Exhaled nitric oxide during normoxic and hypoxic exercise in endurance athletes

Aim: Endogenous nitric oxide (NO) through its relaxing effect on smooth muscle cells may be involved in pulmonary gas exchange as well as in the modulation of the hypoxic pulmonary vasoconstriction. As athletes with exercise-induced hypoxaemia (EIH) present pulmonary gas exchange abnormalities in normoxia that could be even greater in hypoxia, we hypothesized that pulmonary NO may be lower in such athletes with EIH. METHODS: Eleven athletes with EIH [decrease in arterial oxygen blood partial pressure (PaO2) > 12 mmHg] and 9 without EIH (NEIH) exercised at 40%, 60% (10 min) and 90% (5 min) of normoxic maximal power output (Pmax) in normoxia, and at 40% and 60% (10 min) of Pmax in hypoxia (FiO2 = 15%). Exhaled NO concentration during a constant flow exhalation (FENO(0.170)) and arterialized blood gases were measured at every power output. RESULTS: FENO(0.170) decreased from rest to exercise both in normoxia (-27.8 +/- 22.8% at 90% Pmax, P < 0.001) and hypoxia (-23.8 +/- 17.5% at 60% Pmax, P < 0.001). At 90% Pmax in normoxia, EIH athletes showed lower PaO2 (76.7 +/- 5.4 vs. 82.8 +/- 4.4 mmHg, P = 0.013) and greater FENO(0.170) decrement (-37.0 +/- 24.7% vs. -16.6 +/- 14.6%, P = 0.042) than NEIH athletes. During hypoxic exercise, P(a)O(2) and FENO(0.170) decreases were similar in both groups (P > 0.05). CONCLUSION: The present study shows lower pulmonary NO in athletes with gas exchange abnormalities during intense exercise in normoxia, while EIH and NEIH athletes have similar decreases in blood gases and pulmonary NO during hypoxic exercise. Decreased pulmonary NO in such conditions may contribute to ventilation-perfusion inequality and/or increase pulmonary vascular tone in athletes.     

Nitric oxide contributes to resistance of the Brown Norway rat to experimental autoimmune encephalomyelitis

The Brown Norway (BN) rat is reported to be resistant to the induction of experimental autoimmune encephalomyelitis (EAE) and a number of mechanisms have been suggested to explain this resistance. In work reported here we provide evidence that such resistance in the BN rat can be accounted for, at least in part, by their ability to produce higher levels of nitric oxide (NO) than susceptible strains of rats. Spleen cells from the BN rat make significantly more NO following in vitro stimulation than do cells from the Lewis or PVG rat and following in vivo immunization using complete Freund's adjuvant (CFA) the BN rat makes substantially more NO than either susceptible strain. If carbonyl iron is used as adjuvant in vivo there is no increase in NO levels in the BN rat and they are rendered highly susceptible to EAE. Immunizing with CFA simultaneously with neuroantigen and carbonyl iron drives up NO levels and the resistance is restored. EAE produced using carbonyl iron is characterized by extensive macrophage/microglia presence in the central nervous system lesions of the BN rat yet the cytokine profile in the lymph nodes does not differ from that in the EAE Lewis rats.     

Nitric oxide contributes to learning and memory deficits observed in hypothyroid rats during neonatal and juvenile growth

INTRODUCTION:

Severe cognitive impairment follows thyroid hormone deficiency during the neonatal period. The role of nitric oxide (NO) in learning and memory has been widely investigated.

METHODS:

This study aimed to investigate the effect of hypothyroidism during neonatal and juvenile periods on NO metabolites in the hippocampi of rats and on learning and memory. Animals were divided into two groups and treated for 60 days from the first day of lactation. The control group received regular water, whereas animals in a separate group were given water supplemented with 0.03% methimazole to induce hypothyroidism. Male offspring were selected and tested in the Morris water maze. Samples of blood were collected to measure the metabolites of NO, NO₂, NO₃ and thyroxine. The animals were then sacrificed, and their hippocampi were removed to measure the tissue concentrations of NO₂ and NO₃.

