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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 -
Nitric Oxide : Biology and Chemistry Nov 2020Nitric oxide synthase (NOS) inhibition with N(G)-monomethyl-l-arginine (L-NMMA) is often used to assess the role of NO in human cardiovascular function. However, the...
Nitric oxide synthase (NOS) inhibition with N(G)-monomethyl-l-arginine (L-NMMA) is often used to assess the role of NO in human cardiovascular function. However, the window of effect for L-NMMA on human vascular function is unknown, which is critical for designing and interpreting human-based studies. This study utilized the passive leg movement (PLM) assessment of vascular function, which is predominantly NO-mediated, in 7 young male subjects under control conditions, immediately following intra-arterial L-NMMA infusion (0.24 mg⋅dl⋅min), and at 45-60 and 90-105 min post L-NMMA infusion. The leg blood flow (LBF) and leg vascular conductance (LVC) responses to PLM, measured with Doppler ultrasound and expressed as the change from baseline to peak (ΔLBF and ΔLVC) and area under the curve (LBF and LVC), were assessed. PLM-induced robust control ΔLBF (1135 ± 324 ml⋅min) and ΔLVC (10.7 ± 3.6 ml⋅min⋅mmHg) responses that were significantly attenuated (704 ± 196 ml⋅min and 6.7 ± 2 ml⋅min⋅mmHg) immediately following L-NMMA infusion. Likewise, control condition PLM ΔLBF (455 ± 202 ml) and ΔLVC (4.0 ± 1.4 ml⋅mmHg) were significantly attenuated (141 ± 130 ml and 1.3 ± 1.2 ml⋅mmHg) immediately following L-NMMA infusion. However, by 45-60 min post L-NMMA infusion all PLM variables were not significantly different from control, and this was still the case at 90-105 min post L-NMMA infusion. These findings reveal that the potent reduction in NO bioavailability afforded by NOS inhibition with L-NMMA has a window of effect of less than 45-60 min in the human vasculature. These data are particularly important for the commonly employed approach of pharmacologically inhibiting NOS with L-NMMA in the human vasculature.
Topics: Adult; Enzyme Inhibitors; Femoral Artery; Hemodynamics; Humans; Leg; Male; Nitric Oxide; Nitric Oxide Synthase; Regional Blood Flow; Time Factors; Young Adult; omega-N-Methylarginine
PubMed: 32979497
DOI: 10.1016/j.niox.2020.09.001 -
The Journal of Toxicological Sciences 2020Bisphenol A (BPA) interferes the function and development of the central nervous system (CNS), resulting in behavioral abnormalities and memory loss. S-nitrosylation of...
Bisphenol A and rotenone induce S-nitrosylation of protein disulfide isomerase (PDI) and inhibit neurite outgrowth of primary cultured cells of the rat hippocampus and PC12 cells.
Bisphenol A (BPA) interferes the function and development of the central nervous system (CNS), resulting in behavioral abnormalities and memory loss. S-nitrosylation of protein disulfide isomerase (PDI) is increased in brains with sporadic Alzheimer's disease and Parkinson's disease. The aim of the present study was to clarify the role of nitric oxide (NO) in BPA-induced neurotoxicity. Since rotenone induces NO-mediated neurodegeneration through S-nitrosylation of PDI, it was used as a positive control. First, rats were treated with BPA and rotenone, and S-nitrosylation of PDI was detected in rat brain microsomes. BPA and rotenone decreased RNase oxidation activity of PDI concomitant with S-nitrosylation of PDI. Next, to clarify S-nitrosylation of PDI by BPA and rotenone in rat brains, we treated the rat pheochromocytoma cell line PC12 and primary cultured neuron cells from the rat hippocampus with BPA (5 and 10 μM) and rotenone (100 or 200 nM). BPA induced S-nitrosylation of PDI, while NG-monomethyl-L-arginine (L-NMMA), a NOS inhibitor, exerted the opposite effects. Finally, to evaluate the toxicity of BPA in the CNS, we investigated its effects on neurite outgrowth of PC12 and primary cultured neuron cells. BPA inhibited neurite outgrowth of these cells, while L-NMMA reversed this inhibition. The involvement of PDI activity in neurite outgrowth was also examined, and bacitracin, a PDI inhibitor, is shown to decrease neurite outgrowth. Furthermore, the overexpression of PDI, but not a catalytically inactive PDI mutant, enhanced neurite outgrowth. These results suggested that S-nitrosylation of PDI induced by excessive NO caused BPA-induced neurotoxicity.
