Regulation of peptidase activity in a three-dimensional aggregate model of brain tumor vasculature

The abnormal vascular system of brain cancers inappropriately expresses membrane proteins, including proteolytic enzymes, ultimately resulting in blood extravasation. The production of inflammatory mediators, such as cytokines and nitric oxide, and tumor hypoxia have been implicated in these effects. We have previously shown that the activity of aminopeptidase A is increased in the abnormal vascular system of human and rat brain tumors. To study the mechanisms regulating the activities of peptidases in cerebral vasculature in brain tumors, we have developed a three-dimensional model of differentiated rat brain cells in aggregate cultures in which rat brain microvessels were incorporated. The secretion of interleukin-6 (IL-6) in the culture medium of aggregates was used as an indicator of inflammatory activation. Addition to these aggregates of C6 glioma cell medium (C6-CM) conditioned under hypoxic or normoxic conditions or serum mimicked tumor-dependent hypoxia or conditions of dysfunction of brain tumor vasculature. Hypoxic and normoxic C6-CM, but not serum, regulated peptidase activity in aggregates, and in particular it increased the activity of aminopeptidase A determined using histoenzymography. Serum, but not C6-CM, increased IL-6 production, but did not increase aminopeptidase A activity in aggregates. Thus soluble glioma-derived factors, but not serum-derived factors, induce dysfunctions of cerebral vasculature by directly regulating the activity of peptidases, not involving inflammatory activation. Tumor hypoxia is not necessary to modulate peptidase activity.     

Effect of CDP-choline on the biosynthesis of nucleic acids and proteins in brain regions during hypoxia

The effect of CDP-choline on the in vivo incorporation of labeled precursors into DNA, RNA, and proteins in cerebral hemispheres, cerebellum, and brainstem of guinea pigs after hypoxic treatment was studied. The labeling of macromolecules extracted from the various subcellular fractions of these brain regions was also determined. Hypoxic treatment affected macromolecular labeling to a different extent in the three brain regions examined. CDP-choline treatment was not able to reverse the effect of hypoxia on DNA labeling, but it was able to remove the effect of hypoxia on RNA and protein labeling. The action of CDP-choline was particularly evident on the labeling of RNA in nuclei and mitochondria of the cerebellum and on the labeling of proteins in microsomes of the three brain regions examined.     

Involvement of Na+ and Ca2+ channel activation and resultant nitric oxide synthesis in glutamate-mediated hypoxic injury in rat cerebrocortical slices

The role of Na+ and Ca2+ channels in glutamate-mediated hypoxic injury was investigated in slices of the rat cerebral cortex. Hypoxic injury was determined by mitochondrial reduction of 3-(4,5-dimethyl-2-thiazol)-2,5-diphenyltetrazolium bromide after exposure of brain slices to 30-min of hypoxia/glucose deprivation followed by 3-h of reoxygenation. Endogenous glutamate release was markedly elevated during hypoxia/glucose deprivation, but it returned almost to basal level during reoxygenation. Hypoxic injury was prevented by MK-801 or 6-cyano-7-nitroquinoxaline-2,3-dione. Combined treatment with omega-conotoxin GVIA, omega-agatoxin IVA, and tetrodotoxin reversed the hypoxic injury, although none of these agents alone or nifedipine was effective. Moreover, a novel Na+/Ca2+ channel blocker NS-7 [4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy) pyrimidine hydrochloride] significantly inhibited the hypoxic injury. Several inhibitors of nitric oxide synthase also blocked the hypoxic injury. Consistently, nitric oxide synthesis, as estimated from cyclic GMP formation in the extracellular fluids, was enhanced during hypoxia/glucose deprivation. NS-7 and other Na+ and Ca2+ channel blockers suppressed the enhancement of nitric oxide synthesis, although these compounds alone, or in combination, did not reduce hypoxic glutamate release. These findings suggest that hypoxic injury in rat cerebrocortical slices is triggered by glutamate and subsequent enhancement of nitric oxide synthesis through activation of both Na+ and Ca2+ channels. Thus, the simultaneous blockade of both Na+ channel as well as N-type and P/Q-type Ca2+ channels is required to sufficiently reverse the hypoxic injury.     

