慢性间歇性低氧降低急性缺氧对大鼠肺动脉平滑肌细胞膜上电压门控性钾通道的抑制

为了从离子通道水平上探讨机体低氧适应的离子机制,本实验将雄性 SD 大鼠随机分为常氧对照组和慢性间歇性低氧组[氧浓度(10 ± 0.5) %, 间断缺氧每天 8 h]。用酶解法急性分离单个大鼠肺内动脉平滑肌细胞(pulmonary artery smoothmuscle cells, PASMCs),以全细胞膜片钳技术记录 PASMCs 膜上的电压门控性钾通道 (voltage-gated potassium channel, KV) 电流,观察急性缺氧对慢性间歇性低氧大鼠 PASMCs 的 KV 的影响, 为机体适应低氧能力提供实验依据。结果显示:⑴常氧对照组在电流钳下,急性缺氧可使膜电位明显去极化(由-47.2 ±2.6 mV 去极到 -26.7 ±1.2 mV ); 在电压钳下, 急性缺氧可显著抑制 KV电流( 60 mV 时, KV电流密度从 153.4 ± 9.5 pA/pF降到 70.1 ± 10.6 pA/pF), 峰电流的抑制率为(57.6 ± 3.3) %, 电流-电压关系曲线向右下移。⑵慢性间歇性低氧组KV电流密度随低氧时间延长而逐渐减少(慢性低氧10 d后就有显著性意义),电流- 电压关系曲线逐渐右下移。⑶急性缺氧对慢性间歇性低氧大鼠PASMCs KV电流的抑制作用随慢性间歇性低氧时间延长而逐渐减弱。上述观察结果提示慢性间歇性低氧减弱急性缺氧对 KV 的抑制, 这可能是机体低氧适应的一种重要机制。     

Direct effects of acute hypoxia on the reactivity of peripheral arteries of the chicken embryo

In the chicken embryo, acute hypoxemia results in cardiovascular responses, including an increased peripheral resistance. We investigated whether local direct effects of decreased oxygen tension might participate in the arterial response to hypoxemia in the chicken embryo. Femoral arteries of chicken embryos were isolated at 0.9 of incubation time, and the effects of acute hypoxia on contraction and relaxation were determined in vitro. While hypoxia reduced contraction induced by high K(+) to a small extent (-21.8 +/- 5.7%), contractile responses to exogenous norepinephrine (NE) were markedly reduced (-51.1 +/- 3.2%) in 80% of the arterial segments. This effect of hypoxia was not altered by removal of the endothelium, inhibition of NO synthase or cyclooxygenase, or by depolarization plus Ca(2+) channel blockade. When arteries were simultaneously exposed to NE and ACh, hypoxia resulted in contraction (+49.8 +/- 9.3%). Also, relaxing responses to ACh were abolished during acute hypoxia, while the vessels became more sensitive to the relaxing effect of the NO donor sodium nitroprusside (pD(2): 5.81 +/- 0.21 vs. 5.31 +/- 0.27). Thus, in chicken embryo femoral arteries, acute hypoxia blunts agonist-induced contraction of the smooth muscle and inhibits stimulated endothelium-derived relaxation factor release. The consequences of this for in vivo fetal hemodynamics during acute hypoxemia depend on the balance between vasomotor influences of circulating catecholamines and those of the endothelium.     

Chronic hypoxia induces Rho kinase-dependent myogenic tone in small pulmonary arteries

