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• W2140446093 abstract "The incidence of Alzheimer disease is increased following ischemic episodes, and we previously demonstrated that following chronic hypoxia (CH), amyloid β (Aβ) peptide-mediated increases in voltage-gated L-type Ca2+ channel activity contribute to the Ca2+ dyshomeostasis seen in Alzheimer disease. Because in certain cell types mitochondria are responsible for detecting altered O2 levels we examined the role of mitochondrial oxidant production in the regulation of recombinant Ca2+ channel α1C subunits during CH and exposure to Aβ-(1–40). In wild-type (ρ+) HEK 293 cells expressing recombinant L-type α1C subunits, Ca2+ currents were enhanced by prolonged (24 h) exposure to either CH (6% O2) or Aβ-(1–40) (50 nm). By contrast the response to CH was absent in ρ0 cells in which the mitochondrial electron transport chain (ETC) was depleted following long term treatment with ethidium bromide or in ρ+ cells cultured in the presence of 1 μm rotenone. CH was mimicked in ρ0 cells by the exogenous production of O2·¯. by xanthine/xanthine oxidase. Furthermore Aβ-(1–40) enhanced currents in ρ0 cells to a degree similar to that seen in cells with an intact ETC. The antioxidants ascorbate (200 μm) and Trolox (500 μm) ablated the effect of CH in ρ+ cells but were without effect on Aβ-(1–40)-mediated augmentation of Ca2+ current in ρ0 cells. Thus oxidant production in the mitochondrial ETC is a critical factor, acting upstream of amyloid β peptide production in the up-regulation of Ca2+ channels in response to CH. The incidence of Alzheimer disease is increased following ischemic episodes, and we previously demonstrated that following chronic hypoxia (CH), amyloid β (Aβ) peptide-mediated increases in voltage-gated L-type Ca2+ channel activity contribute to the Ca2+ dyshomeostasis seen in Alzheimer disease. Because in certain cell types mitochondria are responsible for detecting altered O2 levels we examined the role of mitochondrial oxidant production in the regulation of recombinant Ca2+ channel α1C subunits during CH and exposure to Aβ-(1–40). In wild-type (ρ+) HEK 293 cells expressing recombinant L-type α1C subunits, Ca2+ currents were enhanced by prolonged (24 h) exposure to either CH (6% O2) or Aβ-(1–40) (50 nm). By contrast the response to CH was absent in ρ0 cells in which the mitochondrial electron transport chain (ETC) was depleted following long term treatment with ethidium bromide or in ρ+ cells cultured in the presence of 1 μm rotenone. CH was mimicked in ρ0 cells by the exogenous production of O2·¯. by xanthine/xanthine oxidase. Furthermore Aβ-(1–40) enhanced currents in ρ0 cells to a degree similar to that seen in cells with an intact ETC. The antioxidants ascorbate (200 μm) and Trolox (500 μm) ablated the effect of CH in ρ+ cells but were without effect on Aβ-(1–40)-mediated augmentation of Ca2+ current in ρ0 cells. Thus oxidant production in the mitochondrial ETC is a critical factor, acting upstream of amyloid β peptide production in the up-regulation of Ca2+ channels in response to CH. Acute regulation of ion channels by hypoxia is a fundamental mechanism by which tissue and cellular responses to this deleterious stimulus are initiated. Acute hypoxic regulation of various ion channel types either initiates or mediates control of a diverse range of cellular functions, including neurosecretion from chemosensory cells, neuronal excitability and the contraction/dilation of vascular smooth muscle (1Lopez-Barneo J. Pardal R. Ortega-Saenz P. Annu. Rev. Physiol. 2001; 63: 259-287Crossref PubMed Scopus (488) Google Scholar). By contrast, responses of ion channels and other cellular proteins to chronic hypoxia (CH) 1The abbreviations used are: CH, chronic hypoxia; Aβ, amyloid β; AβP, amyloid β peptide; HEK, human embryonic kidney; ETC, electron transport chain; ROS, reactive oxygen species; pF, picofarads; X/XO, xanthine/xanthine oxidase. occur on a time scale of hours to days and are generally mediated by the altered expression of proteins following up-regulation of their encoding genes (2Schofield C.J. Ratcliffe P.J. Nat. Rev. Mol. Cell. Biol. 2004; 5: 343-354Crossref PubMed Scopus (1621) Google Scholar, 3Sharp F.R. Bernaudin M. Nat. Rev. Neurosci. 