Cyclooxygenase-Mediated Generation of Free Radicals During Hypoxia in the Cerebral Cortex of Newborn Piglets

Previous studies have demonstrated that free radicals are formed under hypoxic conditions in newborn piglet brain. To test the hypothesis that the cyclooxygenase pathway serves as a source of free radical generation during hypoxia studies were performed on 24 piglets divided into four groups. Six saline (group 3) and six indomethacin treated (group 4) were exposed to hypoxia (FiO2 0.05-0.07) for 60 min. Cerebral hypoxia was documented biochemically by determination of ATP and phosphocreatine. Fluorescent compounds and conjugated dienes were determined as indices of lipid peroxidation. Free radical formation was determined by using n-tert butyl phenyl nitrone (PBN) as a spin trap agent and measuring spin adduct formation in duplicate using a Varian E-109 spectrometer. Groups 1 and 2 (normoxic) showed no spin adduct formation. Group 3 showed a significant increase in spin adduct formation compared to normoxia (372+/-125 vs. 63+/-15, P<0.001). Hypoxic animals pretreated with indomethacin had a spin adduct level of 197+/-132 and were similar to normoxic animals. ATP/PCr levels were the same in groups 3 and 4 denoting the same degree of cerebral hypoxia in all hypoxic animals. Conjugated dienes increased significantly during hypoxia as compared to normoxia (0.142+/-0.017 vs. 0.0+/-0.0) and were decreased insignificantly with indomethacin treatment. Fluorescent compounds were not significantly different among the four groups. Na+,K+-ATPase activity decreased during hypoxia but was not preserved in hypoxic animals pretreated with indomethacin. These data provide direct evidence of the presence of free radicals during hypoxia and the contribution of cyclooxygenase metabolism to their formation.     

Endothelial microvesicles in hypoxic hypoxia diseases

Hypoxic hypoxia, including abnormally low partial pressure of inhaled oxygen, external respiratory dysfunction‐induced respiratory hypoxia and venous blood flow into the arterial blood, is characterized by decreased arterial oxygen partial pressure, resulting in tissue oxygen deficiency. The specific characteristics include reduced arterial oxygen partial pressure and oxygen content. Hypoxic hypoxia diseases (HHDs) have attracted increased attention due to their high morbidity and mortality and mounting evidence showing that hypoxia‐induced oxidative stress, coagulation, inflammation and angiogenesis play extremely important roles in the physiological and pathological processes of HHDs‐related vascular endothelial injury. Interestingly, endothelial microvesicles (EMVs), which can be induced by hypoxia, hypoxia‐induced oxidative stress, coagulation and inflammation in HHDs, have emerged as key mediators of intercellular communication and cellular functions. EMVs shed from activated or apoptotic endothelial cells (ECs) reflect the degree of ECs damage, and elevated EMVs levels are present in several HHDs, including obstructive sleep apnoea syndrome and chronic obstructive pulmonary disease. Furthermore, EMVs have procoagulant, proinflammatory and angiogenic functions that affect the pathological processes of HHDs. This review summarizes the emerging roles of EMVs in the diagnosis, staging, treatment and clinical prognosis of HHDs.     

