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Hypoxia reduces eNOS expression in hypoxic rat placentas.
Physiological Research in press; 2005.
Hypoxia in fetoplacental vessels is believed to be one of the most important factors in the pathogenesis of fetal and neonatal morbidity, such as intrauterine growth retardation.
Chronic hypoxia regulates the expression of endothelial NO synthase in different species and tissues. Typically the expression of endothelial NO synthase (eNOS) rises under chronic conditions. The expression in rat placenta has never been determined in hypoxia. The aim of our study was to analyze the effect of chronic hypoxia on the expression of endothelial NO synthase (eNOS) in rat placenta.
Female rats were exposed to hypoxia (10% O2) for the last 10 days of pregnancy. Control group of rats remaind in normoxia. Experiments were conducted in accordance with the Guide for the Care and Use od Laboratory Animals as adopted and promulgated by U.S. National Institues of Health (agreement number B 67 900). One day before the calculated day of delivery placentas were removed under thiopental anesthesia (60 mg/kg). Placentas were rinsed in saline solution and immediately homogenized. eNOS expression was determined by western blot with immunodetection using rabbit anti eNOS (St. Cruise). The results were quantify by densitometry.
We found significantly lower expression of eNOS in hypoxic placentas compared with normoxic controls.
We suppose, that lower expression of eNOS could contribute to the lower blood flow through the hypoxic placenta and deterioration of fetal blood oxygenation and nutrition.
Supported by GAUK 55/2001/C and GAUK 82/2004/C
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Bíbová J., Hniličková O., Hampl V. and Herget J.:
Perinatal hypoxia aggravates pulmonary hypertension in adulthood.
FASEB Journal 2005.
Chronic hypoxia (CH) induces hypoxic pulmonary hypertension, which appears to result from lung vascular injury in the initial days of hypoxia. Since we have shown that hypoxia at the time of birth can have long-lasting consequences for the pulmonary circulation, we hypothesized in this study that CH-induced lung vascular injury and pulmonary hypertension is aggravated in rats that had experienced perinatal hypoxia (PH). Male rats born in hypoxia (n=5) and hypoxia (n=6) were raised in normoxia and exposed to 10%O2 for 5 days when adult. Baseline perfusion pressure in isolated, constant flow (salt solution with 4% albumin) perfused lungs was higher in PH (14.2±1.1 mmHg) than control (6.9±0.2 mmHg, p<0.001) rats. Perfusion pressure-flow relationship was steeper and shifted to higher pressures in the PH than the control group, indicating increased vascular resistance. In addition, vasoconstrictor reactivity to angiotensin II bolus (0.2 ug) was also increased. We conclude that PH aggravates pulmonary hypertension development upon re-exposure to CH in adulthood.
Supported by and GAUK305/05/0672 and Research project No.1130002.
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Herget J., Vajner L. and Hampl V.:
Collagenase expressing mast cells accummulate in walls of peripheral pulmonary arteries in the initiation of hypoxic pulmonary hypertension.
Journal of Physiology in press: 2005.
Chronic hypoxia results in pulmonary hypertension due to vasoconstriction and structural remodeling of peripheral lung blood vessels. We hypothesize that vascular remodeling is initiated in the walls of prealveolar pulmonary arteries by collagenolytic metalloproteinases (MMP) released from activated mast cells. Distribution of mast cells and their expression of interstitial collagenase, MMP-13, in lung conduit, small muscular, and prealveolar arteries was determined quantitatively in lungs of rats exposed for 4 (n=10) and 20 (n=8) days to hypoxia (10 % 02) or normoxia (n=10). Mast cells were identified using Toluidine Blue staining, and MMP-13 expression was detected using monoclonal antibody. After 4, but not after 20 days of hypoxia, a significant (ANOVA) increase in the number of mast cells and their MMP-13 expression was found within walls of prealveolar arteries. In rats exposed for 20 days, MMP-13 positive mast cells accumulated within the walls of conduit arteries and subpleurally and total number of mast cells was significantly higher than after 4 days exposure and in normoxic controls. These data support the hypothesis that perivascular pulmonary mast cells contribute to the vascular remodeling in hypoxic pulmonary hypertension in rats by releasing interstitial collagenase.
