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Zhongguo Zhong Yao Za Zhi = Zhongguo... Nov 2022This study was designed to determine the inhibitory effect of astragaloside Ⅳ(AS-Ⅳ), a principal bioactive component extracted from the Chinese medicinal Astragali...
This study was designed to determine the inhibitory effect of astragaloside Ⅳ(AS-Ⅳ), a principal bioactive component extracted from the Chinese medicinal Astragali Radix, on the inflammatory response of vascular endothelial cells induced by angiotensin Ⅱ(Ang Ⅱ), the most major pathogenic factor for cardiovascular diseases, and to clarify the role of calcium(Ca~(2+))/phosphatidylinosi-tol-3-kinase(PI3K)/protein kinase B(Akt)/endothelial nitric oxide synthase(eNOS)/nitric oxide(NO) pathway in the process. To be specific, human umbilical vein endothelial cells(HUVECs) were cultured in the presence of AS-Ⅳ with or without the specific inhibitor of NO synthase(NG-monomethyl-L-arginine, L-NMMA), inhibitor of PI3K/Akt signaling pathway(LY294002), or Ca~(2+)-chelating agent(ethylene glycol tetraacetic acid, EGTA) prior to Ang Ⅱ stimulation. The inhibitory effect of AS-Ⅳ on Ang Ⅱ-induced inflammatory response and the involved mechanism was determined with enzyme-linked immunosorbent assay(ELISA), cell-based ELISA assay, Western blot, and monocyte adhesion assay which determined the fluorescently labeled human monocytic cell line(THP-1) adhered to Ang Ⅱ-stimulated endothelial cells. AS-Ⅳ increased the production of NO by HUVECs in a dose-and time-dependent manner(P<0.05) and raised the level of phosphorylated eNOS(P<0.05). The above AS-Ⅳ-induced changes were abolished by pretreatment with L-NMMA, LY294002, or EGTA. Compared with the control group, Ang Ⅱ obviously enhanced the production and release of cytokines(tumor necrosis factor-α, interleukin-6), chemokines(monocyte chemoattractant protein-1) and adhesion molecules(intercellular adhesion molecule-1, vascular cellular adhesion molecule-1), and the number of monocytes adhered to HUVECs(P<0.05), which were accompanied by the enhanced levels of phosphorylated inhibitor of nuclear factor-κBα protein and activities of nuclear factor-κB(NF-κB)(P<0.05). This study also demonstrated that Ang Ⅱ-induced inflammatory response was inhibited by pretreatment with AS-Ⅳ(P<0.05). In addition, the inhibitory effect of AS-Ⅳ was abrogated by pretreatment with L-NMMA, LY294002, or EGTA(P<0.05). This study provides a direct link between AS-Ⅳ and Ca~(2+)/PI3K/Akt/eNOS/NO pathway in AS-Ⅳ-mediated anti-inflammatory actions in endothelial cells exposed to Ang Ⅱ. The results indicate that AS-Ⅳ attenuates endothelial cell-mediated inflammatory response induced by Ang Ⅱ via the activation of Ca~(2+)/PI3K/Akt/eNOS/NO signaling pathway.
Topics: Humans; Angiotensin II; Proto-Oncogene Proteins c-akt; Phosphatidylinositol 3-Kinases; omega-N-Methylarginine; Egtazic Acid; Human Umbilical Vein Endothelial Cells; NF-kappa B; Nitric Oxide; Cells, Cultured
PubMed: 36472009
DOI: 10.19540/j.cnki.cjcmm.20220725.701 -
Amino Acids Feb 2023Protein arginine N-methyltransferases (PRMTs) have emerged as important actors in the eukaryotic stress response with implications in human disease, aging, and cell...