DISCUSSION:

Compared to the control group''s offspring, serum thyroxine levels in the methimazole group''s offspring were significantly lower (P<0.01). In addition, the swim distance and time latency were significantly higher in the methimazole group (P<0.001), and the time spent by this group in the target quadrant (Q1) during the probe trial was significantly lower (P<0.001). There was no significant difference in the plasma levels of NO metabolites between the two groups; however, significantly higher NO metabolite levels in the hippocampi of the methimazole group were observed compared to controls (P<0.05).

CONCLUSION:

These results suggest that the increased NO level in the hippocampus may play a role in the learning and memory deficits observed in childhood hypothyroidism; however, the precise underlying mechanism(s) remains to be elucidated.     

Nitric oxide is not permissive for cutaneous active vasodilatation in humans

The precise role of nitric oxide (NO) in cutaneous active vasodilatation in humans is unknown. We tested the hypothesis that NO is necessary to permit the action of an unknown vasodilator. Specifically, we investigated whether a low-dose infusion of exogenous NO, in the form of sodium nitroprusside (SNP), would fully restore vasodilatation in an area of skin in which endogenous NO was inhibited during hyperthermia. This finding would suggest a 'permissive' role for NO in active vasodilatation. Eight subjects were instrumented with three microdialysis fibres in forearm skin. Sites were randomly assigned to (1) Site A: control site; (2) Site B: NO synthase (NOS) inhibition during established hyperthermia; or (3) Site C: NOS inhibition throughout the protocol. Red blood cell flux was measured using laser-Doppler flowmetry (LDF) and cutaneous vascular conductance (CVC; LDF/mean arterial pressure) was normalized to maximal vasodilatation at each site. In Site B, N ^G-nitro- l -arginine methyl ester ( l -NAME) infusion during hyperthermia reduced CVC by ∼32 % (65 ± 4 % CVC_max vs. 45 ± 4 % CVC_max; P < 0.05). Vasodilatation was not restored to pre-NOS inhibition values in this site following low-dose SNP infusion (55 ± 4 % CVC_max vs. 65 ± 4 % CVC_max; P < 0.05). CVC remained significantly lower than the control site with low-dose SNP infusion in Site C ( P < 0.05). The rise in CVC with low-dose SNP (ΔCVC) was significantly greater in Site B and Site C during hyperthermia compared to normothermia ( P < 0.05). No difference in ΔCVC was observed between hyperthermia and normothermia in the control site (Site A). Thus, NO does not act permissively in cutaneous active vasodilatation in humans but may directly mediate vasodilatation and enhance the effect of an unknown active vasodilator.     

Nitric oxide and prostaglandin pathways interact in the regulation of hypercapnic cerebral vasodilatation

To test whether nitric oxide and prostaglandin pathways interact in hypercapnic cerebral vasodilatation, cerebral blood flow (CBF) was measured in enflurane anaesthetized Sprague–Dawley rats using the hydrogen clearance method. Isometric tension was measured in rat middle cerebral arteries in vitro. The neuronal NO synthase inhibitor 7-nitroindazole (7-NI 60 mg kg^–1 i.p.) reduced the hypercapnic CBF response by 62 ± 7% (but not the hypoxic response) and indomethacin (IMC 6 mg kg^–1 i.v.) reduced the hypercapnic CBF response by 60 ± 5%. Combined application caused only an 80 ± 1% reduction. The attenuation of hypercapnic CBF by IMC was diminished by 7-NI and similarly 7-NI had less effect in the presence of IMC. Spermine-NO (50 μM 0.5 μL min^–1 intracortically) increased eucapnic and hypercapnic CBF in the presence of IMC. In isolated middle cerebral arteries, combined application of sodium nitroprusside (SNP 3 n M ) and prostacyclin (30 n M ) had a synergistic vasodilatory effect. Milrinone (PDE-III inhibitor) also potentiated prostacyclin-mediated vasodilatation. Our results suggest that the NO- and IMC-sensitive pathways involved in the hypercapnic response are distinct, however, both may interact synergistically. A similar synergism was observed between the effects of SNP and prostacyclin.     

