Recognition of intestinal epithelial HIF-1alpha activation by Pseudomonas aeruginosa

Human intestinal epithelial cell monolayers (Caco-2) subjected to hypoxia and reoxygenation release soluble factors into the apical medium that activate the virulence of the opportunistic pathogen Pseudomonas aeruginosa to express the potent barrier-dysregulating protein PA-I lectin/adhesin. In this study, we defined the role of hypoxia-inducible factor (HIF)-1alpha in this response. We tested the ability of medium from Caco-2 cells with forced expression of HIF-1alpha to increase PA-I expression in P. aeruginosa and found that medium from Caco-2 cells overexpressing HIF-1alpha increased PA-I expression compared with medium from control cells (P < 0.001, ANOVA). To identify the components responsible for this response, medium was fractionated by molecular weight and subjected to mass spectroscopy, which identified adenosine as the possible mediator. Both adenosine and its immediate downstream metabolite inosine induced PA-I expression in P. aeruginosa in a dose-dependent fashion. Because inosine was not detectable in the medium of Caco-2 cells exposed to hypoxia or overexpressing HIF-1alpha, we hypothesized that P. aeruginosa itself might metabolize adenosine to inosine. Using mutant and parental strains of P. aeruginosa, we demonstrated that P. aeruginosa metabolized adenosine to inosine via adenosine deaminase and that the conditioned medium enhanced the extracellular accumulation of inosine. Together, these results provide evidence that P. aeruginosa can recognize and respond to extracellular end products of intestinal hypoxia that are released after activation of HIF-1alpha. The ability of P. aeruginosa to metabolize adenosine to inosine may represent a subversive microbial virulence strategy that deprives the epithelium of the cytoprotective actions of adenosine.     

Neuroprotective role of delta-opioid receptors in cortical neurons

We recently demonstrated that delta-opioid receptor (DOR) activation protects cortical neurons against glutamate-induced injury. Because glutamate is a mediator of hypoxic injury in neurons, we hypothesized that DOR is involved in neuroprotection during O2 deprivation and that its activation/inhibition may alter neuronal susceptibility to hypoxic stress. In this work, we tested the effect of opioid receptor activation and inhibition on cultured cortical neurons in hypoxia (1% O2). Cell injury was assessed by lactate dehydrogenase release, morphology-based quantification, and live/dead staining. Our results show that 1) immature neurons (days 4 and 6) were not significantly injured by hypoxia until 72 h of exposure, whereas day 8 neurons were injured after only 24-h hypoxia; 2) DOR inhibition (naltrindole) caused neuronal injury in both day 4 and day 8 normoxic cultures and further augmented hypoxic injury in these neurons; 3) DOR activation ([D-Ala2,D-Leu5]enkephalin) reduced neuronal injury in day 8 cultures after 24 h of normoxic or hypoxic exposure and attenuated naltrindole-induced injury with prolonged exposure; and 4) mu- or kappa-opioid receptor inhibition (beta-funaltrexamine or nor-binaltorphimine) had little effect on neurons in either normoxic or hypoxic conditions. Collectively, these data suggest that DOR plays a crucial role in neuroprotection in normoxic and hypoxic environments.     

Hypoxia-induced pulmonary endothelin-1 expression is unaltered by nitric oxide.