2004; 5: 437-448Crossref PubMed Scopus (444) Google Scholar). However, we recently demonstrated that augmentation of voltage-gated Ca2+ channels by CH can occur in a post-transcriptional manner due to the amyloid β peptide-mediated trafficking of poreforming α1C subunits toward, and/or their retention within, the plasma membrane (4Scragg J.L. Fearon I.M. Boyle J. Ball S.G. Varadi G. Peers C. FASEB J. 2004; 19: 150-152Crossref PubMed Scopus (48) Google Scholar). Furthermore the response of large conductance Ca2+-activated K+ channels to CH may also be mediated by the post-transcriptional trafficking of auxiliary (β) subunits to the plasma membrane (5Hartness M.E. Brazier S.P. Peers C. Bateson A.N. Ashford M.L. Kemp P.J. J. Biol. Chem. 2003; 278: 51422-51432Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). CH is thought to be a predisposing factor in a number of neurological disorders, and there is a body of evidence to support a link between hypoxic/ischemic episodes (such as those that occur during stroke and cardiac arrhythmias) and the prevalence of dementias such as Alzheimer disease (6Moroney J.T. Bagiella E. Desmond D.W. Paik M.C. Stern Y. Tatemichi T.K. Stroke. 1996; 27: 1283-1289Crossref PubMed Scopus (128) Google Scholar). The most commonly described feature of Alzheimer disease is the build-up of fibrillar plaques composed largely of neurotoxic amyloid β peptides (AβPs). These short peptides are produced following the enzymatic cleavage of the amyloid precursor protein by the sequential action of β- and γ-secretases (7Selkoe D.J. Nature. 1999; 399: A23-A31Crossref PubMed Scopus (1526) Google Scholar, 8Vassar R. Citron M. Neuron. 2000; 27: 419-422Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 9Mattson M.P. Physiol. Rev. 1997; 77: 1081-1132Crossref PubMed Scopus (878) Google Scholar), and enhanced production of AβPs following ischemia may occur as a result of increased levels and/or activity of these enzymes (10Smith I.F. Boyle J.P. Green K.N. Pearson H.A. Peers C. J. Neurochem. 2004; 88: 869-877Crossref PubMed Scopus (27) Google Scholar, 11Wen Y. Onyewuchi O. Yang S. Liu R. Simpkins J.W. Brain Res. 2004; 1009: 1-8Crossref PubMed Scopus (174) Google Scholar). Although the neurodegenerative effects of AβPs have yet to be fully elucidated, they are thought to involve disrupted cellular Ca2+ homeostasis (12Mattson M.P. Chan S. J. Mol. Neurosci. 2001; 17: 205-224Crossref PubMed Scopus (153) Google Scholar, 13Mattson M.P. J. Neurovirol. 2002; 8: 539-550Crossref PubMed Scopus (123) Google Scholar). We recently proposed that the AβP-mediated trafficking of L-type Ca2+ channel α1C subunits during CH may contribute to Ca2+ dyshomeostasis and provide a link between ischemic episodes and dementias. Indeed we demonstrated that the effects of CH could be abolished by inhibition of either secretase required for AβP formation (4Scragg J.L. Fearon I.M. Boyle J. Ball S.G. Varadi G. Peers C. FASEB J. 2004; 19: 150-152Crossref PubMed Scopus (48) Google Scholar). In the present study, we sought to investigate the mechanisms underlying CH and AβP-mediated regulation of the L-type channel. In particular we examined the role of oxidant production in the mitochondrial electron transport chain (ETC) in the augmentation of recombinant Ca2+ currents by CH. We demonstrated that during CH altered mitochondrial function and reactive oxygen species (ROS) production precede the production of AβPs and Ca2+ current enhancement and demonstrated a role for the ETC in CH-induced Ca2+ dyshomeostasis. Stable Transfection of HEK 293 Cells—Experiments were conducted in HEK 293 cells stably expressing the human cardiac L-type Ca2+ channel α1C subunit (14Schultz D. Mikala G. Yatani A. Engle D.B. Iles D.E. Segers B. Sinke R.J. Weghuis D.O. Klockner U. Wakamori M. Wang J-J. Melvin D. Varadi G. Schwartz A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6228-6232Crossref PubMed Scopus (123) Google Scholar). The α1C subunit cDNA clone was a kind gift from Dr. Gyula Varadi (University of Cincinnati). After splitting the previous day and seeding at ∼60% confluency, wild-type HEK 293 cells were