Nitric oxide contributes to induction of innate immune responses to gram-negative bacteria in Drosophila

Studies in mammals uncovered important signaling roles of nitric oxide (NO), and contributions to innate immunity. Suggestions of conservation led us to explore the involvement of NO in Drosophila innate immunity. Inhibition of nitric oxide synthase (NOS) increased larval sensitivity to gram-negative bacterial infection, and abrogated induction of the antimicrobial peptide Diptericin. NOS was up-regulated after infection. Antimicrobial peptide reporters revealed that NO triggered an immune response in uninfected larvae. NO induction of Diptericin reporters in the fat body required immune deficiency (imd) and domino. These findings show that NOS activity is required for a robust innate immune response to gram-negative bacteria, NOS is induced by infection, and NO is sufficient to trigger response in the absence of infection. We propose that NO mediates an early step of the signal transduction pathway, inducing the innate immune response upon natural infection with gram-negative bacteria.     

Rapidity of responding to a hypoxic challenge during exercise

The ability to modify power output (PO) in response to a changing stimulus during exercise is crucial for optimizing performance involving an integration system involving a performance template and feedback from peripheral receptors. The rapidity with which PO is modified has not been established, but would be of interest relative to understanding how PO is regulated. The objective is to determine the rapidity of changes in PO in response to a hypoxic challenge, and if change in PO is linked to changes in arterial O₂ saturation (S _aO₂). Well-trained cyclists performed randomly ordered 5-km time trials. Subjects began the trials breathing room air and switched to hypoxic (HYPOXIC, F_IO₂ = 0.15) or room (CONTROL, F_IO₂ = 0.21) air at 2 km, then to room air at 4 km. The time delay to begin decreasing S _aO₂ and PO and to recover S _aO₂ and PO on to room air was compared, along with the half time (t _1/2) during the HYPOXIC trial. Mean S _aO₂ and PO between 2 and 4 km were significantly different between CONTROL and HYPOXIC (94 ± 2 vs. 83 ± 2% and 285 ± 16 vs. 245 ± 19 W, respectively). There was no difference between the time delay for S _aO₂ (31.5 ± 12.8 s) and in PO (25.8 ± 14.4 s) or the recovery of S _aO₂ (29.0 ± 7.7 s) and PO (21.5 ± 12.4 s). The half time for decreases in S _aO₂ (56.6 ± 14.4 s) and in PO (62.7 ± 20.8 s) was not significantly different. Modifications of PO due to the abrupt administration of hypoxic air are related to the development of arterial hypoxemia, and begin within ~30 s.     

Nitric oxide deficiency contributes to impairment of airway relaxation in cystic fibrosis mice

The pulmonary disease of cystic fibrosis (CF) is characterized by persistent airway obstruction, which has been attributed to chronic endobronchial infection and inflammation. The levels of exhaled nitric oxide (NO) are reduced in CF patients, which could contribute to bronchial obstruction through dysregulated constriction of airway smooth muscle. Because airway epithelium from CF mice has been shown to have reduced expression of inducible NO synthase, we examined airway responsiveness and relaxation in isolated tracheas of CF mice. Airway relaxation as measured by percent relaxation of precontracted tracheal segments to electrical field stimulation (EFS) and substance P, a nonadrenergic, noncholinergic substance, was significantly impaired in CF mice. The airway relaxation in response to prostaglandin E2 was similar in CF and non-CF animals. Treatment with the NO synthase inhibitor NG-nitro-L-arginine methylester reduced tracheal relaxation induced by EFS in wild-type animals but had virtually no effect in the CF mice. Conversely, exogenous NO and L-arginine, a NO substrate, reversed the relaxation defect in CF airway. We conclude that the relative absence of NO compromises airways relaxation in CF, and may contribute to the bronchial obstruction seen in the disease.     

Nitric oxide and prostaglandin pathways interact in the regulation of hypercapnic cerebral vasodilatation.