HYPOXIC SURVIVAL OF NORMOGLYCAEMIC YOUNG ADULT AND ADULT MICE IN RELATION TO CEREBRAL METABOLIC RATES12

The hypoxic tolerance and the cerebral metabolic rates (CMR) of young adult mice (20 to 25 g, 4 to 5 weeks old) and adult mice (30 g and above, 6 to 7 weeks old), respectively, were determined and their interrelationship was evaluated. CMRs increased from 25 mmol - P/kg.min to 38 mmol/kg.min as the animals grew older from young to full adulthood. Concurrently the tolerance to aerogcnic hypoxia (5% O₂-95%j N₂) declined. The effects of hypoxia on the cerebral energy metabolism were greater in adult than in young adult animals. It is concluded that the full metabolic maturation of the brain is reached in adult animals only. They become more dependent on an adequate oxygen supply as the aerobic activity of the energy metabolism of the brain is further increasing. Hypoxic gasping occurred while the pool of cerebral energy reserves was still far from being depleted. A failure to utilize energy reserves rather than their exhaustion is suggested as the ultimate cause of death from hypoxia. An acid-soluble form of glycogen or related polyglucan was found in addition to the usual amounts of insoluble glycogen. It was utilizcd rapidly during hypoxia and ischaemic anoxia and it may, therefore, constitute an additional source of carbohydrate substrates in thc brain.     

The seasonal characteristics of the circadian rhythm of hypoxic resistance and convulsive resistance]

Hypoxic and convulsive resistances have daily rhythms, the pattern of which depends on the year season. Latent period of occurrence of epileptic seizures and time of life at the "height" of 11,000 m above the sea level undergo similar changes in autumn and spring. In winter minimal similarity between daily dynamics of each of these resistances and analogous ones in other seasons is observed, but rhythms of tolerance to hypoxia are maximally synchronized with the rhythms of convulsive resistance. In autumn hypoxic and convulsive resistances are minimal. Maximums of these indices are observed in different seasons: for tolerance to hypoxia it is summer, for convulsive resistance--spring.     

Brain adaptation to chronic hypobaric hypoxia in rats.

Rats were exposed to hypobaric hypoxia (0.5 atm) for up to 3 wk. Hypoxic rats failed to gain weight but maintained normal brain water and ion content. Blood hematocrit was increased by 48% to a level of 71% after 3 wk of hypoxia compared with littermate controls. Brain blood flow was increased by an average of 38% in rats exposed to 15 min of 10% normobaric oxygen and by 23% after 3 h but was not different from normobaric normoxic rats after 3 wk of hypoxia. Sucrose space, as a measure of brain plasma volume, was not changed under any hypoxic conditions. The mean brain microvessel density was increased by 76% in the frontopolar cerebral cortex, 46% in the frontal motor cortex, 54% in the frontal sensory cortex, 65% in the parietal motor cortex, 68% in the parietal sensory cortex, 68% in the hippocampal CA1 region, 57% in the hippocampal CA3 region, 26% in the striatum, and 56% in the cerebellum. The results indicate that hypoxia elicits three main responses that affect brain oxygen availability. The acute effect of hypoxia is an increase in regional blood flow, which returns to control levels on continued hypoxic exposure. Longer-term effects of continued moderate hypoxic exposure are erythropoiesis and a decrease in intercapillary distance as a result of angiogenesis. The rise in hematocrit and the increase in microvessel density together increase oxygen availability to the brain to within normal limits, although this does not imply that tissue PO2 is restored to normal.     

Hypoxic regulation of the fetal cerebral circulation.

Fetal cerebrovascular responses to acute hypoxia are fundamentally different from those observed in the adult cerebral circulation. The magnitude of hypoxic vasodilatation in the fetal brain increases with postnatal age although fetal cerebrovascular responses to acute hypoxia can be complicated by age-dependent depressions of blood pressure and ventilation. Acute hypoxia promotes adenosine release, which depresses fetal cerebral oxygen consumption through action of adenosine on neuronal A1 receptors and vasodilatation through activation of A2 receptors on cerebral arteries. The vascular effect of adenosine can account for approximately half the vasodilatation observed in response to hypoxia. Hypoxia-induced release of nitric oxide and opioids can account for much of the adenosine-independent cerebral vasodilatation observed in response to hypoxia in the fetus. Direct effects of hypoxia on cerebral arteries account for the remaining fraction, although the vascular endothelium contributes relatively little to hypoxic vasodilatation in the immature cerebral circulation. In contrast to acute hypoxia, fetal cerebral blood flow tends to normalize during acclimatization to chronic hypoxia even though cardiac output is depressed. However, uncompensated chronic hypoxia in the fetus can produce significant changes in brain structure and function, alteration of respiratory drive and fluid balance, and increased incidence of intracranial hemorrhage and periventricular leukomalacia. At the level of the fetal cerebral arteries, chronic hypoxia increases protein content and depresses norepinephrine release, contractility, and receptor densities associated with contraction but also attenuates endothelial vasodilator capacity and decreases the ability of ATP-sensitive and calcium-sensitive potassium channels to promote vasorelaxation. Overall, fetal cerebrovascular adaptations to chronic hypoxia appear prioritized to conserve energy while preserving basic contractility. Many gaps remain in our understanding of how the effects of acute and chronic hypoxia are mediated in fetal cerebral arteries, but studies of adult cerebral arteries have produced many powerful pharmacological and molecular tools that are simply awaiting application in studies of fetal cerebral artery responses to hypoxia.     