Myogenic tone in the pulmonary vasculature of normoxic adult animals is minimal or nonexistent. Whereas chronic hypoxia (CH) increases basal tone in pulmonary arteries, it is unclear if a portion of this elevated tone is due to development of myogenicity. Since basal arterial RhoA activity and Rho kinase (ROK) expression are augmented by CH, we hypothesized that CH elicits myogenic reactivity in pulmonary arteries through ROK-dependent vascular smooth muscle (VSM) Ca(2+) sensitization. To test this hypothesis, we assessed the contribution of ROK to basal tone and pressure-induced vasoconstriction in endothelium-disrupted pulmonary arteries [50-300 microm inner diameter (ID)] from control and CH [4 wk at 0.5 atmosphere (atm)] rats. Arteries were loaded with fura-2 AM to continuously monitor VSM intracellular Ca(2+) concentration ([Ca(2+)](i)). Basal VSM [Ca(2+)](i) was not different between groups. The ROK inhibitor, HA-1077 (100 nM to 30 microM), caused a concentration-dependent reduction of basal tone in CH arteries but had no effect in control vessels. In contrast, PKC inhibition with GF109203X (1 microM) did not alter basal tone. Furthermore, significant vasoconstriction in response to stepwise increases in intraluminal pressure (5-45 mmHg) was observed at 12, 15, 25, and 35 mmHg in arteries (50-200 microm ID) from CH rats. This myogenic reactivity was abolished by HA-1077 (10 microM) but not by GF109203X. VSM [Ca(2+)](i) was unaltered by HA-1077, GF109203X, or increases in pressure in either group. Myogenicity was not observed in larger vessels (200-300 microm ID). We conclude that CH induces myogenic tone in small pulmonary arteries through ROK-dependent myofilament Ca(2+) sensitization.     

Chronic hypoxia decreases K(V) channel expression and function in pulmonary artery myocytes

Activity of voltage-gated K+ (KV) channels regulates membrane potential (E(m)) and cytosolic free Ca2+ concentration ([Ca2+](cyt)). A rise in ([Ca2+](cyt))in pulmonary artery (PA) smooth muscle cells (SMCs) triggers pulmonary vasoconstriction and stimulates PASMC proliferation. Chronic hypoxia (PO(2) 30-35 mmHg for 60-72 h) decreased mRNA expression of KV channel alpha-subunits (Kv1.1, Kv1.5, Kv2.1, Kv4.3, and Kv9.3) in PASMCs but not in mesenteric artery (MA) SMCs. Consistently, chronic hypoxia attenuated protein expression of Kv1.1, Kv1.5, and Kv2.1; reduced KV current [I(KV)]; caused E(m) depolarization; and increased ([Ca2+](cyt)) in PASMCs but negligibly affected KV channel expression, increased I(KV), and induced hyperpolarization in MASMCs. These results demonstrate that chronic hypoxia selectively downregulates KV channel expression, reduces I(KV), and induces E(m) depolarization in PASMCs. The subsequent rise in ([Ca2+](cyt)) plays a critical role in the development of pulmonary vasoconstriction and medial hypertrophy. The divergent effects of hypoxia on KV channel alpha-subunit mRNA expression in PASMCs and MASMCs may result from different mechanisms involved in the regulation of KV channel gene expression.     

Chronic hypoxia increases fetoplacental vascular resistance and vasoconstrictor reactivity in the rat

An increase in fetoplacental vascular resistance caused by hypoxia is considered one of the key factors of placental hypoperfusion and fetal undernutrition leading to intrauterine growth restriction (IUGR), one of the serious problems in current neonatology. However, although acute hypoxia has been shown to cause fetoplacental vasoconstriction, the effects of more sustained hypoxic exposure are unknown. This study was designed to test the hypothesis that chronic hypoxia elicits elevations in fetoplacental resistance, that this effect is not completely reversible by acute reoxygenation, and that it is accompanied by increased acute vasoconstrictor reactivity of the fetoplacental vasculature. We measured fetoplacental vascular resistance as well as acute vasoconstrictor reactivity in isolated perfused placentae from rats exposed to hypoxia (10% O(2)) during the last week of a 3-wk pregnancy. We found that chronic hypoxia shifted the relationship between perfusion pressure and flow rate toward higher pressure values (by approximately 20%). This increased vascular resistance was refractory to a high dose of sodium nitroprusside, implying the involvement of other factors than increased vascular tone. Chronic hypoxia also increased vasoconstrictor responses to angiotensin II (by approximately 75%) and to acute hypoxic challenges (by >150%). We conclude that chronic prenatal hypoxia causes a sustained elevation of fetoplacental vascular resistance and vasoconstrictor reactivity that are likely to produce placental hypoperfusion and fetal undernutrition in vivo.     