Supported by grant GAČR 304/02/1348.
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Hodyc D., Hnilicková O., Hampl V. and Herget J.:
Heparinization but not ventilation during warm ischemia improves lung function in non-heart-beating donors.
FASEB Journal in press: 2005.
Lungs retrieved from non-heart-beating donors (NHBD) may alleviate the critical shortage of suitable organs for transplantation. We tested the role of pre-arrest heparinization and ventilation during storage for lung viability and respiratory functions.
Adult male Wistar rats were divided into 5 experimental and 1 control group. All experimental groups underwent the protocol of NHBD lung harvesting: 1 hour of warm ischemia after pentobarbital euthanasia followed with 90 min of cold ischemia. The groups were: I: nitrogen ventilated during warm ischemia, heparin added, II: room-air ventilated, heparin, III: room-air ventilated, no heparin, IV: non-ventilated, heparin, V: non-ventilated, no heparin, VI: control group without warm and cold ischemia. Using isolated lungs we measured perfusion pressure, weight, and arterio - venous difference in O2 partial pressure (D PO2).
All ventilated groups showed significantly worse viability with development of pulmonary oedema. Perfusion pressure and lung weight did not differ between controls and non-ventilated, heparinized group, while perfusion pressure of non-heparinized lungs was significantly elevated. D PO2 of control group was significantly higher than in both ventilated groups.
Pre-arrest heparinization without ventilation during warm ischemia is a suitable technique for NHBD lungs preservation.
Supported by IGA MZCR ND 7453-3
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Hodyc D., Hniličková O., Hampl V. and Herget J.:
Pre-arrest administration of cell permeable ros scavenger – tempol, reduces warm ischemic damage of lung function in non-heart-beating donors.
Journal of Physiology in press: 2005.
Background:
To use lungs retrieved from non-heart-beating donors (NHBD) in a safe way there is necessary to design an optimal method of their preservation during warm ischemia. One of the most deteriorative effects of warm ischemia is caused by ROS activity. Therefore we investigated a possible protective effect of cell permeable ROS scavenger, Tempol, on the function of NHBD grafts.
Methods:
4 groups (n = 6) of Wistar male rats underwent experimental protocol of lung harvesting from NHBD. After pre-arrest administration of heparin, rats were euthanized by pentobarbital and then left untouched for 60 minutes (warm ischemia) followed by 90 minutes of cold ischemia provided by intrapleural in-situ topical cooling. Group I: non-ventilated during warm ischemia, group II: non-ventilated, Tempol (100mg/kg b.w.) added pre-arrestly intraperitonealy, group III: room-air ventilated, group IV: room-air ventilated, Tempol. Controls were: group V (n = 6) and group VI (n = 6) with Tempol added, in both lungs harvested immediately under anaesthesia by sodium thiopental (4mg/kg added intraperitonealy) without warm and cold ischemia.
For functional assessment of all groups we used preparation of isolated ventilated rat lungs perfused with salt solution with Ficoll (4g/100ml) and meclofenamate (17 x 10-6 M). Perfusion pressure, lung weight gain and arterio – venous difference in oxygen partial pressure (PO2) were measured in time periods of 30, 90 120 and 180 minutes after beginning of perfusion. To model in-vivo conditions for oxygen transport, the perfusate was equilibrated with hypoxic gas mixture before entering the preparation.
For statistical evaluation we used ANOVA for repeated measures, Games/Howell post hoc test, p0,05.
