Adenosine released by astrocytes contributes to hypoxia-induced modulation of synaptic transmission

Astrocytes play a critical role in brain homeostasis controlling the local environment in normal as well as in pathological conditions, such as during hypoxic/ischemic insult. Since astrocytes have recently been identified as a source for a wide variety of gliotransmitters that modulate synaptic activity, we investigated whether the hypoxia-induced excitatory synaptic depression might be mediated by adenosine release from astrocytes. We used electrophysiological and Ca2+ imaging techniques in hippocampal slices and transgenic mice, in which ATP released from astrocytes is specifically impaired, as well as chemiluminescent and fluorescence photometric Ca2+ techniques in purified cultured astrocytes. In hippocampal slices, hypoxia induced a transient depression of excitatory synaptic transmission mediated by activation of presynaptic A1 adenosine receptors. The glia-specific metabolic inhibitor fluorocitrate (FC) was as effective as the A1 adenosine receptor antagonist CPT in preventing the hypoxia-induced excitatory synaptic transmission reduction. Furthermore, FC abolished the extracellular adenosine concentration increase during hypoxia in astrocyte cultures. Several lines of evidence suggest that the increase of extracellular adenosine levels during hypoxia does not result from extracellular ATP or cAMP catabolism, and that astrocytes directly release adenosine in response to hypoxia. Adenosine release is negatively modulated by external or internal Ca2+ concentrations. Moreover, adenosine transport inhibitors did not modify the hypoxia-induced effects, suggesting that adenosine was not released by facilitated transport. We conclude that during hypoxia, astrocytes contribute to regulate the excitatory synaptic transmission through the release of adenosine, which acting on A1 adenosine receptors reduces presynaptic transmitter release. Therefore, adenosine release from astrocytes serves as a protective mechanism by down regulating the synaptic activity level during demanding conditions such as transient hypoxia.     

星形胶质细胞体外缺血缺氧损伤后炎症模型的建立及应用

目的 建立SD大鼠星形胶质细胞缺血缺氧损伤后炎症模型,探讨亚低温对星形胶质细胞缺血缺氧及损伤后炎症反应的影响.方法 体外原代培养新生SD大鼠星形胶质细胞,免疫荧光染色检测胶质纤维酸性蛋白(GFAP)进行鉴定.实验分正常对照组(C组)、单纯缺氧组(H组)和缺氧+白细胞组(H+W组),以无糖DMEM培养基、5%CO2+95%N2混合气体培养4 h诱导细胞缺氧模型.H+W组加入1 mLSD大鼠外周血白细胞(1×106/mL),C组加入等量培养液.将3组细胞分别置入37℃、34℃、32℃、30 ℃ CO2孵箱中作用24 h,应用速率法和LIVE/DEAD染色分别检测3组细胞在不同温度下乳酸脱氢酶(LDH)释放率的变化和形态学变化.结果 缺氧4 h即可造成星形胶质细胞的损伤.37℃时C组、H组、H+W组细胞LDH释放率依次升高,差异有统计学意义(P<0.05);与37℃相比,亚低温状态(34℃、32℃)下H组、H+W组细胞LDH释放率均降低;但在30℃时,则有明显升高,与32℃相比差异具统计学意义(P<0.05).结论 亚低温状态可明显降低星形胶质细胞的缺血缺氧性损伤及炎症性损伤,其机制可能并非通过单纯的抑制代谢来实现的.     

Fructose-1,6-bisphosphate stabilizes brain intracellular calcium during hypoxia in rats.