Protein arginine N-methyltransferases (PRMTs) have emerged as important actors in the eukaryotic stress response with implications in human disease, aging, and cell signaling. Intracellular free methylarginines contribute to cellular stress through their interaction with nitric oxide synthase (NOS). The arginine-dependent production of nitric oxide (NO), which is strongly inhibited by methylarginines, serves as a protective small molecule against oxidative stress in eukaryotic cells. NO signaling is highly conserved between higher and lower eukaryotes, although a canonical NOS homologue has yet to be identified in yeast. Since stress signaling pathways are well conserved among eukaryotes, yeast is an ideal model organism to study the implications of PRMTs and methylarginines during stress. We sought to explore the roles and fates of methylarginines in Saccharomyces cerevisiae. We starved methyltransferase-, autophagy-, and permease-related yeast knockouts by incubating them in water and monitored methylarginine production. We found that under starvation, methylarginines are expelled from yeast cells. We found that autophagy-deficient cells have an impaired ability to efflux methylarginines, which suggests that methylarginine-containing proteins are degraded via autophagy. For the first time, we determine that yeast take up methylarginines less readily than arginine, and we show that methylarginines impact yeast NO production. This study reveals that yeast circumvent a potential methylarginine toxicity by expelling them after autophagic degradation of arginine-modified proteins.
Topics: Humans; omega-N-Methylarginine; Saccharomyces cerevisiae; Nitric Oxide; Arginine; Nitric Oxide Synthase; Nutrients
PubMed: 36454288
DOI: 10.1007/s00726-022-03220-x -
World Journal of Gastroenterology Aug 2022The mechanisms underlying gastrointestinal (GI) dysmotility with ulcerative colitis (UC) have not been fully elucidated. The enteric nervous system (ENS) plays an...
BACKGROUND
The mechanisms underlying gastrointestinal (GI) dysmotility with ulcerative colitis (UC) have not been fully elucidated. The enteric nervous system (ENS) plays an essential role in the GI motility. As a vital neurotransmitter in the ENS, the gas neurotransmitter nitric oxide (NO) may impact the colonic motility. In this study, dextran sulfate sodium (DSS)-induced UC rat model was used for investigating the effects of NO by examining the effects of rate-limiting enzyme nitric oxide synthase (NOS) changes on the colonic motility as well as the role of the ENS in the colonic motility during UC.
AIM
To reveal the relationship between the effects of NOS expression changes in NOS-containing nitrergic neurons and the colonic motility in a rat UC model.
METHODS
Male rats ( = 8/each group) were randomly divided into a control (CG), a UC group (EG1), a UC + thrombin derived polypeptide 508 trifluoroacetic acid (TP508TFA; an NOS agonist) group (EG2), and a UC + NG-monomethyl-L-arginine monoacetate (L-NMMA; an NOS inhibitor) group (EG3). UC was induced by administering 5.5% DSS in drinking water without any other treatment (EG1), while the EG2 and EG3 were gavaged with TP508 TFA and L-NMMA, respectively. The disease activity index (DAI) and histological assessment were recorded for each group, whereas the changes in the proportion of colonic nitrergic neurons were counted using immunofluorescence histochemical staining, Western blot, and enzyme linked immunosorbent assay, respectively. In addition, the contractile tension changes in the circular and longitudinal muscles of the rat colon were investigated using an organ bath system.
RESULTS
The proportion of NOS-positive neurons within the colonic myenteric plexus (MP), the relative expression of NOS, and the NOS concentration in serum and colonic tissues were significantly elevated in EG1, EG2, and EG3 compared with CG rats. In UC rats, stimulation with agonists and inhibitors led to variable degrees of increase or decrease for each indicator in the EG2 and EG3. When the rats in EGs developed UC, the mean contraction tension of the colonic smooth muscle detected was higher in the EG1, EG2, and EG3 than in the CG group. Compared with the EG1, the contraction amplitude and mean contraction tension of the circular and longitudinal muscles of the colon in the EG2 and EG3 were enhanced and attenuated, respectively. Thus, during UC, regulation of the expression of NOS within the MP improved the intestinal motility, thereby favoring the recovery of intestinal functions.
CONCLUSION
In UC rats, an increased number of nitrergic neurons in the colonic MP leads to the attenuation of colonic motor function. To intervene NOS activity might modulate the function of nitrergic neurons in the colonic MP and prevent colonic motor dysfunction. These results might provide clues for a novel approach to alleviate diarrhea symptoms of UC patients.