To test whether nitric oxide and prostaglandin pathways interact in hypercapnic cerebral vasodilatation, cerebral blood flow (CBF) was measured in enflurane anaesthetized Sprague-Dawley rats using the hydrogen clearance method. Isometric tension was measured in rat middle cerebral arteries in vitro. The neuronal NO synthase inhibitor 7-nitroindazole (7-NI 60 mg kg-1 i.p.) reduced the hypercapnic CBF response by 62 +/- 7% (but not the hypoxic response) and indomethacin (IMC 6 mg kg-1 i.v.) reduced the hypercapnic CBF response by 60 +/- 5%. Combined application caused only an 80 +/- 1% reduction. The attenuation of hypercapnic CBF by IMC was diminished by 7-NI and similarly 7-NI had less effect in the presence of IMC. Spermine-NO (50 microM 0.5 microL min-1 intracortically) increased eucapnic and hypercapnic CBF in the presence of IMC. In isolated middle cerebral arteries, combined application of sodium nitroprusside (SNP 3 nM) and prostacyclin (30 nM) had a synergistic vasodilatory effect. Milrinone (PDE-III inhibitor) also potentiated prostacyclin-mediated vasodilatation. Our results suggest that the NO- and IMC-sensitive pathways involved in the hypercapnic response are distinct, however, both may interact synergistically. A similar synergism was observed between the effects of SNP and prostacyclin.     

Nitric oxide (NO) in expired air at rest and during exercise

Nitric oxide (NO) was analysed in expired air from 27 healthy human subjects. At rest the NO concentration was 10.5 ± 0.9 ng 1^-1 (mean ± SEM) corresponding to 8.6 ± 0.7 parts per billion (ppb). The expired NO concentration did not change when the subjects were switched from breathing NO-free tank gas to room air which contained 7.7 ng 1^-1 NO. Repeated measurements of expired NO with an interval of 1 day showed a mean variation of 2.2 ± 0.7ngl^-1 NO. The NO concentration in the first portion in the expired tidal volume (44%) was insignificantly higher than in the latter expired portion, 6.9 ± 1.9 vs. 5.1 ± 1.0 ng 1^-1 (n= 5). During moderately heavy exercise on an ergometer bicycle (90 W for women, n= 4, 150 W for men, n= 4) the expired concentration of NO decreased, however because of increased minute ventilation, the expired amount of NO almost doubled (from 111 ± 12 to 209 ± 30 ng min^-1). The source of the expired NO is not clear and both the airways and the pulmonary circulation may contribute.     

The pattern of breathing during hypoxic exercise

Summary Breathing pattern was studied in six subjects in normoxia (F_IO₂=0.21) and hypoxia (F_IO₂=0.12) at rest and during incremental work-rate exercise. Ventilation (V) as well as mean inspiratory flow (V_T/T_I) increased with exercise intensity and were augmented in the hypoxic environment, whereas the ratio between inspiratory (T_I) and total (T_tot) breath durations increased with exercise intensity but was unaffected by hypoxia. The relationship of tidal volume (V_T) and inspiratory time duration (T_I) showed linear, coinciding ranges for the normoxic and hypoxic conditions up to V_T/T_I values of about 2.5 l · s⁻¹. At higher V_T/T_I values T_I continued to decrease, whereas V_T tended to level off, an effect which was more evident in the hypoxic condition. The results suggest that the hypoxic augmentation of exercise hyperpnea is primarily brought about by an enhancement of central inspiratory drive, the timing component being largely unaffected by the hypoxic environment, and that at low to moderate levels of exercise hyperpnea inspiratory off-switch mechanisms are essentially unaffected by moderate hypoxia.     

Nitric oxide modulates acetylcholine-induced vasodilatation in the hepatic arterial vasculature of the dual-perfused rat liver.