Results:
We didn’t find any differences between controls (group V and VI). Almost all lungs (5 of 6) retrieved from room-air ventilated rats without Tempol (group III) developed pulmonary oedema by 30 minutes of isolated lung perfusion, in contrast to 100% survival in all other groups. There were no differences in perfusion pressure between these groups. We found significant increase in weight gain in non-ventilated lungs (group I) compared to non-ventilated with Tempol (group II) or room-air ventilated with Tempol (group IV) - see Fig.1. PO2 was significantly lower in non-ventilated lungs (group I) than in controls (group V); in contrast, there were no differences in oxygen transport ability between non-ventilated, room-air ventilated and control groups with Tempol - see Fig. 2.
Conclusion:
Pre-arrest administration of Tempol helps in protection of lung grafts retrieved from NHBD.
Supported by grant: IGA MZCR ND 7453-3, GAUK 45/2005/C
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Lachmannová V., Bíbová J., Miková D., Hniličková O. and Hampl V.:
Nitric oxide production into the exhaled air is elevated by 4 days of hypoxia in the lower, but not the upper, respiratory tract.
Physiological Research 54: 1P; 2005.
The exact role of nitric oxide (NO) in hypoxic pulmonary hypertension is incompletely understood. Exhaled NO provides a non-invasive means of assesing the NO signalling in the respiratory system. Exhaled NO quickly increases during chronic hypoxia. While under normal circumstances the exhaled NO comes mostly from the upper airways, the source of its elevation in hypoxia is unknown. Its determination was the objective of the present study.
Adult male Wistar rats were hept in hypoxic chamber (10% O2; n = 6) or room air (n = 7). After 4 days of hypoxia, all animals were intubated via a tracheostomy under thiopental anaesthesia (50 mg/kg i.p.). To assess total exhaled NO, they were placed into a sealed pletysmograph (2,1 l) and NO accumulation after 10 min was measured with chemiluminescence NO analyzer (CLD 77 AM, EcoPhysis, Switzerland). NO production in the nose and paranasal sinuses is large enough to support discernible diffusion efflux of NO even in the absence of nasal ventilation. To measure NO production into the exhaled air from the lung tissue and lower airways, the exhalate for NO measurement was then collected directly from the tracheal tube into a latex bag (250 ml).
The exhaled NO measured directly from the tracheal tube doubled in hypoxia (1.1 0.1 ppb) compared to the normoxic group (0.60.7 ppb, p=0.001). The difference between the tracheal tube and plethysmograph measurements, reflecting the contribution of the upper airways, did not differ between the hypoxic (1.00.5 ppb) and normoxic (0.90.3 ppb) groups (p=0.6).
Thus, the source of the elevated NO exhalation in hypoxia is the lower respiratory tract. While the contribution of the upper airways remains remarkable, it does not rise in hypoxia.
Supported by LN 00A069
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Baňasová A., Bíbová J., Miková D., Herget J. and Hampl V.:
Chronic hypoxia alters nitric oxide in exhaled breath: newborn rats differ from adults.
Physiological Research 53: 1P; 2004.
While studying the role of endogenous NO in the mechanism of chronic hypoxic pulmonary hypertension, we have found recently that chronic hypoxia elevates NO concentration in exhaled air of adult rats (1). As there are indications that the role of NO in the pulmonary circulation is profoundly different in neonates than in adults (2), we hypothesized that the effect of chronic hypoxia on exhaled NO is different in neonatal than in adult rats.
Rats were placed into a hypoxic chamber (10 % O2) 24 hr after birth and compared to controls kept in room air. Once every 1-2 days, the rats were removed from the chamber for ~ 1hr for NO measurement. Exhaled NO was measured as 15 min NO accumulation in a closed, initially NO-free flask (2.1 l) using a CLD 77 AM chemiluminescence NO analyzer (EcoPhysics, Dürnten, Switzerland).
