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American Journal of Physiology. Heart... Mar 2015In young healthy men, passive leg movement (PLM) elicits a robust nitric oxide (NO)-dependent increase in leg blood flow (LBF), thus providing a novel approach to assess... (Comparative Study)
Comparative Study
UNLABELLED
In young healthy men, passive leg movement (PLM) elicits a robust nitric oxide (NO)-dependent increase in leg blood flow (LBF), thus providing a novel approach to assess NO-mediated vascular function. While the magnitude of the LBF response to PLM is markedly reduced with age, the role of NO in this attenuated response in the elderly is unknown. Therefore, this study sought to determine the contribution of NO in the PLM-induced LBF with age. Fourteen male subjects (7 young, 24 ± 1 yr; and 7 old, 75 ± 3 yr) underwent PLM with and without NO synthase (NOS) inhibition achieved by intra-arterial infusion of N(G)-monomethyl-L-arginine (L-NMMA). LBF was determined second-by-second by Doppler ultrasound, and central hemodynamics were measured by finger photoplethysmography. NOS inhibition blunted the PLM-induced peak increase in LBF in the young (control: 668 ± 106;
L-NMMA
431 ± 95 Δml/min; P = 0.03) but had no effect in the old (control: 266 ± 98;
L-NMMA
251 ± 92 Δml/min; P = 0.59). Likewise, the magnitude of the reduction in the overall (i.e., area under the curve) PLM-induced LBF response to NOS inhibition was less in the old (LBF: -31 ± 18 ml) than the young (LBF: -129 ± 21 ml; P < 0.01). These findings suggest that the age-associated reduction in PLM-induced LBF in the elderly is primarily due to a reduced contribution to vasodilation from NO and therefore support the use of PLM as a novel approach to assess NO-mediated vascular function across the lifespan.
Topics: Adult; Age Factors; Aged; Arterial Pressure; Blood Flow Velocity; Enzyme Inhibitors; Femoral Artery; Heart Rate; Humans; Infusions, Intra-Arterial; Lower Extremity; Male; Muscle Contraction; Muscle, Skeletal; Nitric Oxide; Nitric Oxide Synthase; Regional Blood Flow; Stroke Volume; Ultrasonography; Vasodilation; Young Adult; omega-N-Methylarginine
PubMed: 25576629
DOI: 10.1152/ajpheart.00806.2014 -
PloS One 2015Due to its unique location, the endothelial surface glycocalyx (ESG) at the luminal side of the microvessel wall may serve as a mechano-sensor and transducer of blood...
Due to its unique location, the endothelial surface glycocalyx (ESG) at the luminal side of the microvessel wall may serve as a mechano-sensor and transducer of blood flow and thus regulate endothelial functions. To examine this role of the ESG, we used fluorescence microscopy to measure nitric oxide (NO) production in post-capillary venules and arterioles of rat mesentery under reduced (low) and normal (high) flow conditions, with and without enzyme pretreatment to remove heparan sulfate (HS) of the ESG and in the presence of an endothelial nitric oxide synthase (eNOS) inhibitor, NG-monomethyl-L-arginine (L-NMMA). Rats (SD, 250-300 g) were anesthetized. The mesentery was gently taken out from the abdominal cavity and arranged on the surface of a glass coverslip for the measurement. An individual post-capillary venule or arteriole was cannulated and loaded for 45 min with 5 μM 4, 5-Diaminofluorescein diacetate, a membrane permeable fluorescent indictor for NO, then the NO production was measured for ~10 min under a low flow (~300 μm/s) and for ~60 min under a high flow (~1000 μm/s). In the 15 min after switching to the high flow, DAF-2-NO fluorescence intensity increased to 1.27-fold of its baseline, DAF-2-NO continuously increased under the high flow, to 1.53-fold of its baseline in 60 min. Inhibition of eNOS by 1 mM L-NMMA attenuated the flow-induced NO production to 1.13-fold in 15 min and 1.30-fold of its baseline in 60 min, respectively. In contrast, no significant increase in NO production was observed after switching to the high flow for 60 min when 1 h pretreatment with 50 mU/mL heparanase III to degrade the ESG was applied. Similar NO production was observed in arterioles under low and high flows and under eNOS inhibition. Our results suggest that ESG participates in endothelial cell mechanosensing and transduction through its heparan sulfate to activate eNOS.