BACKGROUND AND PURPOSE: Exogenously administered fructose-1,6-bisphosphate reduces neuronal injury from hypoxic or ischemic brain insults. To test the hypothesis that fructose-1,6-bisphosphate prevents changes in intracellular calcium ([Ca2+]i) and high-energy phosphate levels, we measured [Ca2+]i, intracellular pH (pHi), and adenosine triphosphate in cultured rat cortical astrocytes and cortical brain slices during hypoxia. METHODS: The fluorescent indicators fura-2 and bis-carboxyethyl-carboxyfluorescein were used to simultaneously measure [Ca2+]i and pHi with a fluorometer. RESULTS: Exposure to hypoxia (95% N2, 5% CO2) or 100 microM sodium cyanide produced transient increases in [Ca2+]i in astrocytes and sustained increases in [Ca2+]i in brain slices. Adenosine triphosphate levels fell in slices exposed to hypoxia or cyanide. Fructose-1,6-bisphosphate (3.5 mM) blocked increases in [Ca2+]i and prevented depletion of adenosine triphosphate. Fructose-1,6-bisphosphate also partially prevented adenosine triphosphate depletion in brain slices incubated in glucose-free medium. Iodoacetate (a specific inhibitor of glycolysis) elevated [Ca2+]i and partially prevented these actions of fructose-1,6-bisphosphate. Changes in pHi during hypoxia were not affected by fructose-1,6-bisphosphate. CONCLUSIONS: Fructose-1,6-bisphosphate supports adenosine triphosphate production via stimulation of glycolysis and results in the maintenance of normal [Ca2+]i during hypoxia or hypoglycemia.     

Exposure of cultured primary rat astrocytes to hypoxia results in intracellular glucose depletion and induction of glycolytic enzymes

Based on the neurotrophic properties of astrocytes in response to ischemia, the current work focuses on the mechanism for cultured astrocytes to adapt to a hypoxic environment. Intracellular glucose levels in primary cultured rat astrocytes exposed to hypoxia fell by 30% within 24 h, in parallel with a decrease in glycogen stores. Glycolytic metabolism was crucial for cell survival during hypoxia, as 2-deoxyglucose resulted in rapid ATP depletion and cell death. The mechanism for maintaining glucose levels under these conditions appeared to be mobilization of glycogen stores, rather than increased extracellular uptake of glucose, as gluconolactone (an inhibitor of beta1-4 amyloglucosidase) induced a rapid fall in cellular ATP in cultures subjected to hypoxia, whereas cytochalasin B was without affect. Addition of cycloheximide diminished the viability of astrocytes in hypoxia, suggesting an obligatory role of de-novo gene expression to respond to hypoxia. Consistently, the results of differential display suggested the induction of glycolytic enzymes, including aldolase A (EC 4.1.2.13), hexokinase II (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1), and triosephosphate isomerase (EC 5.3.1.1) in the hypoxic culture. Marked induction of these glycolytic enzymes in hypoxic astrocytes was confirmed by Northern blot analysis. These data provide a theoretical basis to understand the ability of astrocytes to tolerate ischemic condition.     

Hypoxia-induced dysfunctions and injury of astrocytes in primary cell cultures

The effects of severe hypoxia were studied in a primary culture of astrocytes prepared from newborn rat cerebral cortex. Hypoxia was created by placing cultures in an airtight chamber that was flushed with 95% N2/5% CO2 for 15 min before being sealed. The hypoxic environment was maintained constant for up to 24 h. During the first 12 h of hypoxia, astrocytes showed no morphological changes by phase-contrast microscopy. After 18 h of hypoxia, some astrocytes in culture became swollen and started to detach from the culture dish. All cells in the culture were destroyed after 24 h of hypoxia. The lactate dehydrogenase level in the culture medium increased more than tenfold between 12 and 24 h of hypoxia. Glutamate uptake was inhibited 80% by similar hypoxic conditions. The cell volume of astrocytes, as measured by 3-O-methyl-[14C]-D-glucose uptake, was increased. These observations suggested cell membrane dysfunction. The malondialdehyde level of hypoxic cultures increased two-fold after 24 h of hypoxia. Verapamil (0.5 mM), furosemide (1 mM), indomethacin (1 mM), MgCl2 (10 mM), and mannitol (10 mM) reduced but never completely abolished the release of lactate dehydrogenase from hypoxic astrocytes. These data suggest multifactorial causes for severe injury in hypoxic astrocytes.     