Nitric oxide (NO) attenuates hypoxia-induced endothelin (ET)-1 expression in cultured umbilical vein endothelial cells. We hypothesized that NO similarly attenuates hypoxia-induced increases in ET-1 expression in the lungs of intact animals and reasoned that potentially reduced ET-1 levels may contribute to the protective effects of NO against the development of pulmonary hypertension during chronic hypoxia. As expected, hypoxic exposure (24 h, 10% O(2)) increased rat lung ET-1 peptide and prepro-ET-1 mRNA levels. Contrary to our hypothesis, inhaled NO (iNO) did not attenuate hypoxia-induced increases in pulmonary ET-1 peptide or prepro-ET-1 mRNA levels. Because of this surprising finding, we also examined the effects of NO on hypoxia-induced increases in ET peptide levels in cultured cell experiments. Consistent with the results of iNO experiments, administration of the NO donor S-nitroso-N-acetyl-penicillamine to cultured bovine pulmonary endothelial cells did not attenuate increases in ET peptide levels resulting from hypoxic (24 h, 3% O(2)) exposure. In additional experiments, we examined the effects of NO on the activity of a cloned ET-1 promoter fragment containing a functional hypoxia inducible factor-1 binding site in reporter gene experiments. Whereas moderate hypoxia (24 h, 3% O(2)) had no effect on ET-1 promoter activity, activity was increased by severe hypoxic (24 h, 0.5% O(2)) exposure. ET-1 promoter activity after S-nitroso-N-acetyl-penicillamine administration during severe hypoxia was greater than that in normoxic controls, although activity was reduced compared with that in hypoxic controls. These findings suggest that hypoxia-induced pulmonary ET-1 expression is unaffected by NO.     

Increased normoxic ventilation induced by repetitive hypoxia in conscious dogs.

To determine if a long-lasting increase in normoxic ventilatory drive is induced in conscious animals by repetitive hypoxia, we examined the normoxic [arterial O2 saturation (SaO2) > 93%] ventilatory response following successive episodes of 2-min eucapnic hypoxic challenges (SaO2 = 80%) in awake tracheotomized dogs. End-tidal CO2 was maintained at the resting level during and after repetitive hypoxia. The experimental protocol was performed twice in each of five dogs on separate days. To determine if changes in normoxic ventilation occurred between episodes of repetitive hypoxia, data were compared from six periods (epochs) for all experiments. The mean minute ventilation (VI) during three normoxic periods between episodes of intermittent hypoxia was 135, 154, and 169% of control (P < 0.05). VI during a 30-min recovery period was still higher at 183 and 172% of control (P < 0.05). Normoxic VI between hypoxic and recovery periods was significantly higher than the corresponding values in sham experiments. Our results indicate that a long-lasting increase in normoxic ventilation can be evoked in an awake unanesthetized dog by a short exposure to repetitive hypoxia.     

Basal expression of copper transporter 1 in intestinal epithelial cells is regulated by hypoxia-inducible factor 2α

Hypoxia, via stabilization of HIF2α, regulates the expression of the intestinal iron transporters DMT1 and ferroportin. Here we investigated whether the intestinal copper importer Ctr1 was also regulated by hypoxia. Copper uptake and Ctr1 mRNA expression were significantly increased in Caco-2 cells exposed to hypoxia. To determine whether HIF2α was involved in regulation of Ctr1 expression, we employed three models of HIF2α knockdown (chemical suppression of HIF2α translation in Caco-2 cells; HIF2α-siRNA-treated HuTu80 cells; HIF2α-intestinal knockout mice); Ctr1 mRNA expression was decreased in all three models under normoxic conditions. HIF2α translational inhibitor did not alter Ctr1 expression under hypoxic conditions. We conclude that basal expression of Ctr1 is regulated by HIF2α; however, the induction by hypoxia is a HIF2α-independent event.     

Increased myofibrillar protein phosphatase-1 activity impairs rat aortic smooth muscle activation after hypoxia

We hypothesized that increased myofibrillar type 1 protein phosphatase (PP1) catalytic activity contributes to impaired aortic smooth muscle contraction after hypoxia. Our results show that inhibition of PP1 activity with microcystin-LR (50 nmol/l) or okadaic acid (100 nmol/l) increased phenylephrine- and KCl-induced contraction to a greater extent in aortic rings from rats exposed to hypoxia (10% O(2)) for 48 h than in rings from normoxic animals. PP1 inhibition also restored the level of phosphorylation of the 20-kDa myosin light chain (LC(20)) during maximal phenylephrine-induced contraction to that observed in the normoxic control group. Myofibrillar PP1 activity was greater in aortas from rats exposed to hypoxia than in normoxic rats (P < 0.05). Levels of the protein myosin phosphatase-targeting subunit 1 (MYPT1) that mediates myofibrillar localization of PP1 activity were increased in aortas from hypoxic rats (193 +/- 28% of the normoxic control value, P < 0.05) and in human aortic smooth muscle cells after hypoxic (1% O(2)) incubation (182 +/- 18% of the normoxic control value, P < 0.05). Aortic levels of myosin light chain kinase were similar in normoxic and hypoxic groups. In conclusion, after hypoxia, increased MYPT1 protein and myofibrillar PP1 activity impair aortic vasoreactivity through enhanced dephosphorylation of LC(20).     