transfected with 3 μg of pCDNA3.1-α1C using ExGen 500 (Fermentas, Burlington, Ontario, Canada) according to the manufacturer's instructions. Three days post-transfection, the medium was replaced with one containing 400 μg ml–1 G418 (Invitrogen). Selection was applied for 2 weeks after which time individual colonies could be visualized using an inverted microscope (Zeiss) and phase-contrast objectives. Colonies were picked and seeded in wells of a 96-well plate and allowed to reach confluency after which they were transferred to 35-mm dishes for further culture and examination of Ca2+ currents. G418 selection was continued throughout the cloning process and in all subsequent subculturing. Some of the current studies were carried out in the stable cell line described previously (15Fearon I.M. Palmer A.C. Balmforth A.J. Ball S.G. Mikala G. Schwartz A. Peers C. J. Physiol. 1997; 500: 551-556Crossref PubMed Scopus (77) Google Scholar). Culture of HEK 293 Cells—Cells were grown in minimum essential medium with Earle's salts and l-glutamine (Invitrogen) containing 9% (v/v) fetal calf serum (Globepharm, Esher, Surrey, UK), 1% (v/v) nonessential amino acids, gentamicin (50 mg liter–1), 10,000 units liter–1 penicillin G, 10 mg liter–1 streptomycin, 0.25 mg liter–1 amphotericin, and 400 mg liter–1 G418 (all Invitrogen) at 37 °C in a humidified atmosphere of 95% air, 5% CO2. In studies examining chronic hypoxia, cells were placed in a humidified atmosphere of 6% O2, 5% CO2, 89% N2 or 2.5% O2, 5% CO2, 92.5% N2 after which time they were used in studies for no longer than 1 h following removal from the incubator. Creation of Mitochondria-depleted (ρ0) HEK 293 Cells—The mitochondrial DNA-depleted (ρ0 (16King M.P. Attardi G. Science. 1989; 246: 500-503Crossref PubMed Scopus (1455) Google Scholar)) cell line was generated from HEK 293 cells stably expressing the recombinant L-type Ca2+ channel α1C subunit. These cells were initially grown in minimum essential medium (see above) supplemented with 500 ng ml–1 EtBr, 1 mm pyruvate, and 50 μg ml–1 uridine. The concentration of EtBr was increased after the 1st and 2nd months to 1 and 2 μg ml–1, respectively, because at the lower concentration depletion of mtDNA was incomplete. mtDNA levels were examined monthly and compared with wild-type (ρ+) cells grown in media in the absence of EtBr, pyruvate, and uridine. mtDNA levels were examined as follows. DNA was harvested from the cells using the DNeasy tissue kit (Qiagen) and was analyzed and quantitated by spectrophotometry (260/280 nm). Equal amounts of DNA from ρ+ and ρ0 cells were subjected to quantitative PCR using the Brilliant SYBR green QPCR Master Mix (Stratagene), the Mx3000P QPCR machine (Stratagene), and human-specific primer pairs for mtDNA: upstream, 5′-CCT AGG GAT AAC AGC GCA AT-3′; and downstream, 5′-TAG AAG AGC GAT GGT GAG AG-3′; and for β-actin: upstream, 5′-TGG CCG GGA CCT GAC TGA CTA C-3′; and downstream, 5′-CGT GGC CAT CTC TTG CTC GAA G-3′ (31Muller F.L. Liu Y. Van Remmen H. J. Biol. Chem. 2004; 279: 49064-49073Abstract Full Text Full Text PDF PubMed Scopus (778) Google Scholar). mtDNA levels in ρ0 cells after the 1st, 2nd, and 3rd months of growth in EtBr were ∼60, 40, and 2% of wild-type levels, respectively. This was verified by agarose gel electrophoresis of a sample extracted from the PCR during the log phase (20 cycles; see Fig. 3B). Only ρ0 cells in which the mtDNA was reduced to 2% of control levels were used in experiments. Electrophysiology—Pieces of coverslip with attached cells were transferred to a continually perfused (∼2 ml min–1) recording chamber, and whole-cell patch clamp recordings (17Hamill O.P. Marty A. Neher E. Sakmann B. Sigworth F.J. Pfluegers Arch. 1981; 391: 85-100Crossref PubMed Scopus (15148) Google Scholar) were made using patch pipettes of resistance 4–6 megaohms. Cells were perfused with a solution composed of 95 mm NaCl, 5 mm CsCl, 0.6 mm MgC12, 20 mm BaCl2, 5 mm Hepes, 10 mm d-glucose, 20 mm tetraethylammonium chloride (pH adjusted to 7.4 with NaOH), and patch electrodes were filled with a solution composed of 120 mm CsCl, 20 mm tetraethylammonium chloride, 2 mm MgC12, 10 mm EGTA, 10 mm Hepes, 2 mm ATP (pH adjusted