Endogenous erythropoietin from astrocyte protects the oligodendrocyte precursor cell against hypoxic and reoxygenation injury

The hypoxia-responsive cytokine erythropoietin (EPO) provides neuroprotective effects in the damaged brain during ischemic events and neurodegenerative diseases. The purpose of the present study is to evaluate the EPO/EPO receptor (EPOR) endogenous system between astrocyte and oligodendrocyte precursor cell (OPC) under hypoxia. We report here elevated EPO mRNA levels and protein release in cultured astrocytes following hypoxic stimulation by quantitative RT-PCR and ELISA. Furthermore, the EPOR gene expressions were detected in cultured OPCs as in astrocytes and microglias by quantitative RT-PCR. Cell staining revealed the EPOR expression in OPC. To evaluate the protective effect of endogenous EPO from astrocyte to OPCs, EPO/EPOR signaling was blocked by EPO siRNA or EPOR siRNA gene silencing in in vitro study. The suppression of endogenous EPO production in astrocytes by EPO siRNA decreased the protection to OPCs against hypoxic stress. Furthermore, OPC with EPOR siRNA had less cell survival after hypoxic/reoxygenation injury. This suggested that EPO/EPOR signaling from astrocyte to OPC could prevent OPC damage under hypoxic/reoxygenation condition. Our present finding of an interaction between astrocytes and OPCs may lead to a new therapeutic approach to OPCs for use against cellular stress and injury.     

Rat cultured astrocytes release guanine-based purines in basal conditions and after hypoxia/hypoglycemia

Brain ischemia stimulates release from astrocytes of adenine-based purines, particularly adenosine, which is neuroprotective. Guanosine, which has trophic properties that may aid recovery following neurological damage, is present in high local concentrations for several days after focal cerebral ischemia. We investigated whether guanine-based purines, like their adenine-based counterparts, were released from astrocytes and whether their release increased following hypoxia/hypoglycemia. HPLC analysis of culture medium of rat astrocytes showed spontaneous release of endogenous guanine-based purines at a higher rate than their adenine-based counterparts. The concentration of guanosine (≈120 nM) and adenosine (≈43 nM) in the culture medium remained constant, whereas concentrations of adenine and guanine nucleotides, particularly GMP, and their metabolites increased with time. Exposure of the cultures to hypoxia/hypoglycemia for 30 min increased the extracellular concentration of adenine-based purines by 2.5-fold and of guanine-based purines by 3.5-fold. Following hypoxia/hypoglycemia extracellular adenine nucleotide levels increased further. Adenosine concentration increased, but not proportionally to nucleotide levels. Accumulation of adenosine metabolites indicated it was rapidly metabolized. Conversely, the concentrations of extracellular guanine-based nucleotides remained elevated and the concentration of guanosine continued to increase. These data indicate that astrocytes are a major source of guanine-based purines, the release of which is markedly increased following hypoxia/hypoglycemia, permitting them to exert neurotrophic effects. GLIA 25:93–98, 1999. © 1999 Wiley-Liss, Inc.     

Effects of two nucleoside transport inhibitors, dipyridamole and soluflazine, on purine release from the rat cerebral cortex

The effects of two nucleoside transport inhibitors, dipyridamole and soluflazine, on adenosine, inosine and oxypurine release from the normoxic and hypoxic/ischemic rat cerebral cortex have been studied. Dipyridamole (500 μg/kg) enhanced adenosine release during hypoxic/ischemic challenges in comparison with saline-injected controls. It decreased the hypoxia/ischemia-elicited releases of inosine, hypoxanthine and xanthine. Both basal and hypoxia/ischemia-elicited releases of uric acid were elevated. Soluflazine, administered topically or systemically, failed to enhance adenosine release and did not consistently alter the hypoxia/ischemia-evoked releases of inosine, hypoxanthine and xanthine. Basal release of uric acid was elevated. The failure of either drug to elevate the basal or hypoxia/ischemia-evoked releases of adenosine above predrug levels illustrates one of the problems which may be inherent in the use of bidirectional nucleoside transport inhibitors for the manipulation of adenosine levels in the cerebral interstitial fluid.     