Chronic hypoxia induces apoptosis in cardiac myocytes: a possible role for Bcl-2-like proteins

We report in this study that proliferation inhibition of SCC13 cells by calcipotriol was possibly mediated by its inhibitory effect on autocrine activation of EGF receptor. Based on MTT assay, PCNA staining, DAPI staining, and involucrin immunocytochemical staining, we showed that calcipotriol inhibited cell growth and stimulated differentiation but did not induce apoptosis. Western blot analysis of concanavalin-A-bound fraction demonstrated that calcipotriol specifically dephosphorylated 170- and 66-kDa polypeptides from 8 h posttreatment and complete dephosphorylation was observed at 12 h posttreatment. The 170- and 66-kDa polypeptides were confirmed as EGF receptor and Shc, respectively. Calcipotriol-mediated EGF receptor dephosphorylation required the presence of extracellular calcium. Similar kinetics of the dephosphorylation was also observed in HaCaT cells cultured in medium of high calcium concentration. By BrdU labeling, we also showed calcium dependency of calcipotriol for the inhibition of cell proliferation. Therefore, EGF receptor deactivation by calcipotriol might be a mechanism of action for the inhibition of cell proliferation and the stimulation of differentiation in SCC13 cell and HaCaT cells.     

Lack of physiological plasticity in the early chicken embryo exposed to acute hypoxia

By exposing chicken embryos to hypoxia (10%) acutely (2, 4, and 6 hr) during early development (2, 3, and 4 days) we tested the hypothesis that hypoxia has an impact on embryonic growth and impairs cardiac development at the time cardiac morphogenesis is taking place. After the hypoxic perturbation, the embryos were allowed to develop until day 9, when embryo mass, heart mass, and rate of oxygen consumption were recorded. Four-day-old embryos exposed to 6 hr of hypoxia showed an increased mortality (38.9% versus 18% for controls), indicating the immediate effect of hypoxia on survivability. While only 8% of the controls displayed morphological abnormalities, 3- and 4-day-old embryos exposed for 6 hr showed more frequent developmental abnormalities (25% and 30% respectively). No significant differences in embryo or heart mass were found except in 4-day-old embryos exposed for 2 hr. Mass-specific oxygen consumption was not different between controls and embryos exposed to hypoxia at 2 or 3 days of development, but it was increased in 4-day-old embryos exposed for 4 hr (P < 0.05). These results suggest that an acute hypoxic episode does not have an impact when occurring very early in development (days 2 or 3). However, when the hypoxic episode occurs on day 4, survivability is largely decreased. Considering the lack of permanent effects on the surviving embryos, we suggest that the early embryo resorts to a simple strategy of death or survival, and the individual capacity for survival must be based on interindividual differences rather than the existence of compensatory mechanisms. J. Exp. Zool. 286:450-456, 2000.     

Effect of in ovo application of hydroxyapatite nanoparticles on chicken embryo development,oxidative status and bone characteristics

ABSTRACT

Intensive selection in modern lines of fast-growing chickens has caused several skeletal disorders. Therefore, current research is focused on methods to improve the bones of birds. A new potential solution is in ovo technology using nanoparticles with a high specificity for the bone tissue. Thus, the objective of the present study was to evaluate the effect of in ovo application of hydroxyapatite nanoparticles (HA-NP) in different concentrations (50, 100 and 500 μg/ml colloids) on chicken embryo development, with a particular focus on the oxidative status and bone characteristics of the embryo. The results showed that in ovo treatment with HA-NP did not negatively affect hatchability and body weight. However, bone weight was reduced in 500 μg/ml group. The concentrations of calcium, phosphorus and crude ash were not affected. The modulatory effect of HA-NP was observed on the basis of antioxidative markers – superoxide dismutase, total antioxidant status, malondialdehyde in serum and selected tissues. Glutathione concentration in serum suggested higher metabolic stress. Among bone turnover markers, the concentration of osteocalcin was found to be significantly affected by HA-NP injection. Thus, the in ovo application of HA-NP could modify the molecular responses at the stage of embryogenesis but these changes were not reflected in embryo growth and even slowed down bone development. Nevertheless, the question for the follow-up research is whether in ovo administration of HA-NP would affect the antioxidative status and bone turnover resulting in improved bone conditions and body gain in post hatch chickens.     