Topics: Animals; Male; Rats; Colitis, Ulcerative; Colon; Dextran Sulfate; Drinking Water; Gastrointestinal Motility; Nitrergic Neurons; Nitric Oxide; Nitric Oxide Synthase; omega-N-Methylarginine; Thrombin; Trifluoroacetic Acid
PubMed: 36157548
DOI: 10.3748/wjg.v28.i29.3854 -
PloS One 2022The endothelium maintains and regulates vascular homeostasis mainly by balancing interplay between vasorelaxation and vasoconstriction via regulating Nitric Oxide (NO)...
The endothelium maintains and regulates vascular homeostasis mainly by balancing interplay between vasorelaxation and vasoconstriction via regulating Nitric Oxide (NO) availability. Endothelial nitric oxide synthase (eNOS) is one of three NOS isoforms that catalyses the synthesis of NO to regulate endothelial function. However, eNOS's role in the regulation of endothelial function, such as cell proliferation and migration remain unclear. To gain a better understanding, we genetically knocked down eNOS in cultured endothelial cells using sieNOS and evaluated cell proliferation, migration and also tube forming potential in vitro. To our surprise, loss of eNOS significantly induced endothelial cell proliferation, which was associated with significant downregulation of both cell cycle inhibitor p21 and cell proliferation antigen Ki-67. Knockdown of eNOS induced cell migration but inhibited formation of tube-like structures in vitro. Mechanistically, loss of eNOS was associated with activation of MAPK/ERK and inhibition of PI3-K/AKT signaling pathway. On the contrary, pharmacologic inhibition of eNOS by inhibitors L-NAME or L-NMMA, inhibited cell proliferation. Genetic and pharmacologic inhibition of eNOS, both promoted endothelial cell migration but inhibited tube-forming potential. Our findings confirm that eNOS regulate endothelial function by inversely controlling endothelial cell proliferation and migration, and by directly regulating its tube-forming potential. Differential results obtained following pharmacologic versus genetic inhibition of eNOS indicates a more complex mechanism behind eNOS regulation and activity in endothelial cells, warranting further investigation.
Topics: Cells, Cultured; Endothelial Cells; Endothelium; Ki-67 Antigen; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase Type III; Protein Isoforms; Proto-Oncogene Proteins c-akt; omega-N-Methylarginine
PubMed: 36149900
DOI: 10.1371/journal.pone.0274487 -
Clinical Hemorheology and... 2022Exercise-induced impairment of blood fluidity is considered to be associated with thrombosis development. However, the effects of L-arginine on blood fluidity after...
BACKGROUND
Exercise-induced impairment of blood fluidity is considered to be associated with thrombosis development. However, the effects of L-arginine on blood fluidity after exercise remain unclear.
OBJECTIVE
We investigated the mechanisms of impaired blood fluidity after high-intensity exercise, and examined whether L-arginine improves exercise-induced blood fluidity impairment in vitro.
METHODS
Ten healthy male participants performed 15 minutes of ergometer exercise at 70% of their peak oxygen uptake levels. Blood samples were obtained before and after exercise. L-arginine and NG-monomethyl-L-arginine acetate (L-NMMA)-a nitric oxide (NO) synthase inhibitor-were added to the post-exercise blood samples. Using Kikuchi's microchannel method, we measured the blood passage time, percentage of obstructed microchannels, and the number of adherent white blood cells (WBCs) on the microchannel terrace.
RESULTS
Exercise increased the hematocrit levels. The blood passage times, percentage of obstructed microchannels, and the number of adherent WBCs on the microchannel terrace increased after exercise; however, they decreased in a dose-dependent manner after the addition of L-arginine. L-NMMA inhibited the L-arginine-induced decrease in blood passage time.
CONCLUSIONS
High-intensity exercise impairs blood fluidity by inducing hemoconcentration along with increasing platelet aggregation and WBC adhesion. The L-arginine-NO pathway improves blood fluidity impairment after high-intensity exercise in vitro.