缺氧时培养的肺动脉平滑肌细胞血管紧张素Ⅱ自分泌的变化及其机制

缺氧是否通过影响血管平滑肌细胞的自分泌功能而参与缺氧性肺动脉高压的发生尚不清楚。本实验动态了缺氧对培养的新生小牛肺动脉平滑肌细胞（ＰＡＳＭＣｓ）的血管紧张素Ⅱ（ＡＴⅡ）分泌的影响。结果发现：２．５％Ｏ２缺氧导致ＰＡＳＭＣｓ的ＡＴⅡ分泌降低,０％Ｏ２缺氧进一步抑制ＡＴⅡ分泌。常氧条件下,ＮＯ供体ＳＩＮ－１显著的抑制ＡＴⅡ分泌,而ＮＯ合酶凶制剂硝基精氨酸（ＬＮＡ）则能消除缺氧对ＡＴⅡ分泌的抑制作用。０     

Exercise delays the hypoxic thermal response in rats.

Exercise exacerbates acute mountain sickness. In infants and small mammals, hypoxia elicits a decrease in body temperature (Tb) [hypoxic thermal response (HTR)], which may protect against hypoxic tissue damage. We postulated that exercise would counteract the HTR and promote hypoxic tissue damage. Tb was measured by telemetry in rats (n = 28) exercising or sedentary in either normoxia or hypoxia (10% O2, 24 h) at 25 degrees C ambient temperature (Ta). After 24 h of normoxia, rats walked at 10 m/min on a treadmill (30 min exercise, 30 min rest) for 6 h followed by 18 h of rest in either hypoxia or normoxia. Exercising normoxic rats increased Tb ( degrees C) vs. baseline (39.68 +/- 0.99 vs. 38.90 +/- 0.95, mean +/- SD, P < 0.05) and vs. sedentary normoxic rats (38.0 +/- 0.09, P < 0.05). Sedentary hypoxic rats decreased Tb (36.15 +/- 0.97 vs. 38.0 +/- 0.36, P < 0.05) whereas Tb was maintained in the exercising hypoxic rats during the initial 6 h of exercise (37.61 +/- 0.55 vs. 37.72 +/- 1.25, not significant). After exercise, Tb in hypoxic rats reached a nadir similar to that in sedentary hypoxic rats (35.05 +/- 1.69 vs. 35.03 +/- 1.32, respectively). Tb reached its nadir significantly later in exercising hypoxic vs. sedentary hypoxic rats (10.51 +/- 1.61 vs. 5.36 +/- 1.83 h, respectively; P = 0.002). Significantly greater histopathological damage and water contents were observed in brain and lungs in the exercising hypoxic vs. sedentary hypoxic and normoxic rats. Thus exercise early in hypoxia delays but does not prevent the HTR. Counteracting the HTR early in hypoxia by exercise exacerbates brain and lung damage and edema in the absence of ischemia.     

Cardioprotection in chronically hypoxic rabbits persists on exposure to normoxia: role of NOS and KATP channels