Chronic hypoxia elevates intracellular pH and activates Na+/H+ exchange in pulmonary arterial smooth muscle cells

Chronic hypoxia (CH), caused by many lung diseases, results in pulmonary hypertension due, in part, to increased muscularity of small pulmonary vessels. Pulmonary arterial smooth muscle cell (PASMC) proliferation in response to growth factors requires increased intracellular pH (pHi) mediated by activation of Na+/H+ exchange (NHE); however, the effect of CH on PASMC pHi homeostasis is unknown. Thus we measured basal pHi and NHE activity and expression in PASMCs isolated from mice exposed to normoxia or CH (3 wk/10% O2). pHi was measured using the pH-sensitive fluorescent dye BCECF-AM. NHE activity was determined from Na+-dependent recovery from NH4-induced acidosis, and NHE expression was determined by RT-PCR and immunoblot. PASMCs from chronically hypoxic mice exhibited elevated basal pHi and increased NHE activity. NHE1 was the predominate isoform present in mouse PASMCs, and both gene and protein expression of NHE1 was increased following exposure to CH. Our findings indicate that exposure to CH caused increased pHi, NHE activity, and NHE1 expression, changes that may contribute to the development of pulmonary hypertension, in part, via pH-dependent induction of PASMC proliferation.     

Prolonged root hypoxia induces ammonium accumulation and decreases the nutritional quality of tomato fruits

Here we examined the effects of root hypoxia (1-2% oxygen) on the physiology of the plant and on the biochemical composition of fruits in tomato (Solanum lycopersicum cv. Micro-Tom) plants submitted to gradual root hypoxia at first flower anthesis. Root hypoxia enhanced nitrate absorption with a concomitant release of nitrite and ammonium into the medium, a reduction of leaf photosynthetic activity and chlorophyll content, and an acceleration of fruit maturation, but did not affect final fruit size. Quantitative metabolic profiling of mature pericarp extracts by (1)H NMR showed that levels of major metabolites including sugars, organic acids and amino acids were not modified. However, ammonium concentration increased dramatically in fruit flesh, and ascorbate and lycopene concentrations decreased. Our data indicate that the unfavorable effects of root hypoxia on fruit quality cannot be explained by two of the well-known effects of root hypoxia on the plant, namely a decrease in photosynthesis or an excess in ethylene production, but may instead result from disturbances in the supply of either growth regulators or ammonium, by the roots.     

Divergent contractile and structural responses of the murine PKC-epsilon null pulmonary circulation to chronic hypoxia

Loss of PKC-epsilon limits the magnitude of acute hypoxic pulmonary vasoconstriction (HPV) in the mouse. Therefore, we hypothesized that loss of PKC-epsilon would decrease the contractile and/or structural response of the murine pulmonary circulation to chronic hypoxia (Hx). However, the pattern of lung vascular responses to chronic Hx may or may not be predicted by the acute HPV response. Adult PKC-epsilon wild-type (PKC-epsilon(+/+)), heterozygous null, and homozygous null (PKC-epsilon(-/-)) mice were exposed to normoxia or Hx for 5 wk. PKC-epsilon(-/-) mice actually had a greater increase in right ventricular (RV) systolic pressure, RV mass, and hematocrit in response to chronic Hx than PKC-epsilon(+/+) mice. In contrast to the augmented PA pressure and RV hypertrophy, pulmonary vascular remodeling was increased less than expected (i.e., equal to PKC-epsilon(+/+) mice) in both the proximal and distal PKC-epsilon(-/-) pulmonary vasculature. The contribution of increased vascular tone to this pulmonary hypertension (PHTN) was assessed by measuring the acute vasodilator response to nitric oxide (NO). Acute inhalation of NO reversed the increased PA pressure in hypoxic PKC-epsilon(-/-) mice, implying that the exaggerated PHTN may be due to a relative deficiency in nitric oxide synthase (NOS). Despite the higher PA pressure, chronic Hx stimulated less of an increase in lung endothelial (e) and inducible (i) NOS expression in PKC-epsilon(-/-) than PKC-epsilon(+/+) mice. In contrast, expression of nNOS in PKC-epsilon(+/+) mice decreased in response to chronic Hx, while lung levels in PKC-epsilon(-/-) mice remained unchanged. In summary, loss of PKC-epsilon results in increased vascular tone, but not pulmonary vascular remodeling in response to chronic Hx. Blunting of Hx-induced eNOS and iNOS expression may contribute to the increased vascular tone. PKC-epsilon appears to be an important signaling intermediate in the hypoxic regulation of each NOS isoform.     