to 7.2 with CsOH). Cells were voltage-clamped at –80 mV, and whole-cell currents were evoked by step depolarizing the membrane to various test potentials for 100 ms at a frequency of 0.1 Hz. All recordings were made at room temperature (22 ± 2 °C). Current traces were filtered at 5 kHz, digitized at 10 kHz, and stored on a personal computer for later analysis. Capacitative transients were minimized by analogue means (residual transients have been truncated for illustrative purposes), and corrections for leak current were made off line by the appropriate scaling and subtraction of the average leak current evoked by small hyperpolarizing and depolarizing steps (≤5 mV). Current amplitudes were measured at their peaks during each step depolarization. Current densities (pA/pF) were calculated by dividing evoked currents by the capacitance of the cell. In the insets to figures showing individual traces, currents were evoked in cells of similar size as judged by their capacitance. To minimize the effects of variation in Ca2+ currents, in all experiments current amplitudes following either drug or hypoxic treatments were compared with those obtained in control cells on the same experimental day. All analyses and voltage protocols were performed using an Axopatch 200B/Multiclamp 700A amplifier in combination with a Digidata 1200/1322A interface and pCLAMP 8.0/9.0 software (all Axon Instruments). Immunocytochemistry—For immunocytochemical studies cells were grown as above, harvested in phosphate-buffered saline without Ca2+ or Mg2+, and subcultured on glass coverslips at a seeding density of 3 × 104 cells ml–1. After 2 days in culture cells were washed in phosphate-buffered saline and then fixed in 4% paraformaldehyde (pH 7.4 in phosphate-buffered saline). The cells were incubated (4 °C, overnight) in the presence of a monoclonal antibody (provided by Elan Pharmaceuticals) raised against Aβ-(1–5). Incubation was carried out in phosphate-buffered saline with 1% goat serum. Antibody binding was visualized using a Cy3-conjugated anti-rabbit secondary antibody. Coverslips were mounted on slides using Vectashield (Vector Laboratories Ltd.) and examined using a Zeiss LSM 510 laser scanning confocal microscope. Chemicals and Statistical Analyses—EtBr, pyruvate, uridine, ascorbic acid, xanthine, xanthine oxidase, rotenone, and Aβ-(1–40) were from Sigma. In all experiments, the unaggregated form of Aβ-(1–40) was used. The secretase inhibitors γ-VI, γ-X, BSI, and the antioxidant Trolox were obtained from Calbiochem. Results are expressed as means ± S.E., and statistical comparisons were made using unpaired Student's t tests as appropriate. We recently demonstrated that CH augments Ca2+ channel currents in HEK 293 cells stably expressing recombinant α1C subunits via a mechanism involving the production of AβPs (4Scragg J.L. Fearon I.M. Boyle J. Ball S.G. Varadi G. Peers C. FASEB J. 2004; 19: 150-152Crossref PubMed Scopus (48) Google Scholar). In the present study, we demonstrated that this effect was reproducible at lower levels of hypoxia (6% O2 for 24 h). At all activating test potentials, evoked currents (expressed as current densities following normalization for cell size) were larger in CH cells than in cells cultured in normoxia. For example, at a test potential of +10 mV, mean ± S.E. current densities were –7.8 ± 0.8 pA/pF (n = 20) in normoxia and –13.1 ± 2.5 pA/pF (n = 18) in CH (p < 0.05, unpaired Student's t test; Fig. 1A). Similarly current densities were also significantly enhanced following incubation for 24 h in 50 nm Aβ-(1–40) to –11.8 ± 2.01 pA/pF (n = 10; p < 0.05, unpaired Student's t test; Fig. 1B). The reproducibility of this effect in a cell line newly generated using a different expression vector/promoter than that used previously further emphasizes the lack of involvement of transcriptional regulation of the Ca2+ channel during CH. These effects of CH were dependent on AβP production because they were abolished by co-incubating cells in 100 nm BSI or 10 μm γ-VI, inhibitors of β- and γ-secretase, respectively (Fig. 1C). The Role of Antioxidant Production in Hypoxic Augmentation of L-type Ca2+ Currents—Hypoxic augmentation of native L-type Ca2+ channels in PC12 cells is prevented by antioxidants (18Green K.N. Peers C. J. Neurochem. 