Hypoxia from birth increases resistance to myocardial ischemia in infant rabbits. We hypothesized that increased cardioprotection in hearts chronically hypoxic from birth persists following development in a normoxic environment and involves increased activation of nitric oxide synthase (NOS) and ATP-dependent K (K(ATP)) channels. Resistance to myocardial ischemia was determined in rabbits raised from birth to 10 days of age in a normoxic (Fi(O(2)) = 0.21) or hypoxic (Fi(O(2)) = 0.12) environment and subsequently exposed to normoxia for up to 60 days of age. Isolated hearts (n = 8/group) were subjected to 30 min of global ischemia followed by 35 min of reperfusion. At 10 days of age, resistance to myocardial ischemia (percent recovery postischemic recovery left ventricular developed pressure) was higher in chronically hypoxic hearts (68 +/- 4%) than normoxic controls (43 +/- 4%). At 10 days of age, N(G)-nitro-L-arginine methyl ester (200 microM) and glibenclamide (3 microM) abolished the cardioprotective effects of chronic hypoxia (45 +/- 4% and 46 +/- 5%, respectively) but had no effect on normoxic hearts. At 30 days of age resistance to ischemia in normoxic hearts declined (36 +/- 5%). However, in hearts subjected to chronic hypoxia from birth to 10 days and then exposed to normoxia until 30 days of age, resistance to ischemia persisted (63 +/- 4%). L-NAME or glibenclamide abolished cardioprotection in previously hypoxic hearts (37 +/- 4% and 39 +/- 5%, respectively) but had no effect on normoxic hearts. Increased cardioprotection was lost by 60 days. We conclude that cardioprotection conferred by adaptation to hypoxia from birth persists on subsequent exposure to normoxia and is associated with enhanced NOS activity and activation of K(ATP) channels.     

Platelet-activating factor receptor modulates respiratory adaptation to long-term intermittent hypoxia in mice

During hypoxia, release of platelet-activating factor (PAF) and activation of its cognate receptor (PAFR) regulate neural transmission and are required for full expression of peak hypoxic ventilatory response (pHVR) but not hypercapnic ventilatory response. However, it is unclear whether PAFR underlie components of long-term ventilatory adaptations to hypoxia. To examine this issue, adult male PAFR(+/+) and PAFR(-/-) mice were exposed to intermittent hypoxia (IH) consisting of 90 s 21% O(2) and 90 s 10% O(2) for 30 days, and normoxic and hypoxic ventilatory patterns were assessed using whole body plethysmography. Starting at day 14 of IH, normoxic ventilation in PAFR(-/-) was reduced significantly compared with PAFR(+/+) mice (P < 0.001), the latter exhibiting a prominent long-term ventilatory facilitation (LTVF). However, IH-exposed PAFR(-/-) mice had markedly enhanced pHVR and hypoxic ventilatory decline that became similar to those of IH-exposed PAFR(+/+) mice. Thus we postulate that PAFR expression and/or function underlies critical components of IH-induced LTVF but does not play a role in the potentiation of the hypoxic ventilatory response after IH exposures.     

Acid-base balance and lactatemia in acute hypoxia in normoxic or hypoxic albino rats

Albino rats Wistar family were raised since birth in normobaric hypoxic environment (10% O2 in N2). This hypoxic animal group and a normoxic control group were subjected to acute hypoxia in two spaced tests. The rats were exposed for 15 minutes to 7% O2 and later to 5% O2 gas mixture. At the end of the test with 7% O2 the hypoxic animals since birth showed a smaller quantity of blood lactate and their acid-base balance was more acid when compared to control animals. These differences were significant. In the considered metabolic parameters the differences between the 2 groups became not significant at the end of the test with 5% O2. We believe that the found differences in mentioned parameters between hypoxic and normoxic animals, also according to cellular adaptative processes, occurred during the rearing in hypoxic environment. In the test with 5% O2 the seriousness of the hypoxia overcomes the effects of adaptative mechanisms in hypoxic animals since birth. We believe that hypoxic rats since birth represent, limitedly to some aspects, different metabolic models compared to normoxic animals.     

