Brain Lactate Is an Obligatory Aerobic Energy Substrate for Functional Recovery After Hypoxia: Further In Vitro Validation

Abstract: This study used the rat hippocampal slice preparation and the monocarboxylate transporter inhibitor, α-cyano-4-hydroxycinnamate (4-CIN), to assess the obligatory role that lactate plays in fueling the recovery of synaptic function after hypoxia upon reoxygenation. At a concentration of 500 µ M , 4-CIN blocked lactate-supported synaptic function in hippocampal slices under normoxic conditions in 15 min. The inhibitor had no effect on glucose-supported synaptic function. Of control hippocampal slices exposed to 10-min hypoxia, 77.8 ± 6.8% recovered synaptic function after 30-min reoxygenation. Of slices supplemented with 500 µ M 4-CIN, only 15 ± 10.9% recovered synaptic function despite the large amount of lactate formed during the hypoxic period and the abundance of glucose present before, during, and after hypoxia. These results indicate that 4-CIN, when present during hypoxia and reoxygenation, blocks lactate transport from astrocytes, where the bulk of anaerobic lactate is formed, to neurons, where lactate is being utilized aerobically to support recovery of function after hypoxia. These results unequivocally validate that brain lactate is an obligatory aerobic energy substrate for posthypoxia recovery of function.     

The mechanism of neuronal resistance and adaptation to hypoxia

In this work we provide a theoretical explanation for the observations that: (i) young animals are more resistant to hypoxia than adult ones and (ii) repeated exposure to a hypoxic insult increases the tolerance of young animals and isolated brain tissue to that insult. Considered here is the role of taurine, a putative Ca2+ transport modulator, in attenuating Ca2+ influx and overload in brain tissue upon hypoxia. It is proposed that the higher resistance of young animals to hypoxia stems from their higher brain content of taurine as compared with adults. The increased resistance to lack of oxygen upon re-exposure to hypoxia may occur as a result of protein and coenzyme A (CoA) breakdown which leads to the accumulation of products like cystine, cysteine, cysteamine and other sulfur-containing compounds. Upon reoxygenation, these compounds are oxidized to form taurine, which in turn attenuates neuronal Ca2+ accumulation. The sulfur-containing compounds are considered to be natural scavengers of oxygen-derived free radicals which are formed upon reoxygenation and have been implicated as a major component in the process leading to ischemic/hypoxic brain damage. Repeated hypoxic insults bring about the formation of higher levels of taurine and hence the observed adaptation to oxygen lack. The hypothesis presented here is supported by experimental observations in our laboratory and those of others.     

Preconditioning improves function and recovery of single muscle fibers during severe hypoxia and reoxygenation

Reperfusion following prolonged ischemia induces cellular damage in whole skeletal muscle models. Ischemic preconditioning attenuates the deleterious effects. We tested whether individual skeletal muscle fibers would be similarly affected by severe hypoxia and reoxygenation (H/R) in the absence of extracellular factors and whether cellular damage could be alleviated by hypoxic preconditioning. Force and free cytosolic Ca2+ ([Ca2+]c) were monitored in Xenopus single muscle fibers (n = 24) contracting tetanically at 0.2 Hz during 5 min of severe hypoxia and 5 min of reoxygenation. Twelve cells were preconditioned by a shorter bout of H/R 1 h before the experimental trial. In preconditioned cells, force relative to initial maximal values (P/P(o)) and relative peak [Ca2+]c fell (P < 0.05) during 5 min of hypoxia and recovered during reoxygenation. In contrast, P/P(o) and relative peak [Ca2+]c fell more during hypoxia (P < 0.05) and recovered less during reoxygenation (P < 0.05) in control cells. The ratio of force to [Ca2+]c was significantly higher in the preconditioned cells during severe hypoxia, suggesting that changes in [Ca2+]c were not solely responsible for the loss in force. We conclude that 1) isolated skeletal muscle fibers contracting in the absence of extracellular factors are susceptible to H/R injury associated with changes in Ca2+ handling; and 2) hypoxic preconditioning improves contractility, Ca2+ handling, and cell recovery during subsequent hypoxic insult.     