2002; 5: 1043-1051Crossref Scopus (34) Google Scholar, 19Green K.N. Boyle J.P. Peers C. J. Physiol. 2002; 541: 1013-1023Crossref PubMed Scopus (47) Google Scholar). To examine the role of ROS in hypoxic augmentation of recombinant Ca2+ currents we investigated the ability of two structurally unrelated antioxidants to interfere with hypoxic regulation of Ca2+ channel expression. Results presented in Fig. 2 strongly implicate the involvement of ROS in current augmentation in HEK 293 cells. Both antioxidants used in these studies (ascorbic acid or Trolox) fully prevented hypoxic augmentation of Ca2+ currents (Fig. 2, A and B). Thus, for example, when depolarizing to a test potential of +20 mV, current densities in CH cells were significantly decreased (p < 0.0001; unpaired Student's t test) from –15.5 ± 1.1 pA/pF (n = 10) to –7.8 ± 0.9 pA/pF (n = 12) in CH cells treated with 200 μm ascorbic acid. Additionally when depolarizing to a test potential of + 20mV, current densities in CH cells were significantly decreased (p < 0.005; unpaired Student's t test) from –18.3 ± 2.4 pA/pF (n = 10) to –7.2 ± 1.3 pA/pF (n = 7) by 500 μm Trolox. Neither agent had an effect on current densities in control (normoxically incubated) cells (Fig. 2, C and D). Thus, our data to date suggest that chronic hypoxia augmented L-type Ca2+ channel currents in HEK 293 cells via a mechanism that involved ROS production and required the formation of AβPs. Altered Mitochondrial Function Underlies Ca2+ Current Regulation during CH—Our data above clearly demonstrated a role for ROS in augmenting Ca2+ currents during CH, and both mitochondria (20Turrens J.F. J. Physiol. 2003; 552: 335-344Crossref PubMed Scopus (3608) Google Scholar) and AβPs (21Hensley K. Carney J.M. Mattson M.P. Aksenova M. Harris M. Wu J.F. Floyd R.A. Butterfield D.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3270-3274Crossref PubMed Scopus (1091) Google Scholar) possess the ability to produce ROS in normoxia and hypoxia. To investigate the involvement of the mitochondrial ETC as source of ROS, we created a line of α1C subunit-expressing HEK 293 cells depleted of mitochondrial DNA and as such lacking a functional ETC (Refs. 16King M.P. Attardi G. Science. 1989; 246: 500-503Crossref PubMed Scopus (1455) Google Scholar and 22Waypa G.B. Chandel N.S. Schumacker P.T. Circ. Res. 2001; 88: 1259-1266Crossref PubMed Scopus (322) Google Scholar; Fig. 3, A and B). In these ρ0 cells, depolarization-evoked Ca2+ currents were moderately reduced in magnitude compared with those evoked in ρ+ cells (Fig. 3C). Thus, at +10 mV current densities were reduced from –7.8 ± 0.8 pA/pF (n = 20) in ρ+ cells to –6.5 ± 0.6 pA/pF (n = 30; p < 0.05, unpaired Student's t test) in the mitochondria-depleted ρ0 line. This is suggestive of a role of the ETC in promoting basal channel activity and/or expression. To investigate the involvement of the ETC in responses to CH, we exposed ρ+ and ρ0 cells to hypoxia (6% O2) for 24 h. In contrast to the effect of CH on Ca2+ currents in wild-type (ρ+) cells (e.g. see Fig. 1A), exposure to CH for 24 h was without effect on Ca2+ currents in ρ0 cells (Fig. 3D). For example, at a test potential of +10 mV current densities were –6.5 ± 0.6 pA/pF (n = 30) in cells incubated in normoxia and –5.6 ± 0.7 pA/pF (n = 16) in cells incubated under chronically hypoxic conditions (p > 0.05, unpaired Student's t test). Thus, a functional ETC is requisite for transducing a chronically hypoxic stimulus into Ca2+ current enhancement. Because complex I of the mitochondrial ETC has been proposed to act as an O2 sensor in both the carotid body (23Wyatt C.N. Buckler K.J. J. Physiol. 