Topics: Animals; Arterioles; Blood Flow Velocity; Endothelium, Vascular; Female; Fluoresceins; Glycocalyx; Hemodynamics; Intravital Microscopy; Microvessels; Nitric Oxide; Nitric Oxide Synthase Type III; Polysaccharide-Lyases; Rats; Rats, Sprague-Dawley; omega-N-Methylarginine
PubMed: 25575016
DOI: 10.1371/journal.pone.0117133 -
The Journal of Allergy and Clinical... Mar 2015Clinical observations suggest that anaphylaxis is more common in adult women compared with adult men, although the mechanistic basis for this sex bias is not well...
BACKGROUND
Clinical observations suggest that anaphylaxis is more common in adult women compared with adult men, although the mechanistic basis for this sex bias is not well understood.
OBJECTIVES
We sought to document sex-dependent differences in a mouse model of anaphylaxis and explore the role of female sex hormones and the mechanisms responsible.
METHODS
Passive systemic anaphylaxis was induced in female and male mice by using histamine, as well as IgE or IgG receptor aggregation. Anaphylaxis was assessed by monitoring body temperature, release of mast cell mediators and/or hematocrit, and lung weight as a measure of vascular permeability. A combination of ovariectomy, estrogen receptor antagonism, and estrogen administration techniques were used to establish estrogen involvement.
RESULTS
Anaphylactic responses were more pronounced in female than male mice. The enhanced severity of anaphylaxis in female mice was eliminated after pretreatment with an estrogen receptor antagonist or ovariectomy but restored after administration of estradiol in ovariectomized mice, demonstrating that the sex-specific differences are due to the female steroid estradiol. Estrogen did not affect mast cell responsiveness or anaphylaxis onset. Instead, it increased tissue expression of endothelial nitric oxide synthase (eNOS). Blockage of NOS activity with the inhibitor L-NG-nitroarginine methyl ester or genetic eNOS deficiency abolished the sex-related differences.
CONCLUSION
Our study defines a contribution of estrogen through its regulation of eNOS expression and nitric oxide production to vascular hyperpermeability and intensified anaphylactic responses in female mice, providing additional mechanistic insights into risk factors and possible implications for clinical management in the further exploration of human anaphylaxis.
Topics: Anaphylaxis; Animals; Body Temperature; Capillary Permeability; Disease Models, Animal; Estradiol; Estrogen Receptor Antagonists; Female; Gene Expression; Histamine; Humans; Lung; Male; Mast Cells; Mice; Mice, Knockout; Nitric Oxide; Nitric Oxide Synthase Type III; Ovariectomy; Protein Aggregates; Receptors, Estrogen; Receptors, IgE; Receptors, IgG; omega-N-Methylarginine
PubMed: 25553642
DOI: 10.1016/j.jaci.2014.11.003 -
PloS One 2014Nitric oxide (NO) is a molecule involved in many reproductive processes. Its importance during oocyte in vitro maturation (IVM) has been demonstrated in various species...
Nitric oxide (NO) is a molecule involved in many reproductive processes. Its importance during oocyte in vitro maturation (IVM) has been demonstrated in various species although sometimes with contradictory results. The objective of this study was to determine the effect of NO during IVM of cumulus oocyte complexes and its subsequent impact on gamete interaction in porcine species. For this purpose, IVM media were supplemented with three NOS inhibitors: NG-nitro-L-arginine methyl ester (L-NAME), NG-monomethyl-L-arginine (L-NMMA) and aminoguanidine (AG). A NO donor, S-nitrosoglutathione (GSNO), was also used. The effects on the cumulus cell expansion, meiotic resumption, zona pellucida digestion time (ZPdt) and, finally, on in vitro fertilization (IVF) parameters were evaluated. The oocyte S-nitrosoproteins were also studied by in situ nitrosylation. The results showed that after 42 h of IVM, AG, L-NAME and L-NMMA had an inhibitory effect on cumulus cell expansion. Meiotic resumption was suppressed only when AG was added, with 78.7% of the oocytes arrested at the germinal vesicle state (P<0.05). Supplementation of the IVM medium with NOS inhibitors or NO donor did not enhance the efficiency of IVF, but revealed the importance of NO in maturation and subsequent fertilization. Furthermore, protein S-nitrosylation is reported for the first time as a pathway through which NO exerts its effect on porcine IVM; therefore, it would be important to determine which proteins are nitrosylated in the oocyte and their functions, in order to throw light on the mechanism of action of NO in oocyte maturation and subsequent fertilization.