Effects of Hypoxia on the Catecholamine Release, Ca2+ Uptake, and Cytosolic Free Ca2+ Concentration in Cultured Bovine Adrenal Chromaffin Cells

The purpose of the present study is to clarify the effects of hypoxia on catecholamine release and its mechanism of action. For this purpose, using cultured bovine adrenal chromaffin cells, we examined the effects of hypoxia on high (55 mM) K(+)-induced increases in catecholamine release, in cytosolic free Ca2+ concentration ([Ca2+]i), and in 45Ca2+ uptake. Experiments were carried out in media preequilibrated with a gas mixture of either 21% O2/79% N2 (control) or 100% N2 (hypoxia). High K(+)-induced catecholamine release was inhibited by hypoxia to approximately 40% of the control value, but on reoxygenation the release returned to control levels. Hypoxia had little effect on ATP concentrations in the cells. In the hypoxic medium, [Ca2+]i (measured using fura-2) gradually increased and reached a plateau of approximately 1.0 microM at 30 min, whereas the level was constant in the control medium (approximately 200 nM). High K(+)-induced increases in [Ca2+]i were inhibited by hypoxia to approximately 30% of the control value. In the cells permeabilized by digitonin, catecholamine release induced by Ca2+ was unaffected by hypoxia. Hypoxia had little effect on basal 45Ca2+ uptake into the cells, but high K(+)-induced 45Ca2+ uptake was inhibited by hypoxia. These results suggest that hypoxia inhibits high K(+)-induced catecholamine release and that this inhibition is mainly the result of the inhibition of high K(+)-induced increases in [Ca2+]i subsequent to the inhibition of Ca2+ influx through voltage-dependent Ca2+ channels.     

Effects of taurine on the electrical and mechanical activities of embryonic chick heart

The effect of taurine (2-aminoethanesulphonic acid) on myocardial slow action potentials (APs) and accompanying contractions was examined in isolated perfused chick hearts and reaggregated cultured cells. Isoproterenol (ISO), histamine (HIS), or tetraethylammonium (TEA) induced slow APs and contractions in hearts whose fast Na+ channels had been inactivated by elevated K+. Taurine (10 mM) not only failed to induce slow APs, but actually decreased ISO (10(-8) M), HIS (10(-4) M), or TEA (10 mM) induced slow APs and contractions transiently (about 30s-2 min after the addition of taurine). The properties of the slow APs recovered to control levels by 7-13 min after the addition of the taurine; at this time, there was an increase in developed tension of the contraction accompanying the slow APs. These results suggest that the positive inotropic action of taurine is not mediated through an increase in the slow inward Ca2+ current. However, the transient depression of Ca2+-dependent slow APs by taurine probably explains the transient negative inotropic effect of taurine.     

Effects of hypoxia on the contractions and lactate release induced by high-K+, ouabain and epinephrine in guinea-pig aorta.

1. The 60 mM K+, 152 mM K+, Na-deficient medium and oubain-induced contractions of aorta were not so affected by severe hypoxia. 2. The 60 mM K+, 152 mM K+, Na(+)-deficient medium-induced responses were greatly reduced by deprivation of external Ca2+ in normoxia. 3. As the concentration of epinephrine increased, the remaining tensions which were expressed as a percentage of the original tensions became progressively greater in hypoxic condition. 4. The percentage of resistant components of the norepinephrine-induced contraction by the lower concentration was further reduced in Ca(2+)-free medium by severe hypoxic condition. 5. The tensions under normoxia and lactate release under severe hypoxia induced by 60 mM K+ or 2.5 x 10(-6) M epinephrine were of the same extent. 6. In conclusion, the inhibition of aortic response to epinephrine with severe hypoxia could not solely be explained by depression of the oxygen supply into the oxidative metabolism. Severe hypoxia did not affect Ca2+ influx through voltage-operated Ca2+ channels, but reduced both receptor-operated Ca2+ influx and intracellular Ca2+ release in the aorta.     

