Delayed cytoprotection induced by hypoxic preconditioning in cultured neonatal rat cardiomyocytes: role of GRP78

Hypoxic preconditioning (HPC) has been well demonstrated to have potent protective effects in many cell types; however, the mechanisms responsible for this phenomenon are not fully understood. Recently, glucose-regulated protein 78 (GRP78), an inducible molecular chaperon, was indicated to be associated with ischemic preconditioning. We hypothesized that HPC protects cardiomyocytes against hypoxia by inducing GRP78 in cultured neonatal rat cardiomyocytes. HPC was induced by exposing cardiomyocytes to brief hypoxia (1% O(2), 30 min) followed by reoxygenation. GRP78 was expressed constitutively in cultured cardiomyocytes and its expression was enhanced at 12 h, peaked at 24 h (207.3+/-23.6% of the baseline), and was sustained for up to 72 h after HPC. Twenty-four hours after HPC, the myocytes were subjected to prolonged hypoxia (1% O(2), 12 h). The lactic dehydrogenase (LDH) release and malondialdehyde (MDA) content were reduced, while cell viability and superoxide dismutase (SOD) activity were increased in the preconditioned cells compared with the non-HPC cells. The GRP78 protein level was higher in cells exposed to both HPC and hypoxia than in the cells exposed to HPC alone or hypoxia alone. Heat shock protein 70 (HSP70) was induced in parallel by late HPC. Transfection of GRP78 antisense oligonucleotides blocked GRP78 expression but not HSP70, resulting in attenuated cardioprotection afforded by late HPC. Furthermore, inducing GRP78 by gene transfer protected cardiomyocytes from hypoxic injury. These findings demonstrate that the induction of GRP78 partially mediates the late HPC, suggesting that GRP78 is a novel mechanism responsible for the late cytoprotection of HPC.     

Hypoxia suppresses astrocyte glutamate transport independently of amyloid formation

Sustained hypoxia alters the expression of numerous proteins and predisposes individuals to Alzheimer’s disease (AD). We have previously shown that hypoxia in vitro alters Ca²⁺ homeostasis in astrocytes and promotes increased production of amyloid β peptides (Aβ) of AD. Indeed, alteration of Ca²⁺ homeostasis requires amyloid formation. Here, we show that electrogenic glutamate uptake by astrocytes is suppressed by hypoxia (1% O₂, 24 h) in a manner that is independent of amyloid β peptide formation. Thus, hypoxic suppression of glutamate uptake and expression levels of glutamate transporter proteins EAAT1 and EAAT2 were not mimicked by exogenous application of amyloid β peptide, or by prevention of endogenous amyloid peptide formation (using inhibitors of either β or γ secretase). Thus, dysfunction in glutamate homeostasis in hypoxic conditions is independent of Aβ production, but will likely contribute to neuronal damage and death associated with AD following hypoxic events.     

Gill chemoreceptors and cardio-respiratory reflexes in the neotropical teleost pacu, <Emphasis Type="Italic">Piaractus mesopotamicus</Emphasis>

This study examined the location and distribution of O₂ chemoreceptors involved in cardio-respiratory responses to hypoxia in the neotropical teleost, the pacu (Piaractus mesopotamicus). Intact fish and fish experiencing progressive gill denervation by selective transection of cranial nerves IX and X were exposed to gradual hypoxia and submitted to intrabuccal and intravenous injections of NaCN while their heart rate, ventilation rate and ventilation amplitude were measured. The chemoreceptors producing reflex bradycardia were confined to, but distributed along all gill arches, and were sensitive to O₂ levels in the water and the blood. Ventilatory responses to all stimuli, though modified, continued following gill denervation, however, indicating the presence of internally and externally oriented receptors along all gill arches and either in the pseudobranch or at extra-branchial sites. Chemoreceptors located on the first pair of gill arches and innervated by the glossopharyngeal nerve appeared to attenuate the cardiac and respiratory responses to hypoxia. The data indicate that the location and distribution of cardio-respiratory O₂ receptors are not identical to those in tambaqui (Colossoma macropomum) despite their similar habitats and close phylogenetic lineage, although the differences between the two species could reduce to nothing more than the presence or absence of the pseudobranch.     