离子通道在肺动脉的分布及在低氧肺血管收缩反应中的作用

低氧性肺血管收缩反应（HPV）是指在急性低氧时,肺泡氧分压降到某一临界值,肺血管发生的快速、可逆的收缩反应,以纠正肺泡通气／灌流的不匹配。HPV的发生与肺动脉平滑肌细胞上K^＋、Ca^2＋、Cl^-通道的状态密切相关,而这些通道在不同部位的肺动脉上分布存在差异,因此不同部位的肺动脉在低氧中所表现的收缩反应程度也不同,本综述将对上述通道在肺动脉上的分布特点及其在HPV中的作用做一总结。     

Chronic hypoxia upregulates pulmonary arterial ASIC1: a novel mechanism of enhanced store-operated Ca2+ entry and receptor-dependent vasoconstriction

Acid-sensing ion channel 1 (ASIC1) is a newly characterized contributor to store-operated Ca(2+) entry (SOCE) in pulmonary vascular smooth muscle (VSM). Since SOCE is implicated in elevated basal VSM intracellular Ca(2+) concentration ([Ca(2+)](i)) and augmented vasoconstriction in chronic hypoxia (CH)-induced pulmonary hypertension, we hypothesized that ASIC1 contributes to these responses. To test this hypothesis, we examined effects of the specific pharmacologic ASIC1a inhibitor, psalmotoxin 1 (PcTX1), on vasoconstrictor and vessel wall [Ca(2+)](i) responses to UTP and KCl (depolarizing stimulus) in fura-2-loaded, pressurized small pulmonary arteries from control and CH (4 wk at 0.5 atm) Wistar rats. PcTX1 had no effect on basal vessel wall [Ca(2+)](i), but attenuated vasoconstriction and increases in vessel wall [Ca(2+)](i) to UTP in arteries from control and CH rats; normalizing responses between groups. In contrast, responses to the depolarizing stimulus, KCl, were unaffected by CH exposure or PcTX1. Upon examining potential Ca(2+) influx mechanisms, we found that PcTX1 prevented augmented SOCE following CH. Exposure to CH resulted in a significant increase in pulmonary arterial ASIC1 protein. This study supports a novel role of ASIC1 in elevated receptor-stimulated vasoconstriction following CH which is likely mediated through increased ASIC1 expression and SOCE.     

Chronic myocardial infarction induces phenotypic and functional remodeling in the guinea pig cardiac plexus

Chronic myocardial infarction (CMI) is associated with remodeling of the ventricle and evokes adaption in the cardiac neurohumoral control systems. To evaluate the remodeling of the intrinsic cardiac nervous system following myocardial infarction, the dorsal descending coronary artery was ligated in the guinea pig heart and the animals were allowed to recover for 7-9 wk. Thereafter, atrial neurons of the intrinsic cardiac plexus were isolated for electrophysiological and immunohistochemical analyses. Intracellular voltage recordings from intrinsic cardiac neurons demonstrated no significant changes in passive membrane properties or action potential configuration compared with age-matched controls and sham-operated animals. The intrinsic cardiac neurons from chronic infarcted hearts did demonstrate an increase in evoked action potential (AP) frequency (as determined by the number of APs produced with depolarizing stimuli) and an increase in responses to exogenously applied histamine compared with sham and age-matched controls. Conversely, pituitary adenylate cyclase-activating polypeptide (PACAP)-induced increases in intrinsic cardiac neuron-evoked AP frequency were similar between control and CMI animals. Immunohistochemical analysis demonstrated a threefold increase in percentage of neurons immunoreactive for neuronal nitric oxide synthase (NOS) in CMI animals compared with control and the additional expression of inducible NOS by some neurons, which was not evident in control animals. Finally, the density of mast cells within the intrinsic cardiac plexus was increased threefold in preparations from CMI animals. These results indicate that CMI induces a differential remodeling of intrinsic cardiac neurons and functional upregulation of neuronal responsiveness to specific neuromodulators.     