Unusual sensitivity of cytosolic free Ca2+ to changes in extracellular Ca2+ in rat C-cells

An essential function of C-cells is to monitor extracellular Ca2+ concentration ([Ca2+]e) and to respond to changes in [Ca2+]e by regulating hormone secretion. Using the calcitonin-secreting rat C-cell line rMTC 44-2, we have investigated a possible tight linkage between [Ca2+]e and cytosolic free Ca2+ ([Ca/+]i). We have demonstrated, using the Ca2+ indicator Quin 2, that the [Ca2+]i is particularly sensitive to changes in [Ca2+]e. Sequential increases in [Ca2+]e as small as 0.1 mM evoke clear elevations in [Ca2+]i. In contrast, other cell types tested did not alter their [Ca2+]i in response to increasing [Ca2+]e even to levels as high as 4.0 mM. Sequential 1.0 mM increments in [Ca2+]e caused the [Ca2+]i to rise from a base line of 357 +/- 20 nM Ca2+i at 1.0 mM Ca2+e to a maximum of 1066 +/- 149 nM Ca2+i at 5.0 mM Ca2+e. [Ca2+]e above 2.0 mM produced a biphasic response in [Ca2+]i consisting of an immediate (less than 5 s) spike followed by a decay to a new plateau. Treatment of rMTC 44-2 cells with either 50 mM K+ or 100 nM ionomycin at 1.0 mM Ca2+e caused an immediate spike in [Ca2+]i to micromolar levels. Pretreatment with EGTA or verapamil inhibited completely the increase in [Ca2+]i induced by 50 mM K+. However, pretreatment with EGTA only slightly attenuated the spike phase in [Ca2+]i produced by ionomycin, demonstrating that ionomycin released intracellular stores of calcium. We conclude that rMTC 44-2 cells regulate [Ca2+]i by monitoring small physiological changes in [Ca2+]e, the primary secretagogue for C-cells.     

Protection against cell damage due to hypoxia and reoxygenation: The role of taurine and the involved mechanisms

Summary The effect of taurine on cell viability and metabolism of human colon and porcine renal cells was investigated during and after hypoxia. Taurine administered during hypoxia markedly reduced cellular deterioration due to hypoxia and reoxygenation and led to a significantly greater recovery of cellular function following the hypoxic insult. The responsible mechanisms for the beneficial effects were an improvement in osmotic status and calcium homeostasis and an induction in cellular growth despite oxygen deficiency and reoxygenation. Free oxygen radical generation and lipid membrane peroxidation were not reduced by taurine. Taurine acted as a potent endogenous agent with multifactorial effects against cellular damage due to hypoxia and reoxygenation.     

Suppression of Ca2+ oscillations in cultured rat hepatocytes by chemical hypoxia

The model of "chemical hypoxia" with KCN plus iodoacetic acid mimics the ATP depletion and reductive stress of hypoxia. Here, we examined the effects of chemical hypoxia on cytosolic free Na+ and Ca2+ in single cultured rat hepatocytes by multiparameter digitized video microscopy and ratio imaging of sodium-binding furan indicator (SBFI) and Fura-2. Intracellular Na+ increased from about 10 mM to more than 100 mM after 20 min of chemical hypoxia, whereas cytosolic free Ca2+ remained virtually unchanged. In normoxic hepatocytes, phenylephrine (50 microM) and Arg-vasopressin (20-40 nM) induced Ca2+ oscillations in 70 and 40% of cells, respectively. These Ca2+ oscillations were suppressed after one spike following the onset of chemical hypoxia. Phenylephrine and vasopressin also increased inositol phosphate formation by 22 and 147%, respectively. This effect was suppressed by KCN plus iodoacetate. Intracellular acidosis is characteristic of chemical hypoxia. Intracellular acidosis induced by 40 mM Na-acetate suppressed Ca2+ oscillations but did not inhibit hormone-induced inositol phosphate formation. Cytosolic alkalinization also suppressed Ca2+ oscillations. However, prevention of intracellular acidosis with monensin (10 microM) did not prevent suppression of Ca2+ oscillations during chemical hypoxia. Mitochondrial depolarization with uncoupler did not change free Ca2+ levels during chemical hypoxia, indicating that mitochondria do not regulate free Ca2+ during chemical hypoxia. From these results, we conclude: 1) chemical hypoxia does not block Na+ influx across the plasma membrane; 2) Chemical hypoxia inhibits hormone-stimulated Ca2+ flux pathways across cellular membranes by two different mechanisms: (a) by ATP depletion, which disrupts hormone-myo-inositol 1,4,5-triphosphate coupling, and (b) by intracellular acidosis, which inhibits myo-inositol 1,4,5-triphosphate-stimulated Ca2+ release from intracellular stores; 3) during ATP depletion by chemical hypoxia, mitochondria do not take up Ca2+ to maintain cytosolic free Ca2+ at low concentrations.     