Topics: Animals; Enzyme Inhibitors; Female; Fertilization in Vitro; Guanidines; In Vitro Oocyte Maturation Techniques; Meiosis; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Oocytes; Protein S; Swine; omega-N-Methylarginine
PubMed: 25542028
DOI: 10.1371/journal.pone.0115044 -
Blood Jan 2015Nitric oxide (NO) exerts vasodilatatory, antiplatelet, antioxidant, and antiproliferative effects. Endothelium-derived NO has been shown to be of crucial importance in...
Nitric oxide (NO) exerts vasodilatatory, antiplatelet, antioxidant, and antiproliferative effects. Endothelium-derived NO has been shown to be of crucial importance in cardiovascular protection, whereas evidence that NO is synthesized by platelets and regulates platelet function is still controversial. By using a sensitive and specific fluorescent probe, 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM), we visualized NO production in individual platelets undergoing adhesion on a collagen substrate under flow conditions. NO production, monitored in real time, was dependent on the shear rates applied, increasing with the raising of the shear rates. Furthermore, NO production increased in the presence of l-arginine (nitric-oxide synthase [NOS] substrate), and it decreased in the presence of L-NG-monomethyl arginine (L-NMMA) (NOS inhibitor) but not of D-NG-monomethyl arginine (D-NMMA) (L-NMMA-inactive enantiomer). Platelet deposition, measured with mepacrine-labeled platelets, was inversely related to NO production. A correlation was evident between Ca(++) elevation and NO production, suggesting that platelet NO formation is triggered by intracytoplasmic Ca(++) elevation. Simultaneous measurement of NO and Ca(++) indicated that NO production in individual platelets is preceded by Ca(++) elevations, with a lag phase of 33 ± 9.5 s. Our studies provide the first direct demonstration of platelet NO production triggered by the interaction with an activating surface under flow and suggest that intraplatelet Ca(++) elevation elicits the production of NO which, in turn, modulates thrombus size.
Topics: Animals; Blood Flow Velocity; Blood Platelets; Calcium; Collagen; Enzyme Inhibitors; Fluoresceins; Male; Mice; Mice, Knockout; Nitric Oxide; Platelet Adhesiveness; Quinacrine; omega-N-Methylarginine
PubMed: 25480660
DOI: 10.1182/blood-2014-06-579474 -
Diabetes & Vascular Disease Research Jan 2015The interaction between platelets and endothelium in vivo is a complex phenomenon. Our aim was to develop an in vitro system that mimics the in vivo environment and...
The interaction between platelets and endothelium in vivo is a complex phenomenon. Our aim was to develop an in vitro system that mimics the in vivo environment and investigate platelet function in a common pathological condition. Human umbilical vein endothelial cells were used and platelets from 28 type 2 diabetes patients were studied under shear stress conditions. Mean coefficient of variation of platelet aggregation was 10% in dynamic conditions in the presence of endothelium. Endothelial cells increased the concentration of inductor needed to achieve 50% platelet aggregation to adenosine diphosphate from 2.6 ± 1.3 in static conditions to 3.7 ± 1.3 µM in dynamic conditions. A similar pattern was observed when collagen was used for platelet activation. Incubation of endothelium with a nitric oxide inhibitor abolished this effect, indicating platelet inhibitory effect of endothelial cells is nitric oxide mediated. Platelet reactivity of healthy controls was less influenced by the presence of endothelial cells and displayed reduced basal platelet reactivity compared with platelets from diabetes patients. We show that platelet aggregation in diabetes as commonly reported in vitro may not fully reflect the in vivo pathophysiological process. Future studies are warranted to investigate other pathological conditions and analyse the effects of antiplatelet agents using this system.
Topics: Blood Platelet Disorders; Cells, Cultured; Diabetes Mellitus, Type 2; Down-Regulation; Endothelium, Vascular; Enzyme Inhibitors; Feasibility Studies; Female; Human Umbilical Vein Endothelial Cells; Humans; Male; Middle Aged; Nitric Oxide Synthase Type III; Platelet Activation; Platelet Adhesiveness; Platelet Aggregation; Platelet Function Tests; Reproducibility of Results; omega-N-Methylarginine
PubMed: 25349181
DOI: 10.1177/1479164114553784 -
Biological Research Sep 2014Nitric oxide (NO) has been shown to be important in sperm function, and the concentration of NO appears to determine these effects. Studies have demonstrated both...