The role of nitric oxide in the modulation of hepatic arterial vascular reactivity was investigated in an isolated dual-perfused rat liver preparation. Twelve male Wistar rats (200-250 g) were anaesthetized with sodium pentobarbitone (60 mg kg-1 i.p.). The livers were then excised and perfused in vitro through hepatic arterial and portal venous cannulae at constant flow rates. Concentration-dependent dose-response curves to acetylcholine (10(-8)-10(-5) M), sodium nitroprusside (10-6(-5) x 10(-4) M), and adenosine triphosphate (ATP) (10(-8)-10(-5) M) in the hepatic artery were constructed after the tone was raised by addition of methoxamine (3 micorM L(-1)). Acetylcholine-induced vasodilatation in the hepatic artery was significantly attenuated with inhibition of nitric oxide synthase by using NG-nitro-L-arginine methyl ester (30 microM), Emax = 51.7 +/- 2.8 vs. 32.5 +/- 3.1 mmHg, before vs. after NG-nitro-L-arginine methyl ester, respectively. ATP-induced hepatic arterial vasoconstriction which was significantly enhanced with L-NAME, Emax = 94.0 +/- 9.3 vs. 127.0 +/- 8.0 mmHg, before vs. after NG-nitro-L-arginine methyl ester, respectively. Sodium nitroprusside-induced hepatic arterial vasodilatation remained unchanged with NG-nitro-L-arginine methyl ester, Emax = 57.0 +/- 3.4 vs. 57.0 +/- 4.1, before vs. after NG-nitro-L-arginine methyl ester, respectively. The data from the present study suggest that acetylcholine-induced vasodilatation in the intrahepatic arterial vasculature of the rat liver is at least, in part, mediated by the release of nitric oxide. In addition, ATP-induced hepatic arterial vasoconstriction is also modulated by the release of nitric oxide (*P < 0.05, Student's paired t-test).     

Nitric oxide (NO) does not contribute to the generation or action of adenosine during exercise hyperaemia in rat hindlimb

Exercise hyperaemia is partly mediated by adenosine A_2A-receptors. Adenosine can evoke nitric oxide (NO) release via endothelial A_2A-receptors, but the role for NO in exercise hyperaemia is controversial. We have investigated the contribution of NO to hyperaemia evoked by isometric twitch contractions in its own right and in interaction with adenosine. In three groups of anaesthetized rats the effect of A_2A-receptor inhibition with ZM241385 on femoral vascular conductance (FVC) and hindlimb O₂ consumption     at rest and during isometric twitch contractions (4 Hz) was tested (i) after NO synthase inhibition with l- NAME, and when FVC had been restored by infusion of (ii) an NO donor (SNAP) or (iii) cell-permeant cGMP. Exercise hyperaemia was significantly reduced (32%) by l- NAME and further significantly attenuated by ZM241385 (60% from control). After restoring FVC with SNAP or 8-bromo-cGMP, l- NAME did not affect exercise hyperaemia, but ZM241385 still significantly reduced the hyperaemia by 25%. There was no evidence that NO limited muscle     during contraction. These results indicate that NO is not required for adenosine release during contraction and that adenosine released during contraction does not depend on new synthesis of NO to produce vasodilatation. They also substantiate our general hypothesis that the mechanisms by which adenosine contributes to muscle vasodilatation during systemic hypoxia and exercise are different: we propose that, during muscle contraction, adenosine is released from skeletal muscle fibres independently of NO and acts directly on A_2A-receptors on the vascular smooth muscle to cause vasodilatation.     

Does nitric oxide allow endothelial cells to sense hypoxia and mediate hypoxic vasodilatation?in vivo and in vitro studies