Metabolic depression: a response of cancer cells to hypoxia?

Hypoxic tumours have the worst prognosis because they are the most aggressive and the most likely to metastasize. This may be because these aggressive cancers have a hypoxic core which generates signals that activate angiogenesis which enables the supply of nutrients and oxygen to a rapidly growing outer oxidative shell. The hypoxic core is a crucial element of this hypothesis, as is the fact that the cells in the hypoxic core are inherently adapted to survive hypoxia. We reasoned therefore that cancer cells exposed to hypoxia/anoxia should show the hallmarks of adaptation to hypoxia/anoxia, i.e. a down-regulation of protein synthesis and a reverse Pasteur effect. We tested this hypothesis in transformed (MCF-7) and normal (HME) human mammary epithelial cells, by exposing both cell types to a range of oxygen concentrations, including anoxia. We find that indeed protein synthesis is down-regulated in the MCF-7, but not in the HME cells in response to anoxia. The data on glycolysis are not as clear-cut, but in the light of similar previous measurements on hypoxia-tolerant animals, is still consistent with the hypothesis.     

Function of the rat carotid body chemoreceptors in ageing

Some age-related deficits in the ventilatory responses have been attributed to a decline in the functionality of the carotid body (CB) arterial chemoreceptors, but a systematic study of the CB function in ageing is lacking. In rats aged 3-24 months, we have performed quantitative morphometry on specific chemoreceptor tissue, assessed the function of chemoreceptor cells by measuring the content, synthesis and release of catecholamines (a chemoreceptor cell neurotransmitter) in normoxia and hypoxia, and determined the functional activity of the intact organ by measuring chemosensory activity in the carotid sinus nerve (CSN) in normoxia, hypoxia and hypercapnic acidosis. We found that with age CBs enlarge, but at the same time there is a concomitant decrease in the percentage of chemoreceptor tissue. CB content and turnover time for their catecholamines increase with age. Hypoxic stimulation of chemoreceptor cells elicits a smaller release of catecholamines in rats after 12 months of age, but a non-specific depolarizing stimulus elicits a comparable release at all ages. In parallel, there was a marked decrease in the responsiveness to hypoxia, but not to an acidic-hypercapnic stimulus, assessed as chemosensory activity in the CSN. We conclude that in aged mammals chemoreceptor cells become hypofunctional, leading to a decreased peripheral drive of ventilation.     

Morphofunctional Assessment of Tissue Hypoxia Parameters in Lacunar Stroke in Humans

Human Physiology - Tissue hypoxia, developing as a consequence of vascular pathology, is a typical pathological process that underlies numerous disorders, including cerebrovascular disease. Hypoxic...     

Cerebral hypoperfusion during hypoxic exercise following two different hypoxic exposures: independence from changes in dynamic autoregulation and reactivity