2004; 556: 175-191Crossref PubMed Scopus (148) Google Scholar) and the lung vasculature (22Waypa G.B. Chandel N.S. Schumacker P.T. Circ. Res. 2001; 88: 1259-1266Crossref PubMed Scopus (322) Google Scholar), we examined Ca2+ current densities in ρ+ cells co-incubated with the complex I inhibitor rotenone during either normoxia or CH. Consistent with the ρ0 data, rotenone abolished the enhancement of Ca2+ currents caused by chronic hypoxia (Fig. 3E). For example, at a test potential of +10 mV, current densities were –9.2 ± 3.7 pA/pF (n = 11) in normoxic cells, –23.0 ± 3.7 pA/pF (n = 12) in chronically hypoxic cells, and –9.4 ± 3.1 pA/pF (n = 8) in cells incubated under chronically hypoxic conditions in the presence of 1 μm rotenone (Fig. 3F). This effect of rotenone was not due to a nonspecific action because 1 μm rotenone was without effect on Ca2+ current densities in cells incubated under normoxic conditions with the inhibitor (Fig. 3F). Oxidant Production Precedes AβP Production during CH— Ca2+ current augmentation during CH is dependent on the production of AβPs (4Scragg J.L. Fearon I.M. Boyle J. Ball S.G. Varadi G. Peers C. FASEB J. 2004; 19: 150-152Crossref PubMed Scopus (48) Google Scholar). However, the above experiments in addition to our previous data (4Scragg J.L. Fearon I.M. Boyle J. Ball S.G. Varadi G. Peers C. FASEB J. 2004; 19: 150-152Crossref PubMed Scopus (48) Google Scholar) were unable to discern whether AβP production is causative of (24Abramov A.Y. Canevari L. Duchen M.R. J. Neurosci. 2004; 24: 565-575Crossref PubMed Scopus (472) Google Scholar) or a consequence of (25Smith M.A. Drew K.L. Nunomura A. Takeda A. Hirai K. Zhu X. Atwood C.S. Raina A.K. Rottkamp C.A. Sayre L.M. Friedland R.P. Perry G. Neurochem. Int. 2002; 40: 527-531Crossref PubMed Scopus (66) Google Scholar) mitochondrial dysfunction. To address this, we examined Ca2+ currents in ρ0 cells incubated overnight in 50 nm Aβ-(1–40). Similar to that observed in ρ+ cells, mean Ca2+ current densities were significantly increased following this intervention (Fig. 4A) such that at a test potential of +10 mV current densities were –6.5 ± 0.6 pA/pF (n = 30) in control cells and –9.3 ± 1.0 pA/pF (n = 12) following incubation in Aβ-(1–40) (p < 0.05, unpaired Student's t test). Thus, because in ρ0 cells Ca2+ currents were augmented by Aβ-(1–40) but not by CH, altered mitochondrial function and ROS production lie upstream of AβP production and Ca2+ current augmentation. In support of this, 200 μm ascorbate was without effect on the Aβ-(1–40)-mediated augmentation of Ca2+ currents in ρ0 cells (Fig. 4B). Current densities at +10 mV were –6.6 ± 1.7 pA/pF (n = 7) under control conditions and –11.4 ± 1.3 pA/pF (n = 9) following exposure to 50 nm Aβ-(1–40) and 200 μm ascorbate (p > 0.05 when comparing currents in Aβ-(1–40)-treated cells in the absence and presence of ascorbate; unpaired Student's t test). Superoxide Production in the ETC Underlies AβP Production during CH—In ETC-deficient ρ0 cells, which lack the ability to produce ROS during hypoxia (26Schroedl C. McClintock D.S. Budinger G.R. Chandel N.S. Am. J. Physiol. 2002; 283: L922-L931Crossref PubMed Scopus (237) Google Scholar), a 6-h incubation in the superoxide-generating xanthine/xanthine oxidase (X/XO) system (100 μm/5 milliunits ml–1) mimicked the response to CH. At all activating test potentials, Ca2+ currents were increased in cells exposed to X/XO when compared with controls (Fig. 5A). At a test potential of +10 mV, this effect was statistically significant (p < 0.05, unpaired Student's t test; Fig. 5B). The increase in Ca2+ current due to X/XO was mediated by enhanced production of AβPs because its effects were abolished by the γ-secretase inhibitors γ-VI (10 μm), γ-X (100 nm), and BSI (100 nm) (Fig. 5B). Furthermore the effect of X/XO was abolished by 400 μm ascorbate (Fig. 5B), indicating that its action was mediated by superoxide production and not by a nonspecific effect. Mean current densities in the presence of either secretase inhibitor or ascorbate were not significantly different from that of the control group (p > 0.05, unpaired Student's t test; Fig. 5B). Because the AβP-dependent effects of CH were reproduced in ρ0 cells by pharmacologically evoked ROS production, these data support the hypothesis that during CH oxidant production within the mitochondrial ETC precedes AβP-mediated augmentation of Ca2+ currents. To provide support for this hypothesis, we carried out immunocytochemical staining of HEK 293 cells using the 3D6 antibody raised against the five N-terminal amino acids of AβP that has been previously shown to selectively recognize AβP species