During the first postnatal week, the exhaled NO was near the detection limit of the analyzer (~1 ppb) in both the normoxic and hypoxic pups. From day 8, exhaled NO was significantly lower in the hypoxic (0.46 ± 0.13 ppb/) than in the normoxic (2.51 ± 0.45 ppb) pups. However, when the rats were 14 days old, exhaled NO in the hypoxic group (6.03 ± 0.67 ppb) exceeded that in the normoxic controls (2.27 ± 0.32 ppb) and remained elevated for the rest of the exposure, resembling the situation in adult rats (1). The exhaled NO remained low in normoxic controls. When the hypoxic exposure was terminated on day 27, exhaled NO fell rapidly to low level similar to that in controls.
We conclude that chronic hypoxia reduces exhaled NO concentration in neonatal rats. By contrast, exhaled NO is elevated by chronic hypoxia in rats older than ~ 2 weeks. This developmental difference is likely to have implications in pulmonary hypertension.
Supported by MSMT CR 111300002.
(1) Bíbová J., et al.: Physiol. Res. 52:24P,2003.
(2) Hampl V., Herget J.: Physiol. Rev. 80:1337-1372,2000.
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Bobková D., Miková D., Hampl V., Kovář J. and Poledne R.:
NO production in an experimental model of hyperlipoproteinemia - apolipoprotein E-defficient mice.
Physiological Research 53: 2P; 2004.
Nitric oxide (NO) is a very important vasorelaxation factor. We tested the effect of diets and unselective and selective inhibition of inducible NO synthase by N ω-nitro-L-arginine methyl ester (L-NAME) and L-N6(1 iminoethyl)lysine (L-NIL), respectively, on the plasma NO concentrations of apolipoprotein E-deficient (apo E KO) mice. Homozygous (-/-) and heterozygous (+/-) apo E KO mice and wild-type mice (+/+) were used. Compared with +/- and +/+ mice, -/- mice have higher cholesterol concentrations (~ 2.3 mmol/L vs. ~11 mmol/L) and develop atherosclerotic changes. Whilst on cholesterol diet, cholesterolemia increased dramatically by ~100% in -/- mice, and only by ~30% in +/- and +/+ mice. When on chow diet, the NO concentrations in +/+ and +/- mice were comparable (1363 ppb and 2065 ppb, respectively) but were lower compared with -/- mice (4079 ppb). On cholesterol diet, NO concentrations rose in all groups; to 3917 ppb in +/+ mice, to 4985 ppb in +/- mice, and to 8868 ppb in -/- mice. Compared with chow diet, the NO concentrations on cholesterol diet with L-NAME decreased in -/- and +/- mice (606 ppb and 715 ppb), but remained unaltered in +/+ mice (1488 ppb). In -/- mice on cholesterol diet, the inhibition of NO production by L-NIL leads to a decrease in NO concentrations to levels (2492 ppb) below those seen on chow diet. We suggest that increasing plasma NO concentrations in -/- mice as an effect of higher NO production via inducible and endothelial NO synthase makes part of the protective mechanism against higher plasma cholesterol concentration and atherosclerotic vascular changes.
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Bíbová J., Baňasová A., Herget J. and Hampl V.:
Slowly reversible normalization of blood pressure by 2-week hypoxia in the ren-2 transgenic rat model of hypertension.
Physiological Research 53: 1P; 2004.
Chronic hypoxia reduces systemic hypertension in several experimental models. However, what happens with systemic arterial blood pressure after termination of the hypoxic exposure is not characterized. Therefore, we aimed to answer 2 questions: 1) Does chronic hypoxia reduce blood pressure also in a novel, genetically well characterized model of hypertension, the transgenic rat harboring mouse ren-2 renin gene; and 2) how does blood pressure change during recovery from chronic hypoxia?
As ren-2 transgenic homozygotes have high mortality, we used heterozygote (+/-) males. Systolic arterial pressure (SAP) was measured in conscious rats by tail plethysmography every few days. Each measurement was repeated 5 times. Starting at 66 days of age, rats were exposed to hypoxia (10% O2) for 2 weeks.