We examined the effects of exposure to 10-12 days intermittent hypercapnia [IHC: 5:5-min hypercapnia (inspired fraction of CO(2) 0.05)-to-normoxia for 90 min (n = 10)], intermittent hypoxia [IH: 5:5-min hypoxia-to-normoxia for 90 min (n = 11)] or 12 days of continuous hypoxia [CH: 1,560 m (n = 7)], or both IH followed by CH on cardiorespiratory and cerebrovascular function during steady-state cycling exercise with and without hypoxia (inspired fraction of oxygen, 0.14). Cerebrovascular reactivity to CO(2) was also monitored. During all procedures, ventilation, end-tidal gases, blood pressure, muscle and cerebral oxygenation (near-infrared spectroscopy), and middle cerebral artery blood flow velocity (MCAv) were measured continuously. Dynamic cerebral autoregulation (CA) was assessed using transfer-function analysis. Hypoxic exercise resulted in increases in ventilation, hypocapnia, heart rate, and cardiac output when compared with normoxic exercise (P < 0.05); these responses were unchanged following IHC but were elevated following the IH and CH exposure (P < 0.05) with no between-intervention differences. Following IH and/or CH exposure, the greater hypocapnia during hypoxic exercise provoked a decrease in MCAv (P < 0.05 vs. preexposure) that was related to lowered cerebral oxygenation (r = 0.54; P < 0.05). Following any intervention, during hypoxic exercise, the apparent impairment in CA, reflected in lowered low-frequency phase between MCAv and BP, and MCAv-CO(2) reactivity, were unaltered. Conversely, during hypoxic exercise following both IH and/or CH, there was less of a decrease in muscle oxygenation (P < 0.05 vs. preexposure). Thus IH or CH induces some adaptation at the muscle level and lowers MCAv and cerebral oxygenation during hypoxic exercise, potentially mediated by the greater hypocapnia, rather than a compromise in CA or MCAv reactivity.     

Cellular disposition of transported polyamines in hypoxic rat lung and pulmonary arteries

The polyamines putrescine, spermidine (SPD), and spermine are a family of low-molecular-weight organic cations essential for cell growth and differentiation and other aspects of signal transduction. Hypoxic pulmonary vascular remodeling is accompanied by depressed lung polyamine synthesis and markedly augmented polyamine uptake. Cell types in which hypoxia induces polyamine transport in intact lung have not been delineated. Accordingly, rat lung and rat main pulmonary arterial explants were incubated with [(14)C]SPD in either normoxic (21% O(2)) or hypoxic (2% O(2)) environments for 24 h. Autoradiographic evaluation confirmed previous studies showing that, in normoxia, alveolar epithelial cells are dominant sites of polyamine uptake. In contrast, hypoxia was accompanied by prominent localization of [(14)C]SPD in conduit, muscularized, and partially muscularized pulmonary arteries, which was not evident in normoxic lung tissue. Hypoxic main pulmonary arterial explants also exhibited substantial increases in [(14)C]SPD uptake relative to control explants, and autoradiography revealed that enhanced uptake was most evident in the medial layer. Main pulmonary arterial explants denuded of endothelium failed to increase polyamine transport in hypoxia. Conversely, medium conditioned by endothelial cells cultured in hypoxic, but not in normoxic, environments enabled hypoxic transport induction in denuded arterial explants. These findings in arterial explants were recapitulated in rat cultured main pulmonary artery cells, including the enhancing effect of a soluble endothelium-derived factor(s) on hypoxic induction of [(14)C]SPD uptake in smooth muscle cells. Viewed collectively, these results show in intact lung tissue that hypoxia enhances polyamine transport in pulmonary artery smooth muscle by a mechanism requiring elaboration of an unknown factor(s) from endothelial cells.     

Effect of peripheral and central alpha-adrenoceptor blockade on cerebral microvascular and blood flow responses to hypoxia.