Dose-Dependent, K+-Stimulated Efflux of Endogenous Taurine from Primary Astrocyte Cultures Is Ca2+-Dependent

The K+-stimulated efflux of endogenous taurine from primary rat cerebellar astrocyte cultures prepared from 7-9-day-old rats was studied at 16-18 days in vitro using HPLC analysis. Taurine efflux was dose-dependent at K+ concentrations between 10 mM and 80 mM, with an EC50 of approximately 50 mM. Maximum stimulation of efflux above basal levels ranged from 56% at 10 mM K+ (204 pmol/min/mg protein) to 470% at 80 mM K+ (960 pmol/min/mg protein). Removal of Ca2+ from the buffer and the addition of either 1 mM EGTA or 10 mM Mg2+ abolished K+-stimulated efflux. Taurine efflux peaked and fell in parallel with the K+ concentration, but with an approximate lag of 3-5 min. The time course and amount of preloaded [3H]taurine released did not differ significantly from that seen for endogenous efflux. Basal taurine efflux varied inversely with the extracellular concentration of Ca2+ over the concentration range 0-5.0 mM. The observed Ca2+ dependence is consistent with a role for Ca2+ in the regulation of taurine release. Furthermore, taurine release from astrocytes in response to elevated K+ may reflect a neuromodulatory role for this amino acid in the CNS.     

Early hypoxic contracture of the myocardium in adult and newborn rats]

The effect of 30 min substrate free hypoxia (H) on isometric tension was studied in isolated myocardium (M) of adult (A) and newborn (N) rats. The perfusion with 50% Na+ H solution caused in AM the development of H contracture which was more than 50% higher than control contracture. H perfusion with 0.1 mM Ca2+, 1.0 mM La3+, and 10.0 mM of caffeine provides the discrimination of control and hypoNa+ contractures. It is assumed that early H contracture in AM is a result of inability of Ca-sequestering system to accumulate intracellular Ca2+ and Ca2+ influxing through the sarcolemma. In myocardium of N rats Na-Ca exchange is proposed as a main source of Ca2+ for H contracture development.     

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.     