Topics: Humans; Male; omega-N-Methylarginine; Nitric Oxide; Arginine; Exercise; Leukocytes; Platelet Aggregation
PubMed: 35599472
DOI: 10.3233/CH-211201 -
Clinical Oral Investigations Jul 2022Methylated arginine metabolites and nitric oxide synthase (NOS) play a critical role in regulating endothelial function. The aim of this study was to determine levels of...
OBJECTIVES
Methylated arginine metabolites and nitric oxide synthase (NOS) play a critical role in regulating endothelial function. The aim of this study was to determine levels of NOS, and methylated arginine metabolites (ADMA, SDMA, homoarginine, arginine, and L-NMMA) and IL-6 in serum and saliva in patients with advanced periodontal diseases and identify their association with clinical parameters.
MATERIALS AND METHODS
The study consisted of two groups: healthy individuals (control: n = 24), and generalized Stage III Grade B periodontitis (P: n = 21). Clinical periodontal parameters (probing pocket depth, bleeding on probing, clinical attachment level) were recorded. IL 6 and NOS levels in saliva and serum were analyzed by enzyme-linked immunosorbent assay (ELISA). ADMA, SDMA, homoArg, arginine, and L-NMMA in saliva and serum were analyzed by liquid chromatography-mass spectrometry (LC MS/MS).
RESULTS
Clinical parameters were significantly higher in the periodontitis group (p < 0.001). In periodontitis group, NOS, ADMA, and arginine levels in saliva were statistically significantly higher than control group (p < 0.05). Serum levels of SDMA were statistically significantly lower, and IL-6 was statistically significantly higher in P group than C group (p < 0.05). ADMA, NOS, and arginine levels were significantly positive correlated with all clinical periodontal parameters (p < 0.05).
CONCLUSIONS
These findings suggest that there is a relationship between severity of periodontal disease and endothelial dysfunction by means of ADMA. Salivary ADMA may be related with periodontal inflammation.
CLINICAL RELEVANCE
ADMA levels in periodontal inflammation are associated with endothelial dysfunction. According to the results of our study, periodontal inflammation is effective on both local and systemic methylated arginine metabolites and nitric oxide synthase levels. This may shed light on the relationship between periodontal disease and systemic status.
Topics: Arginine; Humans; Inflammation; Interleukin-6; Nitric Oxide Synthase; Periodontal Diseases; Periodontitis; Tandem Mass Spectrometry; omega-N-Methylarginine
PubMed: 35426000
DOI: 10.1007/s00784-022-04479-w -
Behavioural Brain Research Mar 2022Nitric oxide (NO)-dependent pathways may play a significant role in the decline of synaptic and cognitive functions in Alzheimer's disease (AD). However, whether NO in...
Nitric oxide impairs spatial learning and memory in a rat model of Alzheimer's disease via disturbance of glutamate response in the hippocampal dentate gyrus during spatial learning.
Nitric oxide (NO)-dependent pathways may play a significant role in the decline of synaptic and cognitive functions in Alzheimer's disease (AD). However, whether NO in the hippocampal dentate gyrus (DG) is involved in the spatial learning and memory impairments of AD by affecting the glutamate (Glu) response during these processes is not well-understood. Here, we prepared an AD rat model by long-term i.p. of D-galactose into ovariectomized rats, and then the effects of L-NMMA (a NO synthase inhibitor) on Glu concentration and amplitude of field excitatory postsynaptic potential (fEPSP) were measured in the DG region during the Morris water maze (MWM) test in freely-moving rats. During the MWM test, compared with the sham group, the escape latency was increased in the place navigation trial, and the percentage of time spent in target quadrant and the number of platform crossings were decreased in the spatial probe trial, in addition, the increase of fEPSP amplitude in the DG was significantly attenuated in AD group rats. L-NMMA significantly attenuated the spatial learning and memory impairment in AD rats, and reversed the inhibitory effect of AD on increase of fEPSP amplitude in the DG during the MWM test. In sham group rats, the Glu level in the DG increased significantly during the MWM test, and this response was markedly enhanced in AD rats. Furthermore, the response of Glu in the DG during spatial learning was recovered by microinjection of L-NMMA into the DG. Our results suggest that NO in the DG impairs spatial learning and memory and related synaptic plasticity in AD rats, by disturbing the Glu response during spatial learning.