and not secreted forms of the amyloid precursor protein (27Johnson-Wood K. Lee M. Motter R. Hu K. Gordon G. Barbour R. Khan K. Gordon M. Tan H. Games D. Leiberburg I. Schenk D. Seubert P. McConlogue L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1550-1556Crossref PubMed Scopus (584) Google Scholar). As shown in Fig. 5C, unlike wild-type (ρ+) HEK 293 cells (4Scragg J.L. Fearon I.M. Boyle J. Ball S.G. Varadi G. Peers C. FASEB J. 2004; 19: 150-152Crossref PubMed Scopus (48) Google Scholar) CH caused no increase in the expression of AβPs in ρ0 cells. However, exogenous production of superoxide via the X/XO system enhanced the expression of AβPs in these cells (Fig. 5C, right). A characteristic feature of Alzheimer disease is neurodegeneration due to the build-up of AβPs subsequent to altered cleavage of precursors of these neurotoxic proteins (28Walter J. Kaether C. Steiner H. Haass C. Curr. Opin. Neurobiol. 2001; 11: 585-590Crossref PubMed Scopus (158) Google Scholar, 29Wolfe M.S. Curr. Top. Dev. Biol. 2003; 54: 233-261Crossref PubMed Google Scholar). Following ischemic episodes such as those that occur during stroke, enhanced activity of the β- and γ-secretases responsible for cleaving amyloid precursor proteins into toxic amyloid peptides (7Selkoe D.J. Nature. 1999; 399: A23-A31Crossref PubMed Scopus (1526) Google Scholar, 8Vassar R. Citron M. Neuron. 2000; 27: 419-422Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 9Mattson M.P. Physiol. Rev. 1997; 77: 1081-1132Crossref PubMed Scopus (878) Google Scholar) leads to their accumulation (10Smith I.F. Boyle J.P. Green K.N. Pearson H.A. Peers C. J. Neurochem. 2004; 88: 869-877Crossref PubMed Scopus (27) Google Scholar, 11Wen Y. Onyewuchi O. Yang S. Liu R. Simpkins J.W. Brain Res. 2004; 1009: 1-8Crossref PubMed Scopus (174) Google Scholar). Subsequently neurodegeneration may occur due to Ca2+-dependent processes and as a consequence of AβP-induced Ca2+ dyshomeostasis. In support of this, recent studies in neuronal PC12 cells have demonstrated that prolonged incubation under hypoxic conditions, an intervention designed to mimic ischemic stroke, enhances both voltage-dependent (L-type) and voltage-independent plasmalemmal Ca2+ entry pathways (19Green K.N. Boyle J.P. Peers C. J. Physiol. 2002; 541: 1013-1023Crossref PubMed Scopus (47) Google Scholar, 30Green K.N. Peers C. J. Neurochem. 2001; 77: 953-956Crossref PubMed Scopus (67) Google Scholar). This enhancement was dependent on CH-induced elevation of AβP levels because the effects of CH on Ca2+ currents were mimicked by exogenously applied AβPs and abrogated by secretase inhibitors or a monoclonal antibody raised against AβP (19Green K.N. Boyle J.P. Peers C. J. Physiol. 2002; 541: 1013-1023Crossref PubMed Scopus (47) Google Scholar, 30Green K.N. Peers C. J. Neurochem. 2001; 77: 953-956Crossref PubMed Scopus (67) Google Scholar). More recently, we have shown that the effect of CH on the voltage-dependent Ca2+ entry pathway could be mimicked in HEK 293 cells stably expressing the α1C subunit of the L-type Ca2+ channel (4Scragg J.L. Fearon I.M. Boyle J. Ball S.G. Varadi G. Peers C. FASEB J. 2004; 19: 150-152Crossref PubMed Scopus (48) Google Scholar). Thus, the effect of CH appeared to be due to a direct interaction of AβPs with the pore-forming subunit, and we extended this finding to demonstrate that Ca2+ current enhancement was due to AβP-mediated enhanced trafficking toward, or retention within, the plasma membrane (4Scragg J.L. Fearon I.M. Boyle J. Ball S.G. Varadi G. Peers C. FASEB J. 2004; 19: 150-152Crossref PubMed Scopus (48) Google Scholar). In PC12 cells, it has been shown previously that CH enhancement of Ca2+ currents was abrogated by antioxidants (18Green K.N. Peers C. J. Neurochem. 2002; 5: 1043-1051Crossref Scopus (34) Google Scholar), giving evidence for the altered production of highly reactive oxidant molecules in mediating the effects of CH. In accordance with this, in the current studies we demonstrated that the enhancement of L-type Ca2+ currents by CH was abrogated by concomitant incubation with the antioxidants ascorbic acid or Trolox. This effect could not be attributed to a nonspecific effect of these agents because they were without effect on Ca2+ currents under normoxic conditions. Because ROS production due to inefficiencies in the mitochondrial ETC has been implicated in hypoxic signaling in numerous cell types (20Turrens J.F. J. Physiol. 