BACKGROUND
Nitric oxide (NO) has been shown to be important in sperm function, and the concentration of NO appears to determine these effects. Studies have demonstrated both positive and negative effects of NO on sperm function, but have not been able to provide a clear link between NO concentration and the extent of exposure to NO. To study the relationship between nitric oxide and sperm capacitation in vitro, and to provide a theoretical basis for the use of NO-related preparations in improving sperm motility for in vitro fertilization, we investigated the effects of NO concentration and time duration at these concentrations on in vitro sperm capacitation in both normal and abnormal sperm groups. We manipulated NO concentrations and the time duration of these concentrations using sodium nitroprusside (an NO donor) and NG-monomethyl-L-argenine (an NO synthase inhibitor).
RESULTS
Compared to the normal sperm group, the abnormal sperm group had a longer basal time to reach the appropriate concentration of NO (p < 0.001), and the duration of time at this concentration was longer for the abnormal sperm group (p < 0.001). Both the basal time and the duration of time were significantly correlated with sperm viability and percentage of progressive sperm (p < 0.001). The experimental group had a significantly higher percentage of progressive sperm than the control group (p < 0.001).
CONCLUSIONS
We hypothesize that there is a certain regularity to both NO concentration and its duration of time in regards to sperm capacitation, and that an adequate duration of time at the appropriate NO concentration is beneficial to sperm motility.
Topics: Adult; Cell Survival; Fertilization in Vitro; Humans; In Vitro Techniques; Male; Nitric Oxide; Nitric Oxide Synthase; Nitroprusside; Sperm Capacitation; Sperm Motility; Time Factors; omega-N-Methylarginine
PubMed: 25299622
DOI: 10.1186/0717-6287-47-44 -
PloS One 2014The actin-sequestering protein thymosin beta-4 (Tβ4) is involved in various cellular and physiological processes such as proliferation, motility, growth and metastasis....
The actin-sequestering protein thymosin beta-4 (Tβ4) is involved in various cellular and physiological processes such as proliferation, motility, growth and metastasis. Nitric oxide (NO) promotes tumor invasiveness and metastasis by activating various enzymes. Herein, we investigated whether hypoxia-inducible NO regulates Tβ4 expression and cancer cell migration using HeLa cervical cancer cells. NO production and Tβ4 expression were increased in a hypoxic condition. The treatment with N-(β-D-Glucopyranosyl)-N2-acetyl-S-nitroso-D, L-penicillaminamide (SNAP-1), to generate NO, enhanced the transcription of Tβ4 and cancer cell migration. SNAP-1-induced cell migration was decreased by the inhibition of Tβ4 with small interference (si) RNA. In a hypoxic condition, treatment with N(G)-monomethyl-L-arginine (L-NMMA), nitric oxide synthase (NOS) inhibitor, reduced Tβ4 transcriptional activity, and hypoxia-inducible factor (HIF)-1α. Hypoxia-induced cancer cell migration was also decreased by L-NMMA treatment. In a normoxic condition, Tβ4 transcriptional activity was decreased in the cells incubated in the presence of L-NMMA after co-transfection with Tβ4 promoter and GST-conjugated HIF-1α. Collectively, these results suggest that NO could regulate the expression of Tβ4 by direct or indirect effect of HIF-1α on Tβ4 promoter.
Topics: Actins; Cell Movement; Gene Expression Regulation; HeLa Cells; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Nitric Oxide; Nitric Oxide Synthase; Promoter Regions, Genetic; Protein Binding; RNA Interference; RNA, Small Interfering; Thymosin; omega-N-Methylarginine
PubMed: 25271630
DOI: 10.1371/journal.pone.0106532 -
PloS One 2014Pharmacological inhibition of arginase and remote ischemic perconditioning (RIPerc) are known to protect the heart against ischemia/reperfusion (IR) injury.
BACKGROUND
Pharmacological inhibition of arginase and remote ischemic perconditioning (RIPerc) are known to protect the heart against ischemia/reperfusion (IR) injury.
PURPOSE
The objective of this study was to investigate whether (1) peroxynitrite-mediated RhoA/Rho associated kinase (ROCK) signaling pathway contributes to arginase upregulation following myocardial IR; (2) the inhibition of this pathway is involved as a cardioprotective mechanism of remote ischemic perconditioning and (3) the influence of diabetes on these mechanisms.