Topics: Animals; Benzhydryl Compounds; Brain; Depression, Chemical; Hippocampus; Male; Neuronal Outgrowth; Neurotoxins; Nitric Oxide; Oxidation-Reduction; PC12 Cells; Phenols; Protein Disulfide-Isomerases; Rats; Rats, Sprague-Dawley; Ribonucleases; Rotenone; omega-N-Methylarginine
PubMed: 33268678
DOI: 10.2131/jts.45.783 -
Redox Biology Sep 2019L-N-Nitro arginine methyl ester (L-NAME) has been widely applied for several decades in both basic and clinical research as an antagonist of nitric oxide synthase (NOS)....
L-N-Nitro arginine methyl ester (L-NAME) has been widely applied for several decades in both basic and clinical research as an antagonist of nitric oxide synthase (NOS). Herein, we show that L-NAME slowly releases NO from its guanidino nitro group. Daily pretreatment of rats with L-NAME potentiated mesenteric vasodilation induced by nitrodilators such as nitroglycerin, but not by NO. Release of NO also occurred with the NOS-inactive enantiomer D-NAME, but not with L-arginine or another NOS inhibitor L-NMMA, consistent with the presence or absence of a nitro group in their structure and their nitrodilator-potentiating effects. Metabolic conversion of the nitro group to NO-related breakdown products was confirmed using isotopically-labeled L-NAME. Consistent with Fenton chemistry, transition metals and reactive oxygen species accelerated the release of NO from L-NAME. Both NO production from L-NAME and its nitrodilator-potentiating effects were augmented under inflammation. NO release by L-NAME can confound its intended NOS-inhibiting effects, possibly by contributing to a putative intracellular NO store in the vasculature.
Topics: Animals; Arginine; Enzyme Inhibitors; Female; Mesenteric Arteries; Mice; Myography; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Nitroglycerin; RAW 264.7 Cells; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Sheep; Stereoisomerism; Vasodilation; Vasodilator Agents; omega-N-Methylarginine
PubMed: 31200239
DOI: 10.1016/j.redox.2019.101238 -
Microcirculation (New York, N.Y. : 1994) Apr 2020The aim of this study was to investigate whether the effects on local blood flow and metabolic changes observed in the skin after an endogenous systemic increase in... (Clinical Trial)
Clinical Trial
OBJECTIVE
The aim of this study was to investigate whether the effects on local blood flow and metabolic changes observed in the skin after an endogenous systemic increase in insulin are mediated by the endothelial nitric oxide pathway, by administering the nitric oxide synthase inhibitor N -monomethyl l-arginine using microdialysis.
METHODS
Microdialysis catheters, perfused with N -monomethyl l-arginine and with a control solution, were inserted intracutaneously in 12 human subjects, who received an oral glucose load to induce a systemic hyperinsulinemia. During microdialysis, the local blood flow was measured by urea clearance and by laser speckle contrast imaging, and glucose metabolites were measured.
RESULTS
After oral glucose intake, microvascular blood flow and glucose metabolism were both significantly suppressed in the N -monomethyl l-arginine catheter compared to the control catheter (urea clearance: P < .006, glucose dialysate concentration: P < .035). No significant effect of N -monomethyl l-arginine on microvascular blood flow was observed with laser speckle contrast imaging (P = .81).
CONCLUSION
Local delivery of N -monomethyl l-arginine to the skin by microdialysis reduces microvascular blood flow and glucose delivery in the skin after oral glucose intake, presumably by decreasing local insulin-mediated vasodilation.
Topics: Adult; Blood Flow Velocity; Blood Glucose; Female; Glucose Tolerance Test; Humans; Male; Microcirculation; Microdialysis; Regional Blood Flow; omega-N-Methylarginine
PubMed: 31628700
DOI: 10.1111/micc.12597 -
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 -
Tissue & Cell Dec 2021Puerarin regulates the osteoblast differentiation of umbilical cord mesenchymal stem cells. This study, hereby, explored the effects of puerarin on the osteogenic...