缺氧诱导心肌细胞凋亡与Caspase-3激活及细胞内钙超载的关系

目的: 研究缺氧对心肌细胞Caspases的激活效应与细胞内钙的关系.方法: 将培养的心肌细胞暴露于95% N2/5% CO2混合气体培养室进行缺氧处理,缺氧0、30 min、1、3、6、12、24 h后,应用激光共聚焦显微镜检测缺氧早期心肌细胞胞浆游离钙的变化;应用细胞内钙螯合剂、Caspase-1与Caspase-3特异性抑制剂预处理心肌细胞后缺氧12 h,检测DNA片段化与Caspase-3活性;Western blot、RT-PCR方法分别检测细胞线粒体与胞浆中Cyt c蛋白的变化以及Caspase-3 mRNA表达的改变.结果: 缺氧后30 min心肌细胞胞浆游离钙即显著增高,1 h达到峰值.缺氧3 h线粒体Cyt c无显著变化,而胞浆中Cyt c开始增多,至缺氧12 h,线粒体中Cyt c仅见一弱阳性条带,而胞浆呈强阳性反应,缺氧24 h 线粒体中已不能检测到Cyt c.缺氧3 h心肌细胞Caspase-3 mRNA上调,在观察的24 h内持续处于高表达状态.分别应用Caspase-3抑制剂和细胞内钙螯合剂预处理心肌细胞后均可使心肌细胞Caspase-3活性降低并完全阻断DNA片段化发生,细胞内钙螯合剂与Caspase-3抑制剂联合应用使凋亡细胞百分率降低的程度比分别单独使用两者更明显.结论: 缺氧可致心肌细胞线粒体Cyt c释放至胞浆,导致Caspase途径激活,从而导致细胞凋亡.缺氧所致心肌细胞钙超载在时序上早于线粒体Cyt c释放与Caspase-3的激活,螯合细胞内钙可阻断缺氧诱导的Caspase-3激活,并拮抗心肌细胞凋亡的发生,因此细胞钙超载是启动线粒体Cyt c释放与Caspase-3激活的一个早期重要分子事件.     

Action of tetanus toxin on cholinergic neuroblastoma X glioma hybrid cells: selective blockade of Ca spikes

We examined the effect of tetanus toxin on clonal neuroblastoma X glioma hybrid cells, NG108-15, by intracellular microelectrode studies of passive membrane electrical properties and action potentials generated under various conditions. Binding of tetanus toxin to the surface of the cells was demonstrated by indirect immunofluorescent staining but no morphological alteration was observed in tetanus toxin-treated cells under a phase contrast microscope. These is no significant difference between the tetanus toxin-treated and untreated cells in their passive electrical membrane properties, i.e. resting membrane potentials, input resistances, time constants and input capacities. Cells in 120 mM Na+, 2 mM Ca2+ salt solution showed Na spikes, and cells in high Ca2+ (30 mM), Na+-free salt solution showed Ca spikes in response to depolarizing current pulses. While the Na spike was not affected by tetanus toxin, the Ca spike was blocked by the toxin. The minimum dose of tetanus toxin for maximum suppression of the peak potential level of the Ca spike was 250 ng/ml. Addition of tetraethyl ammonium (TEA) to extracellular fluid enhanced the Ca spike in untreated cells. In toxin-treated cells, TEA did not alter the effect of tetanus toxin on the Ca spike. Blockade of the Ca spike by tetanus toxin could be detected even at low extracellular Ca2+ concentration (10 mM) by adding TEA to the extracellular fluid and adjusting the membrane potential to a steady hyperpolarized level (-80 mV) to ensure optimal and uniform electrical responses. The usefulness of NG108-15 hybrid cells for in vitro investigations on the mechanism of action of tetanus toxin was discussed.     

Aminoglycoside antibiotics affect hippocampal LTP: a comparative study with the N-type calcium antagonist omega-conotoxin-GVIA.

The in vitro activity of N-type calcium antagonists such as omega-conotoxin-GVIA and the aminoglycoside antibiotics neomycin and streptomycin was studied in rat hippocampal slices. The effects of the drugs were tested on basal CA1 synaptic transmission and on the hippocampal long-term potentiation (LTP) induced by tetanic electrical stimulation and by increasing (4mM) the calcium concentration. Omega-conotoxin-GVIA, neomycin and streptomycin were able to significantly reduce the amplitude of the CA1 population spike at 1 microM, 0.5 mM and 1 mM, respectively. In addition, the drugs affected the induction and maintenance of the CA1 tetanic and calcium-induced LTP at concentrations which did not modify the magnitude of the control CA1 population spike. Omega-conotoxin-GVIA (0.5 microM), neomycin (0.3 mM) and streptomycin (0.7 mM) perfused for 60 min, before inducing LTP, prevented the subsequent increase of the CA1 population spike in all the experiments. The same concentrations of these drugs perfused for 60-min after a previously established LTP significantly reduced the amplitude of the CA1 population spike. The results promote a role for the N-type calcium channels and for the release of neurotransmitters in both the induction and the maintenance of hippocampal LTP.     