Topics: Alzheimer Disease; Animals; Behavior, Animal; Dentate Gyrus; Disease Models, Animal; Enzyme Inhibitors; Excitatory Postsynaptic Potentials; Female; Glutamic Acid; Maze Learning; Nitric Oxide; Nitric Oxide Synthase; Ovariectomy; Rats; Rats, Sprague-Dawley; omega-N-Methylarginine
PubMed: 35033612
DOI: 10.1016/j.bbr.2022.113750 -
Clinical Laboratory Jan 2022In this study, we aimed to show that methylated arginines are the predictors of non-clinical atherosclerotic cardiovascular complications in metal workers exposed to Cd.
BACKGROUND
In this study, we aimed to show that methylated arginines are the predictors of non-clinical atherosclerotic cardiovascular complications in metal workers exposed to Cd.
METHODS
The 80 Cd-exposed metal workers and 80 non-exposed workers (control) included in the study were available for measuring arginine, ADMA, SDMA, and L-NMMA levels.
RESULTS
The average urine Cd levels (CdU) found were 1.03 ± 0.8 µg/g creatinine (0.84 ± 0.65 µg/L) ranging from 0.01 to 3.00 µg/g creatinine in the control group and 5.41 ± 5.2 µg/g creatinine (4.29 ± 3.81 µg/L) ranged from 0.11 to 27.2 µg/g creatinine in metal workers. On the other hand, the median ratios of the different groups (exposed and control) were found to be 449.35 and 483.88 for arginine/ADMA and 1.28 and 1.33 SDMA/ADMA, respectively.
CONCLUSIONS
The present study was undertaken to investigate the relationship between cadmium exposure and methylated arginines such as ADMA/SDMA/L-NMMA parameters which is important for the early detection atherosclerotic cardiovascular diseases.
Topics: Arginine; Atherosclerosis; Cadmium; Creatinine; Humans; omega-N-Methylarginine
PubMed: 35023667
DOI: 10.7754/Clin.Lab.2021.210504 -
Science Translational Medicine Dec 2021The inducible nitric oxide signaling (iNOS) pathway is associated with poor prognosis in triple-negative breast cancer (TNBC). Prior studies using in vivo models showed...
The inducible nitric oxide signaling (iNOS) pathway is associated with poor prognosis in triple-negative breast cancer (TNBC). Prior studies using in vivo models showed that inhibition of the iNOS signaling pathway using the pan-NOS inhibitor NG-monomethyl-l-arginine (L-NMMA) reduced tumor growth and enhanced survival in patients with TNBC. Here, we report a first-in-class phase 1/2 trial of L-NMMA combined with taxane for treating patients with chemorefractory, locally advanced breast cancer (LABC) or metastatic TNBC. We also examined immune cell correlates of chemotherapy response. 35 patients with metastatic TNBC were recruited: 15 in the phase 1 trial and 24 in the phase 2 trial (including 4 recommended phase 2 dose patients from the phase 1 trial). The overall response rate was 45.8% (11 of 24): 81.8% (9 of 11) for patients with LABC and 15.4% (2 of 13) for patients with metastatic TNBC. Among the patients with LABC, three patients had a pathological complete response at surgery (27.3%). Grade ≥3 toxicity was noted in 21% of patients; however, no adverse events were attributed to L-NMMA. Immune cells analyzed by CyTOF indicated that chemotherapy nonresponders showed greater expression of markers associated with M2 macrophage polarization and increased concentrations of circulating IL-6 and IL-10 cytokines. In contrast, chemotherapy responders showed an increase in CD15 neutrophils in blood, as well as a decrease in arginase (a marker of protumor N2 neutrophils) in tumor biopsies obtained at the end of treatment. L-NMMA combined with taxane warrants further investigation in larger clinical studies of patients with breast cancer.