Puerarin regulates the osteoblast differentiation of umbilical cord mesenchymal stem cells. This study, hereby, explored the effects of puerarin on the osteogenic differentiation of dental follicle cells (DFCs) for the first time. Rat DFCs (rDFCs) were isolated and identified. After the rDFCs were treated by Puerarin and cultured in osteogenic induction medium, the viability, osteogenic differentiation, and the activities of alkaline phosphatase (ALP) and nitric oxide (NO) were detected. Besides, the secretion of cyclic guanosine monophosphate (cGMP) and expressions of collagen I, osteocalcin (OC), osteopontin (OPN), runt-related transcription factor 2 (RUNX2), soluble guanylate cyclase (SGC), and protein kinase G 1 (PKG-1) were further determined or quantified. Puerarin enhanced the viability and osteogenic differentiation, and increased the activities of ALP, NO, and cGMP and the expressions of Collagen I, OC, OPN, RUNX2, SGC, and PKG-1 in rDFCs. After the co-treatment with puerarin and L-NMMA (NO synthase inhibitor), the promotive effects of Puerarin on cell viability, osteogenic differentiation, and the expressions of collagen I, OC, OPN, RUNX2, SGC, and PKG-1 in rDFCs were reversed by L-NMMA. Puerarin boosted the osteogenic differentiation of rDFCs by activating the NO pathway.
Topics: Alkaline Phosphatase; Animals; Animals, Newborn; Cell Differentiation; Cell Survival; Cells, Cultured; Collagen Type I; Core Binding Factor Alpha 1 Subunit; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Dental Sac; Guanylate Cyclase; Isoflavones; Nitric Oxide; Osteocalcin; Osteogenesis; Osteopontin; Rats, Sprague-Dawley; Solubility; omega-N-Methylarginine; Rats
PubMed: 34371290
DOI: 10.1016/j.tice.2021.101601 -
The American Journal of Pathology Dec 2021Bone homeostasis depends on the balance between bone resorption by osteoclasts (OCs) and bone formation by osteoblasts. Bone resorption can become excessive under...
Bone homeostasis depends on the balance between bone resorption by osteoclasts (OCs) and bone formation by osteoblasts. Bone resorption can become excessive under various pathologic conditions, including rheumatoid arthritis. Previous studies have shown that OC formation is promoted under hypoxia. However, the precise mechanisms behind OC formation under hypoxia have not been elucidated. The present study investigated the role of inducible nitric oxide synthase (iNOS) in OC differentiation under hypoxia. Primary bone marrow cells obtained from mice were stimulated with receptor activator of NF-κB ligand and macrophage colony-stimulating factor to induce OC differentiation. The number of OCs increased in culture under hypoxia (oxygen concentration, 5%) compared with that under normoxia (oxygen concentration, 20%). iNOS gene and protein expression increased in culture under hypoxia. Addition of an iNOS inhibitor under hypoxic conditions suppressed osteoclastogenesis. Addition of a nitric oxide donor to the normoxic culture promoted osteoclastogenesis. Furthermore, insulin-like growth factor 2 expression was significantly altered in both iNOS inhibition experiments and nitric oxide donor experiments. These data might provide clues to therapies for excessive osteoclastogenesis under several hypoxic pathologic conditions, including rheumatoid arthritis.
Topics: Animals; Bone Resorption; Cell Differentiation; Cell Hypoxia; Cells, Cultured; Enzyme Induction; Hypoxia; Male; Mice; Mice, Inbred C57BL; Nitric Oxide Synthase Type II; Osteoclasts; Osteogenesis; Oxygen; Signal Transduction; omega-N-Methylarginine
PubMed: 34560064
DOI: 10.1016/j.ajpath.2021.08.014 -
Journal of the American Heart... Aug 2020Background Basal release of nitric oxide (NO) from the vascular endothelium regulates the tone of muscular arteries and resistance vasculature. Effects of NO on muscular... (Randomized Controlled Trial)
Randomized Controlled Trial
Background Basal release of nitric oxide (NO) from the vascular endothelium regulates the tone of muscular arteries and resistance vasculature. Effects of NO on muscular arteries could be particularly important during exercise when shear stress may stimulate increased NO synthesis. Methods and Results We investigated acute effects of NO synthase inhibition on exercise hemodynamics using N-monomethyl-l-arginine (l-NMMA), a nonselective NO synthase -inhibitor. Healthy volunteers (n=10, 5 female, 19-33 years) participated in a 2-phase randomized crossover study, receiving l-NMMA (6 mg/kg, iv over 5 minutes) or placebo before bicycle exercise (25-150 W for 12 minutes). Blood pressure, cardiac output (measured by dilution of soluble and inert tracers) and femoral artery diameter were measured before, during, and after exercise. At rest, l-NMMA reduced heart rate (by 16.2±4.3 bpm relative to placebo, <0.01), increased peripheral vascular resistance (by 7.0±1.4 mmHg per L/min, <0.001), mean arterial blood pressure (by 8.9±3.5 mmHg, <0.05), and blunted an increase in femoral artery diameter that occurred immediately before exercise (change in diameter: 0.14±0.04 versus 0.32±0.06 mm after l-NMMA and placebo, <0.01). During/after exercise l-NMMA had no significant effect on peripheral resistance, cardiac output, or on femoral artery diameter. Conclusions These results suggest that NO plays little role in modulating muscular artery function during exercise but that it may mediate changes in muscular artery tone immediately before exercise.