Just before the exposure to hypoxia, SAP was elevated in +/- rats (210 _ 3 mmHg) compared to wild-type controls (-/-; 145 _ 1 mmHg). SAP did not change appreciably during hypoxia in the -/- rats. In the +/- group, SAP started to drop in hypoxia, so that by day 3 it was significantly lower (195 _ 4 mmHg) than before the exposure. By day 14 of hypoxia, it did not differ from the normoxic -/- group (166 _ 3 vs. 161 _ 2 mmHg). Measurements in a separate set of rats anesthetized with thiopental confirmed that mean carotid artery pressure was lower in +/- rats at the end of 2-wk hypoxia (111 _ 7 mmHg) than in +/- rats kept in room air (151 _ 3mmHg) and did not differ from that in -/- rats (111 _ 5 mmHg). There were no significant differences among the groups in cardiac output estimated from ascending aorta blood flow measured in open-chest, ventilated animals. After cessation of hypoxia, SAP in the +/- rats rose first quickly (184 _ 2 mmHg on day 3 post hypoxia) and very slowly thereafter. Six weeks after hypoxia, SAP in the +/- rats (200 _ 4 mmHg) still did not quite reach the pre-hypoxic level.
Thus, hypoxia as brief as 2 weeks completely normalizes systemic hypertension in the ren-2 transgenic rat model, and this beneficial effect partly persists for a period several times longer than the exposure itself.
Supported by MSMT CR 111300002.
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Bíbová J., Baňasová A., Miková D., Hampl V. and Herget J.:
Inducible nitric oxide synthase particicates in the mechanism of hypoxic pulmonary hypertension.
Physiological Research 53: 2P; 2004.
Accumulating evidence suggests that a key event in the development of chronic hypoxic pulmonary hypertension is vascular wall injury during the first few days of hypoxia and that nitric oxide (NO) may participate in this process (1). In many tissues, NO produced by endothelial isoform of NO synthase (eNOS) acts as a vasodilator, whereas NO produced by the inducible isoform (iNOS) can cause tissue injury. We hypothesized that NO produced in the pulmonary circulation during the first few days of hypoxia by iNOS contributes to the vascular wall injury and thus promotes the development of pulmonary hypertension, while the eNOS activity throughout the hypoxic exposure may reduce vascular tone and thus limit pulmonary hypertension.
To test this hypothesis, we used NG-nitro-L-arginine methyl ester (L-NAME), a non-selective NOS inhibitor, and L-N6-(1-iminoethyl)lysine (L-NIL), a selective iNOS blocker. Rats were treated with L-NAME (500 mg/l) or L-NIL (8 mg/l) in drinking water either during the first or the last week of a 3-wk hypoxic exposure. Mean pulmonary artery pressure (PAP) was measured after 1 or 3 weeks of hypoxia in spontaneously breathing rats under thiopental anesthesia (40 mg/kg BW i.p).
In rats treated with L-NIL during the first week of hypoxia, PAP was reduced at the end of both 1-wk and 3-wk hypoxic exposure compared to untreated hypoxic controls. L-NIL treatment only during the last week of a 3-wk hypoxia had no significant effect, suggesting that iNOS activity during the first week promoted pulmonary hypertension. In support of this interpretation, rats treated with L-NAME during the first week of hypoxia had reduced PAP at the end of 1-wk hypoxia. However, their PAP was increased at the end of a 3-wk exposure, perhaps due to the known poor reversibility of the effect of L-NAME. PAP was also elevated in rats treated with L-NAME only during the last week of a 3-wk hypoxic exposure, implying that endogenous NO reduces vascular tone in pulmonary hypertension.
We conclude that iNOS activity in the first week of hypoxia contributes to the mechanism of pulmonary hypertension.
Supported by MSMT CR 111300002.
(1) Hampl V., Herget J.: Physiol. Rev. 80:1337-1372, 2000.
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Bíbová J., Hniličková O., Hampl V. and Herget J.:
Perinatal hypoxia results in increase of pulmonary vascular reactivity to hypoxic exposure in adult rats.