Chronic hypoxia abolishes posthypoxia frequency decline in the anesthetized rat

In anesthetized rats, increases in phrenic nerve amplitude and frequency during brief periods of hypoxia are followed by a reduction in phrenic nerve burst frequency [posthypoxia frequency decline (PHFD)]. We investigated the effects of chronic exposure to hypoxia on PHFD and on peripheral and central O2-sensing mechanisms. In Inactin-anesthetized (100 mg/kg) Sprague-Dawley rats, phrenic nerve discharge and arterial pressure responses to 10 s N2 inhalation were recorded after exposure to hypoxia (10 +/- 0.5% O2) for 6-14 days. Compared with rats maintained at normoxia, PHFD was abolished in chronic hypoxic rats. Because of inhibition of PHFD, the increased phrenic burst frequency and amplitude after N2 inhalation persisted for 1.8-2.8 times longer in chronic hypoxic (70 s) compared with normoxic (25-40 s) rats (P < 0.05). After acute bilateral carotid body denervation, N2 inhalation produced a short depression of phrenic nerve discharge in both chronic hypoxic and normoxic rats. However, the degree and duration of depression of phrenic nerve discharge was smaller in chronic hypoxic compared with normoxic rats (P < 0.05). We conclude that after exposure to chronic hypoxia, a reduction in PHFD contributes to an increased duration of the acute hypoxic ventilatory response in anesthetized rats. Furthermore, after exposure to chronic hypoxia, the central network responsible for respiration is more resistant to the depressant effects of acute hypoxia in anesthetized rats.     

Short-term potentiation of ventilation after different levels of hypoxia.

Short-term potentiation of ventilation (VSTP) may be observed in healthy subjects on sudden termination of an hypoxic stimulus. We hypothesized that the level of hypoxia preceding normoxia would modify the duration and magnitude of the ensuing ventilatory decay. Ten healthy adults were studied on two different occasions, during which they were randomly exposed to isocapnic 6 or 10% O2 for 60 s and then switched to an isocapnic normoxic gas mixture. Both hypoxic gases induced significant ventilatory responses, and mean peak minute ventilation before the isocapnic normoxic switch was higher in 6% O2 (P < 0.001). The fast time constant of the two-exponential equation representing the best fit for ventilatory decay was unaffected by the magnitude of the hypoxic stimulus. However, the slow time constant, which is considered to represent VSTP, was markedly prolonged in 6% compared with 10% O2 [106.7 +/- 11.3 vs. 38. 2 +/- 6.1 (SD) s, respectively; P < 0.0001]. This result indicates that VSTP is stimulus dependent. We conclude that the magnitude of hypoxia preceding a normoxic transient modifies VSTP characteristics. We speculate that the interdependence function of ventilatory stimulus and short-term potentiation is crucial for preservation of system stability during transitions from high to low ventilatory drives.     

Mechanisms of aortic smooth muscle hyporeactivity after prolonged hypoxia in rats.

The aim of this study was to determine whether the effects of hypoxia on aortic contractility reflect a decrease in smooth muscle activation [phosphorylation of the 20-kDa myosin regulatory light chain (LC(20))], the capacity for myofibrillar ATP hydrolysis (mATPase activity), or both. Our results indicate that, in endothelium-denuded aortic rings from rats exposed to hypoxia for 48 h (inspired O(2) concentration = 10%), contractions to phenylephrine and potassium chloride (KCl) are impaired compared with rings from normoxic rats. The proportion of phosphorylated to total LC(20) during aortic contraction induced by 10(-5) M phenylephrine was reduced after hypoxia (51.4 +/- 5.4% in normoxic control rats vs. 32.5 +/- 4.7% in hypoxic rats, P < 0.01). Aortic mATPase activity was also decreased (maximum ATPase rate = 29.6 +/- 3.4 and 20.7 +/- 3.7 nmol. min(-1). mg protein(-1) in control and hypoxic rats, respectively, P < 0.05). Neither proliferation nor dedifferentiation of aortic smooth muscle was evident in this model; immunostaining for smooth muscle expression of the proliferating cell nuclear antigen was negative and smooth muscle-specific isoforms of myosin heavy chains, h-caldesmon, and calponin were increased, not decreased, after hypoxic exposure. Decreased aortic reactivity after hypoxia is associated with both impairment of smooth muscle activation and diminished capacity of the actomyosin complex, once activated, to hydrolyze ATP. These changes cannot be attributed to smooth muscle dedifferentiation or to reduced contractile protein expression.     