Protective effect of gap junction uncouplers given during hypoxia against reoxygenation injury in isolated rat hearts

It has been shown that cell-to-cell chemical coupling may persist during severe myocardial hypoxia or ischemia. We aimed to analyze the effects of different, chemically unrelated gap junction uncouplers on the progression of ischemic injury in hypoxic myocardium. First, we analyzed the effects of heptanol, 18alpha-glycyrrhetinic acid, and palmitoleic acid on intracellular Ca2+ concentration during simulated hypoxia (2 mM NaCN) in isolated cardiomyocytes. Next, we analyzed their effects on developed and diastolic tension and electrical impedance in 47 isolated rat hearts submitted to 40 min of hypoxia and reoxygenation. All treatments were applied only during the hypoxic period. Cell injury was determined by lactate dehydrogenase (LDH) release. Heptanol, but not 18alpha-glycyrrhetinic acid nor palmitoleic acid, attenuated the increase in cytosolic Ca2+ concentration induced by simulated ischemia in cardiomyocytes and delayed rigor development (rigor onset at 7.31 +/- 0.71 min in controls vs. 14.76 +/- 1.44 in heptanol-treated hearts, P < 0.001) and the onset of the marked changes in electrical impedance (tissue resistivity: 4.02 +/- 0.29 vs. 7.75 +/- 1.84 min, P = 0.016) in hypoxic rat hearts. LDH release from hypoxic hearts was minimal and was not significantly modified by drugs. However, all gap junction uncouplers, given during hypoxia, attenuated LDH release during subsequent reoxygenation. Dose-response analysis showed that increasing heptanol concentration beyond the level associated with maximal effects on cell coupling resulted in further protection against hypoxic injury. In conclusion, gap junction uncoupling during hypoxia has a protective effect on cell death occurring upon subsequent reoxygenation, and heptanol has, in addition, a marked protective effect independent of its uncoupling actions.     

Pulmonary vascular tone is increased by a voltage-dependent calcium channel potentiator

The mechanism of hypoxia-induced pulmonary vasoconstriction remains unknown. To explore the possible dependence of the hypoxic response on voltage-activated calcium (Ca2+) channels, the effects of BAY K 8644 (BAY), a voltage-dependent Ca2+ channel potentiator, were observed on the pulmonary vascular response to hypoxia of both the intact anesthetized dog and the perfused isolated rat lung. In six rat lungs given BAY (1 X 10(-6)M), hypoxia increased mean pulmonary arterial pressure (Ppa) to 30.5 +/- 1.7 (SEM) Torr compared with 14.8 +/- 1.2 Torr for six untreated rat lungs (P less than 0.01). After nifedipine, the maximum Ppa during hypoxia fell 14.1 +/- 2.4 Torr from the previous hypoxic challenge in the BAY-stimulated rats (P less than 0.01). BAY (1.2 X 10(-7) mol/kg) given during normoxia in seven dogs increased pulmonary vascular resistance 2.5 +/- 0.3 to 5.0 +/- 1.2 Torr X 1(-1) X min (P less than 0.05), and systemic vascular resistance 55 +/- 4.9 to 126 +/- 20.7 Torr X 1(-1) X min (P less than 0.05). Systemic mean arterial pressure rose 68 Torr, whereas Ppa remained unchanged. Administration of BAY during hypoxia produced an increase in Ppa: 28 +/- 1.5 to 33 +/- 1.9 Torr (P less than 0.05). Thus BAY, a Ca2+ channel potentiator, enhances the hypoxic pulmonary response in vitro and in vivo. This, together with the effect of nifedipine on BAY potentiation, suggests that increased Ca2+ channel activity may be important in the mechanism of hypoxic pulmonary vasoconstriction.     