Hypoxia-evoked vasodilatation is a fundamental regulatory mechanism that is often attributed to adenosine. The identity of the O₂ sensor is unknown. Nitric oxide (NO) inhibits endothelial mitochondrial respiration and ATP generation by competing with O₂ for its binding site on cytochrome oxidase. We proposed that in vivo this interaction allows endothelial cells to release adenosine when O₂ tension falls or NO concentration increases. Using anaesthetised rats, we confirmed that the increase in femoral vascular conductance (FVC, hindlimb vasodilatation) evoked by systemic hypoxia is attenuated by NO synthesis blockade with l -NAME, but restored when baseline FVC is restored by infusion of NO donor. This 'restored' hypoxic response, like the control hypoxic response, is inhibited by the adenosine A₁ receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine. Similarly, the FVC increase evoked by adenosine infusion was attenuated by l -NAME but restored by infusion of NO donor. However, when baseline FVC was restored after l -NAME with 8-bromo-cGMP, the FVC increase evoked by adenosine infusion was restored, but not in response to systemic hypoxia, suggesting that adenosine was no longer released by hypoxia. Infusion of NO donor at a given rate after treatment with l -NAME evoked a greater FVC increase during systemic hypoxia than during normoxia, both responses being reduced by 8-cyclopentyl-1,3-dipropylxanthine. Finally, both bradykinin and NO donor released adenosine from superfused endothelial cells in vitro ; l -NAME attenuated only the former response. We propose that in vivo , shear-released NO increases the apparent K _m of endothelial cytochrome oxidase for O₂, allowing the endothelium to act as an O₂ sensor, releasing adenosine in response to moderate falls in O₂.     

Nitric oxide requirement for vasomotor nerve-induced vasodilatation and modulation of resting blood flow in muscle microcirculation

Intravital microscopy of rabbit tenuissimus muscle was used for studies of endogenous nitric oxide as a microvascular regulator in vivo. Derivatives of arginine were administered in order to modulate the formation of nitric oxide from L-arginine. N omega-nitro-L-arginine methylester (L-NAME) (1-100 mg kg-1 i.v.) dose-dependently reduced microvascular diameters. A concomitant blood pressure increase and a decrease in heart rate was observed. The blood pressure increase induced by L-NAME (30 mg kg-1) was reversed by L-arginine (1 g kg-1) but not D-arginine. Vasodilation in response to topical acetylcholine (0.03-3 microM) was significantly inhibited by L-NAME (30 mg kg-1), whereas vasodilation by sodium nitroprusside (300 nM) was not affected. Vasomotor nerve-induced vasodilatation, induced by stimulation of the tenuissimus nerve after neuromuscular blockade by pancuronium in animals pretreated with guanethidine, was significantly attenuated by L-NAME, an effect also reversed by L-arginine. The vasodilatation in response to active contractions of the muscle induced by motor nerve stimulation as well as the vasodilator response elicited by graded perfusion pressure reductions were unaffected by L-NAME or NG-monomethyl-L-arginine (L-NMMA, 10(-4) M) administered topically. Our results indicate that endogenous nitric oxide formed from L-arginine is a modulator of microvascular tone in vivo. Furthermore, the results suggest that endogenous nitric oxide is required for vasomotor nerve-induced vasodilatation, whereas it does not appear to play a role in myogenic vasodilatation or functional hyperaemia in this tissue.     

Nitric oxide synthase activity in rat gastric mucosa contributes to mucin synthesis elicited by calcitonin gene-related peptide

The majority of research for the calcitonin gene-related peptide (CGRP) in the stomach has been devoted to the submucosal blood flow, and only slight attention has been paid to its involvement in the gastric epithelial function. In this study, we examined the age-related change in the CGRP-containing nerves and its effects on the mucus metabolism. We compared the immunoreactivity for CGRP in the gastric mucosa of 7-week-old rats (young) to that of 52-week-old animals (middle-aged). The effects of CGRP on the mucin biosynthesis were compared using the stomachs from both young and middle-aged rats. The nitric oxide synthase (NOS) activity was measured in the surface and deep mucosa of the gastric corpus. The density of the CGRP nerve fibers was reduced in both the lamina propria and submucosa of the middle-aged rats compared to the young rats. CGRP stimulated the mucin biosynthesis in the cultured corpus mucosa from the 7-week-old rats, but not from the 52-week-old rats. The total NOS activity of the surface layer in the corpus mucosa was markedly reduced in the middle-aged rats compared to the young rats. These findings demonstrate the age-dependent reduction in the CGRP-induced mucin biosynthesis, as well as in the density of the CGRP fibers in the rat stomach. The decreased NOS activity in the surface layer of the oxyntic mucosa in the aged rats may also be a principal cause for the lack of regulation of the mucin biosynthesis by CGRP.