Adenosine triphosphate accelerates recovery from hypoxic/hypoglycemic perturbation of guinea pig hippocampal neurotransmission via a P(2) receptor

The present study was designed to assess the effects of adenosine triphosphate (ATP) on hippocampal neurotransmissions under the normal and hypoxic/hypoglycemic conditions. ATP reversely depressed population spikes (PSs), which were monitored in the dentate gyrus of guinea pig hippocampal slices, in a dose-dependent manner at concentrations ranged from 0.1 micro M to 1 mM. A similar depression was obtained with the P(2) receptor agonist, alpha,beta-methylene ATP (alpha,beta-MeATP), and the effect was inhibited by the P(2) receptor antagonists, suramin and PPADS. The inhibitory action of ATP or alpha,beta-MeATP was inhibited by the gamma-aminobutyric acid(A) (GABA(A)) receptor antagonist, bicuculline, but it was not affected by theophylline, a broad inhibitor of adenosine (P(1)) receptors, tetraethylammonium, a broad inhibitor of K(+) channels, or ecto-protein kinase inhibitors. ATP or alpha,beta-MeATP enhanced GABA release from guinea pig hippocampal slices, that was inhibited by deleting extracellular Ca(2+) or in the presence of tetrodotoxin, while ATP had no effect on GABA release from cultured rat hippocampal astrocytes or postsynaptic GABA-gated channel currents in cultured rat hippocampal neurons. Twenty-minutes deprivation of glucose and oxygen from extracellular solution abolished PSs, the amplitude recovering to about 30% of basal levels 50 min after returning to normal conditions. ATP or alpha,beta-MeATP accelerated the recovery after hypoxic/hypoglycemic insult (approximately 80% of basal levels). Adenosine diphosphate and adenosine monophosphate accelerated the recovery, but to a much lesser extent, and adenosine had no effect. The results of the present study thus suggest that ATP inhibits neuronal activity by enhancing neuronal GABA release via a P(2) receptor, perhaps a P2X receptor, thereby protecting against hypoxic/hypoglycemic perturbation of hippocampal neurotransmission.     

Role of central adenosine in the respiratory and thermoregulatory responses to hypoxia

No reports are available about the role of central adenosine in the respiratory and thermoregulatory responses to hypoxia in conscious rats. We therefore measured ventilation (VE) and body temperature (Tb) before and after intracerebroventricular injection of saline or aminophylline (adenosine antagonist), followed by a 30-min period of hypoxia exposure. Aminophylline did not change VE or Tb during normoxia; however, during hypoxia, it caused a significant increase in VE, and significantly attenuated hypoxic hypothermia. The present data indicate that central adenosine has an inhibitory effect on hypoxic hyperventilation and partially causes hypoxic hypothermia, suggesting that the ventilatory and metabolic interaction during hypoxia does not involve opposing mechanisms.     

Piracetam and vinpocetine exert cytoprotective activity and prevent apoptosis of astrocytes in vitro in hypoxia and reoxygenation

The aim of the present study was to establish whether piracetam (2-pyrrolidon-N-acetamide; PIR) and vinpocetine (a vasoactive vinca alkaloid; VINP) are capable of protecting astrocytes against hypoxic injury. Using the model of astrocyte cell culture we observed the cells treated with PIR and VINP during and after in vitro simulated hypoxia. Cell viability was determined by Live/Dead Viability/Cytotoxicity Assay Kit, LDH release assay and MTT conversion test. Apoptotic cell death was distinguished by a method of Hoechst 33342 staining underfluorescence microscope and caspase-3 colorimetric assay. In addition the intracellular levels of ATP and phosphocreatine (PCr) were evaluated by bioluminescence method. Moreover, the effect of the drugs on the DNA synthesis was evaluated by measuring the incorporation of [3H]thymidine into DNA of astrocytes. PIR (0.01 and 1 mM) and VINP (0.1 and 10 microM) were added to the medium both during 24 h normoxia, 24 h hypoxia or 24 h reoxygenation. Administration of 1 mM PIR or 0.1 microM VINP to the cultures during hypoxia significantly decreases the number of dead and apoptotic cells. The antiapoptic effects of drugs in the above mentioned concentrations was also confirmed by their stimulation of mitochondrial function, the increase of intracellular ATP, and the inhibition of the caspase-3 activity. The prevention of apoptosis was accompanied by the increase in ATP and PCr levels and increase in the proliferation of astrocytes exposed to reoxygenation. The higher concentration of VINP (10 microM) was detrimental in hypoxic conditions. Our experiment proved the significant cytoprotective effect of 1 mM PIR and 0.1 microM VINP on astrocytes in vitro.     