Physiological Research in press: 2004.
Rats born in hypoxia and than raised in normoxia have altered pulmonary vasoreactivity (1). Chronic hypoxia induces hypoxic pulmonary hypertension, which results from lung vascular injury in the first week of hypoxic exposure (2). We hypothesize that chronic hypoxia-induced lung vascular injury is enhanced in rats that experienced the period of perinatal hypoxia. Perinatally hypoxic male Wistar rats (exposed to 12 % O2 one week before and one week after birth and than living in air till the age of 6 months) were compared with control rats born and raised in normoxia. Both groups were exposed to 10 % O2 for 5 days when adult. At the end of the exposure, the lungs were isolated and perfused at constant flow rate with salt solution containing 5 % albumin. Rats born in hypoxia had significantly higher baseline perfusion pressure (perinatal hypoxia 14.2 ± 1.1 torr, controls 6.9 ± 0.2 torr, p<0.001). Vasoconstriction induced by a bolus injection of 0.4 _g angiotensin II was significantly greater in rats exposed to perinatal hypoxia whereas the acute hypoxic pulmonary vasoconstriction (ventilation with 0 % O2) was not altered. The pressure-flow (P/Q) relationships were linear in the studied range (4 – 28 ml/min). Slopes of the P/Q lines were significantly steeper in perinatally hypoxic rats, indicating an increased resistance to perfusion flow increments. Pressure axis intercept of the P/Q lines was also elevated in the experimental group, reflecting an increase in vascular closing pressure.
We conclude that some of the effects of perinatal hypoxia persist in male rats till adulthood. The reactivity of pulmonary vasculature in early phases of exposure to chronic hypoxia is increased in adult rats born in hypoxic environment.
(1) Hampl V and Herget J, Am Rev Respir Dis 142: 619-624, 1990.
(2) Herget J et al, Physiol. Res. 49: 493 - 501, 2000.
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Hampl V., Lachmanová V., Hniličková O. and Herget J.:
Critical role of oxygen radicals in the early phase of hypoxic pulmonary hypertension development.
FASEB Journal 18: A1056; 2004.
The mechanism of hypoxia-induced pulmonary hypertension is unclear. Oxidative injury to the pulmonary vascular wall appears important. To test the hypothesis that oxidative stress associated with the onset of hypoxia plays a key role in the development of pulmonary hypertension, we treated rats with an antioxidant, N-acetylcysteine (NAC, 20 g/l of drinking water), before and during hypoxic exposure (10% O2). Pulmonary hypertension was quantified by measuring pulmonary artery pressure (PAP) in closed chest, anesthetized (thiopental 40 mg/kg BW i.p), spontaneously breathing rats or as a slope of the pressure-flow relationship (P/Q slope) in isolated lungs perfused with Krebs-albumin (4%) solution. Four days of hypoxia significantly increased the P/Q slope. This increase was reduced in rats concurrently treated with NAC and completely prevented in rats given NAC either only for the last 4 days before the hypoxic exposure or both before and during hypoxia. The increase in PAP induced by 4 weeks of hypoxia was reduced in rats treated with NAC at the beginning of the exposure (7 days before and the first 7 days of hypoxia). NAC treatment during a relatively stable phase of pulmonary hypertension (last 2 weeks of 4-week exposure) was less effective in reducing PAP. These data show that oxygen radicals are essential in the pathogenesis of hypoxic pulmonary hypertension, particularly during the beginning of the hypoxic exposure.
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Kunes J., Miková D., Dobesová Z., Bíbová J., Hampl V. and Zicha J.:
Vasodilatory mechanisms in L-NAME hypertension.
Hypertension 44: 566; 2004.
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Banasová A., Bíbová J. and Hampl V.:
Gender difference in pulmonary vascular handling of intracellular calcium is abolished by orchiectomy.
Physiological Research 52: 23P; 2003.