Stiffened erythrocytes augment the pulmonary hemodynamic response to hypoxia

Isolated rat lungs were perfused with suspensions containing normal and stiffened erythrocytes (RBCs) during normoxic and hypoxic ventilation to assess the effect of reduced RBC deformability on the hypoxic pressor response. RBC suspensions were prepared with cells previously incubated in isotonic phosphate-buffered saline with or without 0.0125% glutaraldehyde. The washed RBCs were resuspended in isotonic bicarbonate-buffered saline (with 4% albumin) to hematocrits of approximately 35%. The lungs were perfused with control and experimental cell suspensions in succession while pulmonary arterial pressure was measured during normoxic (21% O2) and hypoxic (3% O2) ventilation. On the attainment of a peak hypoxic pressor response, flow rate was changed so that pressure-flow curves could be constructed for each suspension. RBC deformability was quantified by a filtration technique using 4.7-microns-pore filters. Glutaraldehyde treatment produced a 10% decrease in RBC deformability (P less than 0.05). Over the range of flow rates, Ppa was increased by 15-17% (P less than 0.05) and 26-31% (P less than 0.05) during normoxic and hypoxic ventilation, respectively, when stiffened cells were suspended in the perfusate. The magnitude of the hypoxic pressor response was 50-54% greater with stiffened cells over the three flow rates. In a separate set of experiments, normoxic and hypoxic arterial blood samples from conscious unrestrained rats were used to investigate the effects of acute hypoxia on RBC deformability. Deformability was measured with the same filtration technique. There was no difference in the deformability of hypoxic compared with normoxic RBCs. We conclude that the presence of stiffened RBCs enhances the hemodynamic response to hypoxia but acute hypoxia does not affect RBC deformability.     

Effect of prenatal hypoxia on heat stress-mediated cardioprotection in adult rat heart

Fetal programming has profound effects on cardiovascular function in later adult life. We tested the hypothesis that chronic hypoxic exposure during fetal development downregulates endogenous cardioprotective mechanisms in adult rats. Time-dated pregnant rats were divided between normoxic and hypoxic (10.5% O2 from days 15 to 21 of gestation) groups. The male progeny were studied at 2 mo of age. Rats were subjected to heat stress (42 degrees C for 15 min). After 24 h, hearts were excised and subjected to 30 min of global ischemia and 1 h of reperfusion. Prenatal hypoxia did not change adult rat body weight and heart weight, but significantly increased the cross-sectional area of a left ventricular (LV) myocyte. Heat stress significantly improved postischemic recovery of LV function in normoxic control rats, but not in prenatally hypoxic rats. The infarct size in the LV resulting from ischemia-reperfusion was reduced by the heat stress pretreatment in control rats, but not in prenatally hypoxic rats. In accordance, heat stress significantly increased LV myocardial content of heat shock protein 70 only in normoxic control rats. In addition, there was a significant decrease in the LV myocardial content of the PKC-epsilon isoform in prenatally hypoxic rats compared with control rats. We conclude that prenatal hypoxia causes in utero programming of hsp70 gene in the LV, leading to an inhibition of its response to heat stress and a loss of cardioprotection in later adult life.     

缺氧对体外培养的肺动脉平滑肌细胞形态及增殖的影响

本实验应用形态学、免疫组化染色,＾３Ｈ－ＴｄＲ掺入、流式细胞等技术探讨缺氧（〈１％Ｏ２和２．５％Ｏ２）对肺动脉平滑肌细胞（ＰＡＳＭ）形态及增殖的影响。结果表明：缺氧２４ｈ使ＰＡＳＭ表型从收缩型向合成型转换,胞浆内ＳＭ－ａ ａｃｔｉｎ减少,线粒体和粗面内质网增多,缺氧４８ｈ以后线粒体肿胀,空泡化,内质网扩张,胞浆内出现髓鞘样结构。流式细胞分析显示缺氧ＰＡＳＭ Ｇ２／Ｍ期细胞比例明显增多（Ｐ〈０．００