Neuroprotective mechanisms of DNA methyltransferase in a mouse hippocampal neuronal cell line after hypoxic preconditioning

Hypoxic preconditioning has been shown to improve hypoxic tolerance in mice, accompanied by the downregulation of DNA methyltransferases(DNMTs) in the brain. However, the roles played by DNMTs in the multiple neuroprotective mechanisms associated with hypoxic preconditioning remain poorly understood. This study aimed to establish an in vitro model of hypoxic preconditioning, using a cultured mouse hippocampal neuronal cell line(HT22 cells), to examine the effects of DNMTs on the endogenous neuroprotective mechanisms that occur during hypoxic preconditioning. HT22 cells were divided into a control group, which received no exposure to hypoxia, a hypoxia group, which was exposed to hypoxia once, and a hypoxic preconditioning group, which was exposed to four cycles of hypoxia. To test the ability of hypoxic preadaptation to induce hypoxic tolerance, cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium assay. Cell viability improved in the hypoxic preconditioning group compared with that in the hypoxia group. The effects of hypoxic preconditioning on the cell cycle and apoptosis in HT22 cells were examined by western blot assay and flow cytometry. Compared with the hypoxia group, the expression levels of caspase-3 and spectrin, which are markers of early apoptosis and S-phase arrest, respectively, noticeably reduced in the hypoxic preconditioning group. Finally, enzyme-linked immunosorbent assay, real-time polymerase chain reaction, and western blot assay were used to investigate the changes in DNMT expression and activity during hypoxic preconditioning. The results showed that compared with the control group, hypoxic preconditioning downregulated the expression levels of DNMT3A and DNMT3B mRNA and protein in HT22 cells and decreased the activities of total DNMTs and DNMT3B. In conclusion, hypoxic preconditioning may exert anti-hypoxic neuroprotective effects, maintaining HT22 cell viability and inhibiting cell apoptosis. These neuroprotective mechanisms may be associated with the inhibition of DNMT3A and DNMT3B.     

Adenosine A1 receptor activation preferentially protects cultured cerebellar neurons versus astrocytes against hypoxia-induced death

Administration of adenosine A1 receptor agonists in vivo is neuroprotective in various stroke models. Experiments using either mixed cultures of neurons and astrocytes or brain slices, in which several cell types are present, have demonstrated that activation of A1 receptors also is protective against hypoxia and/or hypoglycemia in vitro. In this study, we have examined the effect of the A1 agonist cyclopentyladenosine (CPA) on cellular damage, measured by efflux of lactate dehydrogenase (LDH), in highly enriched primary cultures of either neurons or astrocytes exposed to different metabolic insults. CPA reduced neuronal LDH release induced by a combination of hypoxia and substrate deprivation (“simulated ischemia”; IC₅₀=28 nM) or by hypoxia alone (IC₅₀=170 nM). In contrast, CPA had no effect on neuronal damage induced by substrate deprivation alone, nor did it affect ischemic death to astrocytes. The neuroprotective effects of CPA during simulated ischemia and hypoxia were reversed by the A1 antagonist 1, 3-dipropyl-8-cyclopentylxanthine (DPCPX). These data demonstrate that activation of an adenosine A1 receptor on neurons, but not astrocytes, is protective against cellular damage or death induced specifically by hypoxia as opposed to other metabolic insults such as hypoglycemia.     

Astrocyte-derived VEGF mediates survival and tube stabilization of hypoxic brain microvascular endothelial cells in vitro

Chronic sublethal hypoxia has been associated with changes in neurovascular behavior, mediated, in part, by induction of vascular endothelial growth factor-A (VEGF-A(165)). In this report we demonstrate that RBE4 cells (derived from rodent cerebral microvasculature), when cultured in three-dimensional collagen gels: (1) Are induced to undergo increased tube formation in response to VEGF-A(165) in a dose-dependent manner; (2) undergo apoptosis under mild hypoxic conditions; (3) are rescued from the effects of hypoxia by the addition of exogenous VEGF-A(165) in a dose-dependent and inhibitable manner or by co-culture with primary newborn rat astrocytes, which are induced to express increased amounts of VEGF-A in hypoxic conditions. Further, we demonstrate that: (4) The observed astrocyte-produced, VEGF-mediated protection from apoptosis (survival) is inhibitable with soluble recombinant VEGF receptor-1 (sFlt), and is associated with a robust induction of MAPK tyrosine phosphorylation. These findings illustrate the importance of VEGF in the process of neurovascular survival in response to injury in developing brain and provide insight into the signaling pathways involved.     