新生猪缺氧后心肌连接蛋白Cx43表达的变化

目的探讨新生猪缺氧后心肌连接蛋白Cx43 mRNA和蛋白含量的变化。方法对照组(C组):新生猪6头,未吸入低氧;缺氧组(H组):新生猪6头,吸入低氧(FiO2=0.1)维持1 h。于缺氧结束后5 h,免疫荧光染色检测心肌组织细胞缝隙连接蛋白Cx43蛋白的表达,实时荧光定量逆转录多聚酶链式反应(RT-PCR)检测心肌组织Cx43 mRNA的表达。结果H组心肌组织Cx43蛋白的表达显著低于C组(P<0.01),但两组心肌组织Cx43 mRNA表达差异无统计学意义(P>0.05)。结论缺氧可导致心肌Cx43蛋白的表达或分布发生变化。     

Differential Expression of Apoptotic Proteins Following Hypoxia-induced CREB Phosphorylation in the Cerebral Cortex of Newborn Piglets

The present study investigates the correlation between the hypoxia-induced phosphorylation of cyclic AMP response element binding protein and the expression of apoptotic proteins (proapoptotic proteins Bax and Bad and antiapoptotic proteins Bcl-2 and Bcl-xl) during hypoxia in the cerebral cortex of newborn piglets. Piglets were divided into normoxic (Nx) and hypoxic (Hx, FiO₂ = 0.06 for 1 h) groups. Cerebral tissue hypoxia was documented by ATP and phosphocreatine (PCr) levels. Ser¹³³ phosphorylation of cyclic AMP response element binding (CREB) protein was determined by Western blot analysis using a specific anti-phosphorylated Ser¹³³-CREB protein antibody. The expression of apoptotic proteins was determined by using specific anti-Bax, anti-Bad, anti-Bcl-2 and anti-Bcl-xl antibodies. ATP and PCr values (μmoles/g brain) in Hx were significantly different from Nx (ATP: 4.40 ± 0.39 in Nx vs. 1.19 ± 0.44 in Hx, P < 0.05 vs. Nx; PCr: 3.60 ± 0.40 in Nx vs. 0.70 ± 0.31 in Hx, P < 0.05 vs. Nx). Ser¹³³ phosphorylated CREB protein (OD × mm²) was 74.55 ± 4.75 in Nx and 127.13 ± 19.36 in Hx (P < 0.05 vs. Nx). The expression of proapoptotic proteins Bax and Bad increased and strongly correlated with the increase in CREB protein phosphorylation (correlation coefficient r = 0.82 and r = 0.85, respectively). The expression of antiapoptotic proteins Bcl-2 and Bcl-xl did not show correlation with CREB protein phosphorylation. We conclude that cerebral hypoxia results in differential regulation of CREB protein-mediated expression of proapoptotic and antiapoptotic proteins in the cerebral cortex of newborn piglets. We propose that the increased expression of proapoptotic vs antiapoptotic genes will lead to an increased potential for apoptotic programmed cell death in the Hx newborn brain.     

PKC α-mediated CREB activation is oxygen and age-dependent in rat myocardial tissue

Both hypoxia and aging affect the morphology and the function of rat myocardial tissue. Moreover the heart tries to counteract the impaired function by activating specific signalling cascades. Here we report the involvement of CREB protein in “in vivo” response to hypoxic challenge and during aging in rat hearts. CREB is activated in parallel to HIF-1α nuclear translocation in the young after hypoxia exposure followed by reoxygenation, while this kind of response is not so dramatic in the old, neither in terms of CREB activation, neither in terms of HIF-1α expression and translocation, suggesting in the old the existence of an impaired oxygen-sensing mechanism or an adaptation of the cells to hypoxia. Moreover in the young a PKC α/Erk pathway seems to be involved in the activation of HIF-1α along with CREB, suggesting an attempt of the young to counteract the damage evoked by hypoxia, while in the old a PKC α/p38 MAPK/CREB pathway could determine the occurrence of both aging and aged cell hypoxia response.     