METHODS
Anesthetized rats were subjected to 30 min left coronary artery ligation followed by 2 h reperfusion and included in two protocols. In protocol 1 rats were randomized to 1) control IR, 2) RIPerc induced by bilateral femoral artery occlusion for 15 min during myocardial ischemia, 3) RIPerc and administration of the nitric oxide synthase inhibitor NG-monomethyl-L-arginine (L-NMMA), 4) administration of the ROCK inhibitor hydroxyfasudil or 5) the peroxynitrite decomposition catalyst FeTPPS. In protocol 2 non-diabetic and type 1 diabetic rats were randomosed to IR or RIPerc as described above.
RESULTS
Infarct size was significantly reduced in rats treated with FeTPPS, hydroxyfasudil and RIPerc compared to controls (P<0.001). FeTPPS attenuated both ROCK and arginase activity (P<0.001 vs. control). Similarly, RIPerc reduced arginase and ROCK activity, peroxynitrite formation and enhanced phospho-eNOS expression (P<0.05 vs. control). The cardioprotective effect of RIPerc was abolished by L-NMMA. The protective effect of RIPerc and its associated changes in arginase and ROCK activity were abolished in diabetes.
CONCLUSION
Arginase is activated by peroxynitrite/ROCK signaling cascade in myocardial IR. RIPerc protects against IR injury via a mechanism involving inhibition of this pathway and enhanced eNOS activation. The beneficial effect and associated molecular changes of RIPerc is abolished in type 1 diabetes.
Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Arginase; Diabetes Mellitus, Experimental; Enzyme Inhibitors; Ischemic Preconditioning; Male; Myocardial Reperfusion Injury; Peroxynitrous Acid; Rats; Rats, Sprague-Dawley; Signal Transduction; omega-N-Methylarginine; rho-Associated Kinases
PubMed: 25140754
DOI: 10.1371/journal.pone.0104731 -
Hypertension (Dallas, Tex. : 1979) Oct 2014Obesity is an important risk factor for the development of insulin resistance. Initial compensatory mechanisms include an increase in insulin levels, which are thought... (Randomized Controlled Trial)
Randomized Controlled Trial
Obesity is an important risk factor for the development of insulin resistance. Initial compensatory mechanisms include an increase in insulin levels, which are thought to induce sympathetic activation in an attempt to restore energy balance. We have previously shown, however, that sympathetic activity has no beneficial effect on resting energy expenditure in obesity. On the contrary, we hypothesize that sympathetic activation contributes to insulin resistance. To test this hypothesis, we determined insulin sensitivity using a standard hyperinsulinemic euglycemic clamp protocol in obese subjects randomly assigned in a crossover design 1 month apart to receive saline (intact day) or trimetaphan (4 mg/min IV, autonomic blocked day). Whole-body glucose uptake (MBW in mg/kg per minute) was used as index of maximal muscle glucose use. During autonomic blockade, we clamped blood pressure with a concomitant titrated intravenous infusion of the nitric oxide synthase inhibitor N-monomethyl-L-arginine. Of the 21 obese subjects (43±2 years; 35±2 kg/m(2) body mass index) studied, 14 were insulin resistant; they were more obese, had higher plasma glucose and insulin, and had higher muscle sympathetic nerve activity (23.3±1.5 versus 17.2±2.1 burst/min; P=0.03) when compared with insulin-sensitive subjects. Glucose use improved during autonomic blockade in insulin-resistant subjects (MBW 3.8±0.3 blocked versus 3.1±0.3 mg/kg per minute intact; P=0.025), with no effect in the insulin-sensitive group. These findings support the concept that sympathetic activation contributes to insulin resistance in obesity and may result in a feedback loop whereby the compensatory increase in insulin levels contributes to greater sympathetic activation.
Topics: Adult; Autonomic Nervous System; Blood Glucose; Blood Pressure; Cross-Over Studies; Enzyme Inhibitors; Female; Ganglionic Blockers; Glucose Clamp Technique; Humans; Insulin; Insulin Resistance; Male; Middle Aged; Muscles; Nitric Oxide Synthase; Obesity; Trimethaphan; omega-N-Methylarginine
PubMed: 25001269
DOI: 10.1161/HYPERTENSIONAHA.114.03738