α), Insr (insulin receptor), Slca2 (GLUT1), Sod1, or Sod3 (all p&gt;0.05). Lastly, nest building scores were higher in the OVX-SC (4.3±0.5 AU) compared with sham-SF (3.2±1.2 AU, p=0.02) and OVX-SF (3.2±1.0 AU, p=0.04) mice. In summary, we found that ovariectomy impairs endothelial function in cerebral arteries by reducing nitric oxide bioavailability, but a diet containing soy mitigates these effects of ovariectomy. In addition, a diet containing soy leads to better instinctual nest-building behavior in mice. These findings highlight the importance of considering dietary soy when performing vascular and behavior tests in mice, particularly when interested in the effects of sex hormones. Luvaas Family Fund This is the full abstract presented at the American Physiology Summit 2023 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.