Recently, we found gender differences in pulmonary vasoconstrictive responses mediated by calcium release from endoplasmic reticulum. The present study was therefore designed to look for the source of this variability. Adult male and female Wistar rats were either gonadectomized in deep ether anesthesia or left intact. Four weeks later, ventilated lungs were isolated under thiopental anesthesia and perfused ex vivo with Krebs-albumin solution. To minimize possible confounding influence of endothelial factors, the perfusate contained cyclooxygenase and nitric oxide synthase blockers. Thapsigargin or its solvent (DMSO) were added to the perfusate. Thapsigargin is a highly selective inhibitor of endoplasmic reticulum calcium ATP-ase, and is known to irreversibly deplete the reticulum of calcium. After measuring the vasoconstrictor responses to angiotensin II (A-II, 0.2mg bolus) and hypoxia (0% O2), the concentration of thapsigargin in the perfusate was increased from 10-9M to 10-8M and the measurements were repeated. While 10-9M thapsigargin had little effect on pulmonary vasoconstrion in all groups, 10-8M significantly inhibited the responses to A-II and hypoxia in females but not in males. Ovariectomy did not alter this finding. However, unlike in intact males, in lungs of orchiectomized males thapsigargin inhibited the responses to both A-II and hypoxia. Thus, the effect of thapsigargin in lungs of castrated males was similar to that in all females. We conclude that calcium from the endoplasmic reticulum participates in the pulmonary vasoconstriction much less in males than in females and that the testes are responsible for this gender difference.
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Bíbová J., Miková D., Baňasová A., Herget J. and Hampl V.:
Time profile of breath NO release during three week hypoxia.
Physiological Research 52: 24P; 2003.
Nitric oxide concentration may serve as a marker of NO production in lungs and airways. Expression of NO synthase (NOS) in airways and lung vessels increases in chronic hypoxia. Exposure to chronic hypoxia increases NO synthesis in lung vessels. As the effects of NO depend on its concentration, the current study was designed to determine a time profile of NO release during a 3 week exposure of rats to hypoxia.
Adult male Wistar rats were exposed to hypoxia (10%O2) for up to 19 days. NO concentration in exhaled breath was measured on days 1, 4, 11, 19 of the exposure. For each measurement, the rat was removed from the hypoxic chamber, placed in a body pletysmograph for 20 min to measure NO exhalation and then returned to the hypoxic chamber. Exhaled NO was measured by sampling the air from the pletysmograph into a CLD 77 AM chemiluminescence analyzer (EcoPhysis, Duernten, Switerland) at the end of the 20-min sojourn of the rat in the pletysmograph.
Exhaled NO rose dramatically during the first 4 days of the hypoxic exposure (from 0.183 ± 0.011 to 3,6 ± 0.925 ppb/min). With continuing hypoxia, the exhaled NO production fell to a level still higher than that found in normoxia (0.906 ± 0.225 ppb/min on day 7). Thereafter, the NO production did not change any more (0.769 ± 0.182 ppb/min on day 19).
We conclude that chronic hypoxia elevates NO production into the exhaled breath. This rise in breath NO levels are most prominent during the first week of hypoxia.
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Bíbová J., Miková D., Baňasová A., Herget J. and Hampl V.:
Inducible nitric oxide synthase contributes to the development of chronic hypoxic pulmonary hypertension.
FASEB Journal 17: A408; 2003.