Immunohistochemical detection of hypoxia in mouse liver tissues treated with pimonidazole using “in vivo cryotechnique”

To evaluate hypoxic cells in live mouse liver tissues, immunohistochemistry for protein adducts of reductively activated pimonidazole (PARaPi) was performed using the “in vivo cryotechnique (IVCT)” followed by freeze-substitution fixation. This method was used because cryotechniques have some merits for examining biological events in living animal organs with improved time-resolution compared to conventional perfusion and/or immersion chemical fixation. Pimonidazole was intraperitoneally injected into living mice, and then after various times of hypoxia, their livers were quickly frozen by IVCT. The frozen liver tissues were freeze-substituted in acetone containing 2% paraformaldehyde, and routinely embedded in paraffin wax. De-paraffinized sections were immunostained for PARaPi. In liver tissues of mice without hypoxia, almost no immunostained cells were detected. However, in liver tissues with 30 s of hypoxia, some hepatocytes in the pericentral zones were strongly immunostained. After 60 s of hypoxia, many hepatocytes were immunostained with various degrees of staining intensity in all lobular zones, indicating different reactivities of pimonidazole in the hepatocytes to hypoxia. At this time, the general immunoreactivity also appeared to be stronger around the central veins than other portal areas. Although many hepatocytes were immunostained for PARaPi in the liver tissues with perfusion fixation via heart, those with perfusion via portal vein were not immunostained. Thus, IVCT is useful to detect time-dependent hypoxic states with pimonidazole treatment in living animal organs.     

Glutathione peroxidase 8 is transcriptionally regulated by HIFα and modulates growth factor signaling in HeLa cells

GPx8 is a mammalian Cys-glutathione peroxidase of the endoplasmic reticulum membrane, involved in protein folding. Its regulation is mostly unknown. We addressed both the functionality of two hypoxia-response elements (HREs) within the promoter, GPx8 HRE1 and GPx8 HRE2, and the GPx8 physiological role. In HeLa cells, treatment with HIFα stabilizers, such as diethyl succinate (DES) or 2-2′-bipyridyl (BP), induces GPx8 expression at both mRNA and protein level. Luciferase activity of pGL3^GPx8wt, containing a fragment of the GPx8 promoter including the two HREs, is also induced by DES/BP or by overexpressing either individual HIFα subunit. Mutating GPx8 HRE1 within pGL3^GPx8wt resulted in a significantly higher inhibition of luciferase activity than mutating GPx8 HRE2. Electrophoretic mobility-shift assay showed that both HREs exhibit enhanced binding to a nuclear extract from DES/BP-treated cells, with stronger binding by GPx8 HRE1. In DES-treated cells transfected with pGL3^GPx8wt or mutants thereof, silencing of HIF2α, but not HIF1α, abolishes luciferase activity. Thus GPx8 is a novel HIF target preferentially responding to HIF2α binding at its two novel functional GPx8 HREs, with GPx8 HRE1 playing the major role. Fibroblast growth factor (FGF) treatment increases GPx8 mRNA expression, and reporter gene experiments indicate that induction occurs via HIF. Comparing the effects of depleting GPx8 on the downstream effectors of FGF or insulin signaling revealed that absence of GPx8 results in a 16- or 12-fold increase in phosphorylated ERK1/2 by FGF or insulin treatment, respectively. Furthermore, in GPx8-depleted cells, phosphorylation of AKT by insulin treatment increases 2.5-fold. We suggest that induction of GPx8 expression by HIF slows down proliferative signaling during hypoxia and/or growth stimulation through receptor tyrosine kinases.