Topics: Adult; Arterial Pressure; Arteries; Cardiac Output; Cross-Over Studies; Enzyme Inhibitors; Exercise; Exercise Test; Female; Femoral Artery; Humans; Male; Muscle, Skeletal; Nitric Oxide; Nitric Oxide Synthase; Placebos; Pulse Wave Analysis; Vascular Resistance; Vasodilation; Young Adult; omega-N-Methylarginine
PubMed: 32781940
DOI: 10.1161/JAHA.119.013849 -
The Journal of Physiology Nov 2021The importance of nitric oxide (NO) in regulating cerebral blood flow (CBF) remains unresolved, due in part to methodological approaches, which lack a comprehensive... (Randomized Controlled Trial)
Randomized Controlled Trial
The importance of nitric oxide (NO) in regulating cerebral blood flow (CBF) remains unresolved, due in part to methodological approaches, which lack a comprehensive assessment of both global and regional effects. Importantly, NO synthase (NOS) expression and activity appear greater in some anterior brain regions, suggesting region-specific NOS influence on CBF. We hypothesized that NO contributes to basal CBF in healthy adults, in a regionally distinct pattern that predominates in the anterior circulation. Fourteen healthy adults (7 females; 24 ± 5 years) underwent two magnetic resonance imaging (MRI) study visits with saline (placebo) or the NOS inhibitor, L-NMMA, administered in a randomized, single-blind approach. 4D flow MRI quantified total and regional macrovascular CBF, whereas arterial spin labelling (ASL) MRI quantified total and regional microvascular perfusion. L-NMMA (or volume-matched saline) was infused intravenously for 5 min prior to imaging. L-NMMA reduced CBF (L-NMMA: 722 ± 100 vs. placebo: 771 ± 121 ml/min, P = 0.01) with similar relative reductions (5-7%) in anterior and posterior cerebral circulations, due in part to the reduced cross-sectional area of 9 of 11 large cerebral arteries. Global microvascular perfusion (ASL) was reduced by L-NMMA (L-NMMA: 42 ± 7 vs. placebo: 47 ± 8 ml/100g/min, P = 0.02), with 7-11% reductions in both hemispheres of the frontal, parietal and temporal lobes, and in the left occipital lobe. We conclude that NO contributes to macrovascular and microvascular regulation including larger artery resting diameter. Contrary to our hypothesis, the influence of NO on cerebral perfusion appears regionally uniform in healthy young adults. KEY POINTS: Cerebral blood flow (CBF) is vital for brain health, but the signals that are key to regulating CBF remain unclear. Nitric oxide (NO) is produced in the brain, but its importance in regulating CBF remains controversial since prior studies have not studied all regions of the brain simultaneously. Using modern MRI approaches, a drug that inhibits the enzymes that make NO (L-NMMA) reduced CBF by up to 11% in different brain regions. NO helps maintain proper CBF in healthy adults. These data will help us understand whether the reductions in CBF that occur during ageing or cardiovascular disease are related to shifts in NO signalling.
Topics: Adult; Cerebrovascular Circulation; Female; Humans; Male; Nitric Oxide; Nitric Oxide Synthase; Perfusion; Regional Blood Flow; Single-Blind Method; Young Adult; omega-N-Methylarginine
PubMed: 34587648
DOI: 10.1113/JP281975