The role of nitric oxide (NO) in pulmonary hypertension (PH) is controversial. Inhaled NO reduces chronic hypoxic PH in rats, but NO synthesis is elevated and plasma nitrotyrosine is increased in rats with hypoxic PH. We hypothesized that the involvement of NO in the mechanism of PH is biphasic. Initially, increased NO production contributes to the vascular wall injury and thus to PH development. Later, when PH stabilizes, the importance of NO-mediated injury fades away. Adult male Wistar rats were exposed to hypoxia for 7 or 19 days and treated either with non-selective NO synthase (NOS) inhibitor N G-nitro-L-arginine methyester (L-NAME, 500 mg/l) or selective inhibitor of inducible NOS (iNOS) L-N6-(1-iminoethyl)lysine (L-NIL, 8mg/l) in drinking water. In rats exposed to 1-week hypoxia, this treatment started 3 days before and continued till the end of the exposure. In animals exposed to 3-week hypoxia, L-NIL was given from day 11. Pulmonary arterial blood pressure (PAP), elevated by 1-week hypoxia (from 16±1mmHg in normoxic controls to 23±1mmHg in the hypoxic untreated group) was significantly reduced by L-NAME (19±1mmHg) or L-NIL (20±1mmHg). The elevation of PAP by 3-week hypoxia was not significantly affected by L-NIL treatment for the last week. We conclude that NO production by iNOS contributes to the initial development of hypoxic PH.
Supported by GACR 305/97/S070, 305/00/1432, and MSM 111300002.
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Kolár F., Szárszoi O., Neckár J., Pechánová O., Miková D., Hampl V. and Ostádal B.:
Role of nitric oxide and reactive oxygen species in reperfusion-induced arrhytmias and cardioprotection in chronically hypoxic rat hearts.
Physiological Research 52: 52P; 2003.
Adaptation of rats to intermittent high altitude (IHA) hypoxia increases the tolerance of their hearts to all major manifestations of acute ischemia/reperfusion injury. The mechanism of this protective effect remains still unclear. The aim of our study was to analyze the possible role of nitric oxide (NO) and reactive oxygen species (ROS) in the antiarrhythmic protection by IHA hypoxia. Adult male Wistar rats were exposed to IHA hypoxia of 5000 m in a barochamber (4 h/day, 5 days/week, 24-32 exposures). A control group was kept under normoxic conditions (200 m) for the same period of time. The severity of ventricular reperfusion arrhythmias was assessed by a 5-point score on isolated perfused hearts after 15-min regional ischemia. NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 200 µmol/l), NO donor S-nitrosoglutathione (GSNO, 10 µmol/l) and ROS scavengers tempol (1 mmol/l) or melatonin (10 µmol/l) were added to the perfusion solution 5 min before ischemia and were present throughout reperfusion. Concentration of NO and its oxidation products (nitrates, nitrites) in the coronary effluent was measured by a chemiluminiscence method. Parallel groups of animals were used for immunochemical detection of constitutive and inducible isoforms of NO synthase (eNOS and iNOS, respectively). In the normoxic group, the severity of reperfusion arrhythmias was significantly higher (score 4.04 ± 0.27) as compared with chronically hypoxic hearts (1.58 ± 0.38). L-NAME markedly reduced arrhythmias in controls (0.87 ± 0.28) but had no additional protective effect in the hypoxic group. In contrast, GSNO did not influence arrhythmias in controls but significantly increased the arrhythmia score in hypoxic animals (3.90 ± 0.42). Tempol and melatonin reduced reperfusion arrhythmias in the normoxic group (2.46 ± 0.69, 2.82 ± 0.58; respectively) and completely abolished antiarrhythmic protection in the hypoxic hearts (3.73 ± 0.51, 4.00 ± 0.32; respectively). IHA hypoxia increased myocardial expression of iNOS whereas the abundance of eNOS was reduced. Peak concentration of NO in the coronary effluent from reperfused hearts did not differ between the groups but the total production appeared to be increased in the IHA group. Our results suggest that endogenous NO contributes to reperfusion ventricular arrhythmias in isolated hearts of controls but not of chronically hypoxic rats; this difference cannot be explained by lower NO production by the hypoxic hearts. Exogenous NO is however proarrhythmic in the latter group. ROS appear to have a dual effect on cardiac susceptibility to arrhythmias: they are proarrhythmic in controls but play an essential role in the antiarrhythmic mechanism of chronic IHA hypoxia. Supported by GA CR 305/01/0279.
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