Topics: Enzyme Inhibitors; Humans; Nitric Oxide; Nitric Oxide Synthase; Taxoids; Triple Negative Breast Neoplasms; omega-N-Methylarginine
PubMed: 34910551
DOI: 10.1126/scitranslmed.abj5070 -
The Journal of Physiology Mar 2022Cerebrovascular CO reactivity (CVR) is often considered a bioassay of cerebrovascular endothelial function. We recently introduced a test of cerebral shear-mediated...
Cerebrovascular CO reactivity (CVR) is often considered a bioassay of cerebrovascular endothelial function. We recently introduced a test of cerebral shear-mediated dilatation (cSMD) that may better reflect endothelial function. We aimed to determine the nitric oxide (NO)-dependency of CVR and cSMD. Eleven volunteers underwent a steady-state CVR test and transient CO test of cSMD during intravenous infusion of the NO synthase inhibitor N -monomethyl-l-arginine (l-NMMA) or volume-matched saline (placebo; single-blinded and counter-balanced). We measured cerebral blood flow (CBF; duplex ultrasound), intra-arterial blood pressure and . Paired arterial and jugular venous blood sampling allowed for the determination of trans-cerebral NO exchange (ozone-based chemiluminescence). l-NMMA reduced arterial NO by ∼25% versus saline (74.3 ± 39.9 vs. 98.1 ± 34.2 nM; P = 0.03). The steady-state CVR (20.1 ± 11.6 nM/min at baseline vs. 3.2 ± 16.7 nM/min at +9 mmHg ; P = 0.017) and transient cSMD tests (3.4 ± 5.9 nM/min at baseline vs. -1.8 ± 8.2 nM/min at 120 s post-CO ; P = 0.044) shifted trans-cerebral NO exchange towards a greater net release (a negative value indicates release). Although this trans-cerebral NO release was abolished by l-NMMA, CVR did not differ between the saline and l-NMMA trials (57.2 ± 14.6 vs. 54.1 ± 12.1 ml/min/mmHg; P = 0.49), nor did l-NMMA impact peak internal carotid artery dilatation during the steady-state CVR test (6.2 ± 4.5 vs. 6.2 ± 5.0% dilatation; P = 0.960). However, l-NMMA reduced cSMD by ∼37% compared to saline (2.91 ± 1.38 vs. 4.65 ± 2.50%; P = 0.009). Our findings indicate that NO is not an obligatory regulator of steady-state CVR. Further, our novel transient CO test of cSMD is largely NO-dependent and provides an in vivo bioassay of NO-mediated cerebrovascular function in humans. KEY POINTS: Emerging evidence indicates that a transient CO stimulus elicits shear-mediated dilatation of the internal carotid artery, termed cerebral shear-mediated dilatation. Whether or not cerebrovascular reactivity to a steady-state CO stimulus is NO-dependent remains unclear in humans. During both a steady-state cerebrovascular reactivity test and a transient CO test of cerebral shear-mediated dilatation, trans-cerebral nitrite exchange shifted towards a net release indicating cerebrovascular NO production; this response was not evident following intravenous infusion of the non-selective NO synthase inhibitor N -monomethyl-l-arginine. NO synthase blockade did not alter cerebrovascular reactivity in the steady-state CO test; however, cerebral shear-mediated dilatation following a transient CO stimulus was reduced by ∼37% following intravenous infusion of N -monomethyl-l-arginine. NO is not obligatory for cerebrovascular reactivity to CO , but is a key contributor to cerebral shear-mediated dilatation.
Topics: Carbon Dioxide; Cerebrovascular Circulation; Dilatation; Enzyme Inhibitors; Humans; Nitric Oxide; Nitric Oxide Synthase; Nitrogen Dioxide; omega-N-Methylarginine
PubMed: 34904229
DOI: 10.1113/JP282427