The role of adenosine in CBF increases during hypoxia in young vs aged rats

The role of adenosine in the regional cerebral blood flow (rCBF) response to hypoxia was evaluated in young (6 month) and aged (26-28 month) F344 rats using theophylline, an adenosine antagonist. Regional CBF was measured with radioactive microspheres under control anesthetized conditions (70% N2O, 30% O2) and at two levels of hypoxia (CaO2 = 8.7-9.0 ml . 100ml-1 and 3.2-3.7 ml . 100ml-1). Without theophylline infusion, CBF increases were similar between young and aged rats during moderate hypoxia but were increased more in young during severe hypoxia. Intracerebrovascular theophylline infusion significantly attenuated the increase in CBF during both moderate and severe hypoxia and decreased the difference between young and aged rats. Theophylline infusion produced no significant effect on the increase in CBF produced by hypercapnia, indicating the specificity of the treatment for hypoxic induced CBF changes and adenosine release. Intracerebrovascular infusion of adenosine had no effect on CBF, presumably due to the presence of the blood brain barrier. The results suggest that adenosine plays a major role in CBF increases during both moderate and severe hypoxia and in the difference in response to hypoxia between young and aged rats.     

Stimulus duration modulates the interaction between opioids and nitric oxide in hypoxic pial artery dilation

Since recent studies show that pial artery dilation during a 20 or 40 min hypoxic exposure was less than that observed during a 5 or 10 min exposure, stimulus duration determines the nature of the vascular response to hypoxia. Decremented hypoxic pial dilation during longer exposure periods results, at least in part, from decreased release of methionine enkephalin (Met), an opioid known to contribute to dilation during hypoxia. Nitric oxide and cGMP contribute to both release and the vascular response to this opioid. The present study was designed to determine if the stimulus duration modulates the interaction between opioids and NO in hypoxic pial dilation using newborn pigs equipped with a closed cranial window. Elevation of CSF cGMP during hypoxia (Po2 approximately 35 mmHg) was dependent on stimulus duration (435+/-31, 934+/-46, 747+/-25, and 623+/-17 fmol/ml cGMP during normoxia and after 10, 20, and 40 min of hypoxia). Met-induced pial dilation during hypoxia was also stimulus duration dependent (7+/-1, 10+/-1, and 15+/-1, vs. 4+/-1, 6+/-1, and 8+/-2 vs. 2+/-1, 3+/-1, and 5+/-1% for 10(-10), 10(-8), 10(-6) M Met during normox and after 20, and 40 min of hypoxia). Additionally, the release of cGMP by Met during hypoxia was also stimulus duration dependent (1.8+/-0.1 vs. 1.6+/-0.1 vs. 1.3+/-0.1 fold change in CSF cGMP for 10(-8) M Met during normoxia and after 20 and 40 min of hypoxia). These data indicate that the diminished role of Met in pial dilation during longer hypoxic exposure periods results from a diminished capacity of this opioid to elicit dilation. Such impaired dilation is correlated with diminished stimulated cGMP release. These data also suggest that diminished CSF cGMP release during prolonged hypoxia contributes to decreased release of Met during longer hypoxic periods. Therefore, stimulus duration modulates the interaction between opioids and NO in hypoxic pial artery dilation.     