Role of collagen content and cross-linking in large pulmonary arterial stiffening after chronic hypoxia

Chronic hypoxic pulmonary hypertension (HPH) is associated with large pulmonary artery (PA) stiffening, which is correlated with collagen accumulation. However, the mechanisms by which collagen contributes to PA stiffening remain largely unexplored. Moreover, HPH may alter mechanical properties other than stiffness, such as pulse damping capacity, which also affects ventricular workload but is rarely quantified. We hypothesized that collagen content and cross-linking differentially regulate the stiffness and damping capacity of large PAs during HPH progression. The hypothesis was tested with transgenic mice that synthesize collagen type I resistant to collagenase degradation (Col1a1(R/R)). These mice and littermate controls (Col1a1(+/+)) were exposed to hypoxia for 10 days; some were treated with β-aminopropionitrile (BAPN), which prevents new cross-link formation. Isolated PA dynamic mechanical tests were performed, and collagen content and cross-linking were measured. In Col1a1(+/+) mice, HPH increased both collagen content and cross-linking, and BAPN treatment prevented these increases. Similar trends were observed in Col1a1(R/R) mice except that collagen content further increased with BAPN treatment. Mechanical tests showed that in Col1a1(+/+) mice, HPH increased PA stiffness and damping capacity, and these increases were impeded by BAPN treatment. In Col1a1(R/R) mice, HPH led to a smaller but significant increase in PA stiffness and a decrease in damping capacity. These mechanical changes were not affected by BAPN treatment. Vessel-specific correlations for each strain showed that the stiffness and damping capacity were correlated with the total content rather than cross-linking of collagen. Our results suggest that collagen total content is critical to extralobar PA stiffening during HPH.     

Chronic hypoxia enhances the phrenic nerve response to arterial chemoreceptor stimulation in anesthetized rats.

Chronic exposure to hypoxia results in a time-dependent increase in ventilation called ventilatory acclimatization to hypoxia. Increased O(2) sensitivity of arterial chemoreceptors contributes to ventilatory acclimatization to hypoxia, but other mechanisms have also been hypothesized. We designed this experiment to determine whether central nervous system processing of peripheral chemoreceptor input is affected by chronic hypoxic exposure. The carotid sinus nerve was stimulated supramaximally at different frequencies (0.5-20 Hz, 0.2-ms duration) during recording of phrenic nerve activity in two groups of anesthetized, ventilated, vagotomized rats. In the chronically hypoxic group (7 days at 80 Torr inspired PO(2)), phrenic burst frequency (f(R), bursts/min) was significantly higher than in the normoxic control group with carotid sinus nerve stimulation frequencies >5 Hz. In the chronically hypoxic group, peak amplitude of integrated phrenic nerve activity ( integral Phr, percent baseline) or change in integral Phr was significantly greater at stimulation frequencies between 5 and 17 Hz, and minute phrenic activity ( integral Phr x f(R)) was significantly greater at stimulation frequencies >5 Hz. These experiments show that chronic hypoxia facilitates the translation of arterial chemoreceptor afferent input to ventilatory efferent output through a mechanism in the central nervous system.     

Chronic intermittent but not constant hypoxia decreases NAA/Cr ratios in neonatal mouse hippocampus and thalamus

Chronic constant hypoxia (CCH) and chronic intermittent hypoxia (CIH) are known to have deleterious effects on the central nervous system. Because of the difference in the pattern of hypoxic exposure, it is possible that the pathological outcome would vary. The N-acetyl aspartate/creatine (NAA/Cr) ratio is a reliable marker of neuronal integrity, and this can be noninvasively measured by proton nuclear magnetic resonance spectroscopy. P2 CD1 mouse pups with their dams were exposed to either CCH, where the Fi(O(2)) was maintained at 11% continuously or to CIH, where the Fi(O(2)) was varied between 21 and 11% every 4 min. P30 mice exposed to intermittent hypoxia for 4 wk demonstrated a significant decrease in the NAA/Cr ratio in the hippocampus and thalamus, which was reversed by a subsequent exposure to 4 wk of normoxia. Meanwhile, mice exposed to 4 wk of constant hypoxia did not demonstrate any differences in their NAA/Cr ratios from controls in these brain regions. These results indicate that an intermittent pattern of hypoxic exposure may have a more adverse effect on neuronal function and integrity than a continuous one. The reversal of NAA/Cr levels to baseline during the return to normoxia indicates that therapeutic strategies targeted at alleviating the intermittent hypoxic stress in diseases, such as obstructive sleep apnea, have the potential for inducing significant neurocognitive recovery in these patients.