Depolarization Increases Chloride-Dependent Glutamate Sequestration in Synaptic Membranes of Rat Cerebral Cortex

To assess the functions of Cl- -dependent glutamate "binding" (Cl- -dependent glutamate uptake) in synaptic membranes, possible effects of depolarization on the uptake were examined. When rat cerebral cortical slices were preincubated with depolarizing agents such as veratrine (7 micrograms/ml), 10 microM aconitine, 56 mM K+, and 50 microM monensin, [3H]glutamate uptake by the crude synaptic membranes, which were subsequently prepared from the pretreated slices, was increased by 60-85%. Stimulation of the glutamate uptake by predepolarization was dependent on Na+ but not on Ca2+. The bindings of gamma-[3H]aminobutyric acid and 5-[3H]hydroxytryptamine were not significantly affected by the predepolarization. Veratrine pretreatment increased the maximal density of the glutamate uptake sites without affecting the affinity for glutamate. Several characteristics of the uptake sites increased by the veratrine pretreatment coincided with those of Cl- -dependent glutamate uptake sites. Na+-dependent glutamate binding (Na+-dependent glutamate uptake) to the membranes was not affected by pretreatment with veratrine. The content of endogenous glutamate and the noninulin space in the membrane fractions were not changed by the predepolarization. The increase in the glutamate uptake induced by pretreatment with high K+ was reversible: it returned to the control level after a second incubation of the slices in control medium. These results suggest that the Cl- -dependent glutamate sequestration system in synaptic membranes is regulated by the membrane potential.     

Intermittent high altitude hypoxia protects the heart against lethal Ca2+ overload injury

Adaptation to intermittent high altitude (IHA) hypoxia can protect the heart against ischemia-reperfusion injury. In view of the fact that both Ca2+ paradox and ischemia-reperfusion injury are associated with the intracellular Ca2+ overload, we tested the hypothesis that IHA hypoxia may protect hearts against Ca2+ paradox-induced lethal injury if its cardioprotection bases on preventing the development of intracellular Ca2+ overload. Langendorff-perfused hearts from normoxic and IHA hypoxic rats were subjected to Ca2+ paradox (5 min of Ca2+ depletion followed by 30 min of Ca2+ repletion) and the functional, biochemical and pathological changes were investigated. The Ca2+ paradox incapacitated the contractility of the normoxic hearts, whereas the IHA hypoxic hearts significantly preserved contractile activity. Furthermore, the normoxic hearts subjected to Ca2+ paradox exhibited a marked reduction in coronary flow, increase in lactate dehydrogenase release, and severe myocyte damage. In contrast, these changes were significantly prevented in IHA hypoxic hearts. We, then, tested and confirmed our hypothesis that the protective mechanisms are mediated by mitochondria ATP-sensitive potassium channels (mitoKATP) and Ca2+/calmodulin-dependent protein kinase II (CaMKII), as the protective effect of IHA hypoxia was abolished by 5-hydroxydecanoate, a selective mitoKATP blocker, and significantly attenuated by KN-93, a CaMKII inhibitor. In conclusion, our studies offer for the first time that IHA hypoxia confers cardioprotection against the lethal injury of Ca2+ paradox and give biochemical evidence for the protective mechanism of IHA hypoxia. We propose that researches in this area may lead a preventive regimen against myocardial injury associated with Ca2+ overload.     

Protective effect of interleukin-10 on the development of epileptiform activity induced by brief hypoxic episodes in the rat hippocampal slices

The aim of the study was to investigate the effect of interleukin-10 (IL-10) (1 and 10 ng/ml) on the development of epileptiform activity induced by brief hypoxic episodes in CA1 area of rat hippocampal slices. Three three-minute hypoxic episodes induced a sustained decrease in the threshold of evoked population spike (PS) burst and an increase in the number of PSs in the PS response. IL-10 (1 ng/ml) completely abolished the development of epileptiform activity whereas the effect of IL-10 (10 ng/ml) was weaker. The protective effect of IL-10 on the hyperexcitability of the local neuronal network in hippocampal slices indicate that this cytokine can function as an intercellular mediator in the brain. The present results are the first experimental evidence of a protective role of anti-inflammatory IL-10 in the development of epileptiform events induced by brief episodes of hypoxia in the hippocampus.