Inhibitory effect of hypoxic condition on acetylcholine release is partly due to the effect of adenosine released from the tissue

Isolated longitudinal muscle strip with Auerbach's plexus attached was used to study the stimulation-evoked release of 3H-acetylcholine (3H-ACh) under normoxic and hypoxic conditions. Hypoxia reduced the release of ACh. Theophylline, a purinoceptor P1 antagonist and vinpocetine, an antiischemic compound partly reversed the effect of hypoxia. Unlike theophylline, the effect of vinpocetine was not mediated via adenosine action, since it failed to affect the presynaptic action of adenosine, and the effect of theophylline and vinpocetine was additive. When they were added together the effect of hypoxia was almost completely antagonized. Dipyridamole, an adenosine uptake inhibitor, potentiated the effect of hypoxia and the presynaptic inhibitory action of adenosine on ACh release. Evidence was obtained that the effect of hypoxia is at least partly due to adenosine formed from purine nucleotides.     

Hypoxic preconditioning and tolerance via hypoxia inducible factor (HIF) 1alpha-linked induction of P450 2C11 epoxygenase in astrocytes.

The brain's adaptive response to ischemic preconditioning (IPC) is mediated in part via hypoxia inducible factor (HIF)-responsive genes. We previously showed that IPC induces cytochrome P450 2C11 expression in the brain, associated with protection from stroke. Cytochrome P450 2C11 is an arachidonic acid (AA) epoxygenase expressed in astrocytes, which metabolizes AA to epoxyeicosatrienoic acids (EETs). We tested the hypotheses that hypoxic preconditioning (HPC) induces 2C11 expression in astrocytes via HIF-1alpha, and that the P450 epoxygenase pathway contributes to enhanced astrocyte tolerance to ischemia-like injury induced by oxygen-glucose deprivation (OGD). Primary cultured astrocytes were incubated under normoxic or hypoxic conditions for 1, 3, 6, 24, or 48 h, and protein levels of P450 2C11 and HIF-1alpha were measured by Western blotting. Additionally, 2C11 mRNA was measured by Northern blotting, and binding of HIF-1alpha to 2C11 promoter was evaluated using electrophoretic mobility shift assay (EMSA) with 2C11 promoter DNA containing putative HIF-binding sites. Levels of 2C11 mRNA and protein were significantly increased starting at 3 and 6 h of hypoxia, respectively. The increase in 2C11 expression was preceded by an increase in HIF-1alpha protein at 1 h of hypoxia, and EMSA showed a specific and direct interaction between 2C11 promoter DNA and HIF-1alpha in nuclear extracts from astrocytes. HPC and EETs reduced astrocyte cell death, and P450 epoxygenase inhibition prevented protection by HPC. We conclude that HPC induces tolerance in astrocytes, at least in part, via HIF-1alpha-linked upregulation of P450 2C11.     

Adrenoceptor subtype-specific acceleration of the hypoxic depression of excitatory synaptic transmission in area CA1 of the rat hippocampus

The depression of excitatory synaptic transmission by hypoxia in area CA1 of the hippocampus is largely dependent upon the activation of adenosine A(1) receptors on presynaptic glutamatergic terminals. As well as adenosine, norepinephrine levels increase in the hypoxic/ischemic hippocampus. We sought to determine the influence of alpha- and beta-adrenoceptor (AR) activation on the hypoxic depression of synaptic transmission utilizing electrophysiological, pharmacological and adenosine sensor techniques. Norepinephrine depressed synaptic transmission and significantly accelerated the hypoxic depression of synaptic transmission. The alpha-AR agonist 6-fluoronorepinephrine mimicked both of these effects whilst the alpha(2)-AR antagonist yohimbine, but not the alpha(1)-AR antagonist urapidil, prevented the actions of 6-fluoronorepinephrine. In contrast, the beta-AR agonist isoproterenol enhanced synaptic transmission and only accelerated the hypoxic depression of transmission in hypoxia-conditioned slices in which the hypoxic release of adenosine is reduced. The effects of isoproterenol were blocked by the non-selective beta-AR antagonist propranolol and the selective beta(1)-AR antagonist betaxolol. Using an enzyme-based adenosine sensor we observed that the application of the beta-AR agonist resulted in increased extracellular adenosine during repeated hypoxia. Our results suggest that alpha(2)-AR activation facilitates the hypoxic depression of synaptic transmission probably via the known alpha(2)-AR-mediated inhibition of presynaptic calcium channels whereas beta(1)-AR activation does so via increased extracellular adenosine and greater activation of inhibitory adenosine A(1) receptors.