The purpose of this study was to evaluate the effects of vascular and central alpha-adrenoceptor blockade on cerebral blood flow (CBF) and utilization of brain arteriolar and capillary reserve in conscious rats during normoxia and hypoxia (8% O2 in N2). Animals were divided into three groups and administered either saline, N-methyl chlorpromazine (does not cross the blood-brain barrier), or phenoxybenzamine (crosses the blood-brain barrier) in equipotent doses. Neither agent affected regional CBF and the utilization of brain microvascular reserve during normoxia. CBF increased from 70.9 +/- 2.9 (SEM) ml/min/100 g in the control normoxic group to 123.8 +/- 4.2 ml/min/100 g in control hypoxic animals. In control, hypoxic flow to pons and medulla of the brain was higher than to cortex, hypothalamus or thalamus. The percent of arterioles/mm2 perfused increased from 49.6 +/- 2.0% during control normoxia to 65.6 +/- 3.0% during control hypoxia. The percentage of capillaries/mm2 perfused changed similarly. Hypoxic CBF was increased similarly after administration of N-methyl chlorpromazine or phenoxybenzamine. Administration of N-methyl chlorpromazine or phenoxybenzamine eliminated regional differences in hypoxic CBF and the utilization of arterioles, and did not affect capillary response. There was no difference between the effect of N-methyl chlorpromazine and phenoxybenzamine on cerebral microvascular and blood flow responses to hypoxia. It was concluded that peripheral alpha-adrenoceptors affect the distribution of regional microvascular and blood flow responses to hypoxia, and central alpha-adrenoceptors probably do not participate in this effect.     

Leptin protects the cardiac myocyte cultures from hypoxic damage

Leptin, a circulating hormone mainly produced by adipose tissue, regulates fatty acid metabolism and causes multiple systemic biological actions even the regulation of cardiovascular function. It is previously known that leptin is a hypoxia-inducible hormone, that hypoxic conditions increase the expression of this peptide in various tissues such as placenta, pancreas and also in the heart. Since leptin receptors are present in the heart, we hypothesized that whether leptin was a protector response for tissues especially for the heart against the deleterious effects of hypoxia. Cultured cardiomyocytes from newborn rats were initially treated with 3000 ng/ml leptin incubation for 1, 5 and 20 h separately, then subjected to 120 min of hypoxia. Hypoxic damage of myocytes was assayed using the measurements of both lactate dehydrogenase and creatine kinase releases into the medium and performing morphological observations (ultrastructural and immunocytochemical) of plates. The obtained results from leptin treated and non-treated control groups were compared to each other, and these data have demonstrated that 5 h of leptin treatment before hypoxia provides a significant protection for cardiomyocytes against hypoxia. Neither 1- nor 20-h leptin treated groups exhibited sufficient protection against hypoxia. In conclusion, leptin protects the cardiomyocyte cultures from hypoxia, but this effect is selective and evident only in the 5-h treated myocytes.     

棕榈酸对模拟高原低氧大鼠离体脑线粒体解耦联蛋白活性及质子漏的影响

本文旨在通过观察棕榈酸对模拟高原低氧大鼠离体脑线粒体解耦联蛋白(uncoupling proteins,UCPs)活性的影响及脑线粒体质子漏与膜电位的改变,探讨UCPs在介导游离脂肪酸对低氧时线粒体氧化磷酸化功能改变中的作用.将SpragueDawley大鼠随机分为对照组、急性低氧组和慢性低氧组.低氧大鼠于低压舱内模拟海拔5 000 m高原23 h/d作低氧暴露,分别连续低氧3 d和30 d.用差速密度梯度离心法提取脑线粒体,[3H-GTP法测定UCPs含量与活性,TPMP 电极与Clark氧电极结合法测量线粒体质子漏,罗丹明123荧光法测定线粒体膜电位.结果显示,低氧使脑线粒体内UCPs含量与活性升高、质子漏增加、线粒体膜电位降低;同时,低氧暴露降低脑线粒体对棕榈酸的反应性,UCPs活性的改变率低于对照组,且线粒体UCPs含量、质子漏、膜电位变化率亦出现相同趋势.线粒体质子漏与反映UCPs活性的Kd值呈线性负相关(P<0.01 r=-0.906),与反映UCPs含量的Bmax呈线性正相关(P<0.01,r=0.856),与膜电位呈线性负相关(P<0.01,r=-0.880).以上结果提示,低氧导致的脑线粒体质子漏增加及膜电位降低与线粒体内UCPs活性升高有关,同时低氧暴露能降低脑线粒体对棕榈酸的反应性,提示在高原低氧环境下,游离脂肪酸升高在维持线粒体能量代谢中起着自身保护和调节机制.