2003; 552: 335-344Crossref PubMed Scopus (3608) Google Scholar), we explored the hypothesis that mitochondrial ROS production mediates the effects of CH on the L-type channel. To do this, we examined the effects of CH in a line of HEK 293 cells that were depleted of mitochondrial DNA and as such exhibit no functional ETC and lack the ability to produce ROS during hypoxia (26Schroedl C. McClintock D.S. Budinger G.R. Chandel N.S. Am. J. Physiol. 2002; 283: L922-L931Crossref PubMed Scopus (237) Google Scholar). In these cells, CH caused no change in Ca2+ current amplitudes, implicating a role of the ETC in chronic O2 sensing. Similar to acute hypoxia sensing in the carotid body, the location of the O2 sensor may reside within complex I of the mitochondrial ETC (23Wyatt C.N. Buckler K.J. J. Physiol. 2004; 556: 175-191Crossref PubMed Scopus (148) Google Scholar) because the effects of CH were also ablated by the complex I inhibitor rotenone. Thus, the ETC provides a vital link between CH and enhanced Ca2+ currents that to our knowledge may provide the first demonstration that complex I acts as an O2 sensor to regulate ion channel activity during prolonged exposure to hypoxia. The fact that the effects of CH were abrogated by antioxidants suggests that regulation of complex I activity during chronic hypoxia enhances oxidant production, a suggestion in accordance with previous studies (22Waypa G.B. Chandel N.S. Schumacker P.T. Circ. Res. 2001; 88: 1259-1266Crossref PubMed Scopus (322) Google Scholar). Prior to the current study, the temporal contributions of AβPs and ROS to Ca2+ dyshomeostasis remained to be addressed. AβPs themselves are capable of producing ROS in vitro (21Hensley K. Carney J.M. Mattson M.P. Aksenova M. Harris M. Wu J.F. Floyd R.A. Butterfield D.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3270-3274Crossref PubMed Scopus (1091) Google Scholar), and a recent study demonstrated that AβPs are able to induce both mitochondrial dysfunction and increased ROS levels, although this may be cell type-specific because these effects of AβPs were observed in isolated astrocytes but not neurons (24Abramov A.Y. Canevari L. Duchen M.R. J. Neurosci. 2004; 24: 565-575Crossref PubMed Scopus (472) Google Scholar). In contrast, there is a large body of evidence to suggest that compromised mitochondrial function and the ensuing ROS production are responsible for elevating AβP levels as part of a physiological neuroprotective mechanism utilizing the antioxidant properties of AβPs (25Smith M.A. Drew K.L. Nunomura A. Takeda A. Hirai K. Zhu X. Atwood C.S. Raina A.K. Rottkamp C.A. Sayre L.M. Friedland R.P. Perry G. Neurochem. Int. 2002; 40: 527-531Crossref PubMed Scopus (66) Google Scholar). Here we report that Aβ-(1–40) enhanced voltage-gated Ca2+ currents in HEK 293 cells that were depleted of a functional mitochondrial ETC. This is in stark contrast to the lack of effect of CH in these ρ0 cells. The most plausible explanation for this finding is that CH causes altered mitochondrial function, which is a trigger for AβP production and subsequently for the trafficking/membrane retention of α1C subunits (4Scragg J.L. Fearon I.M. Boyle J. Ball S.G. Varadi G. Peers C. FASEB J. 2004; 19: 150-152Crossref PubMed Scopus (48) Google Scholar). Thus, these data provide compelling evidence that, in terms of Ca2+ dyshomeostasis due to CH, altered mitochondrial function temporally precedes the enhanced production of AβPs. Our data utilizing rotenone as a specific inhibitor of complex I suggest a role for this ETC complex in chronic O2 sensing. Along with complex III (31Muller F.L. Liu Y. Van Remmen H. J. Biol. Chem. 2004; 279: 49064-49073Abstract Full Text Full Text PDF PubMed Scopus (778) Google Scholar), the ubisemiquinone site of mitochondrial complex I is a site of superoxide ( (O2·¯)) production (32Lambert A.J. Brand M.D. J. Biol. Chem. 2004; 279: 39414-39420Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar). To probe the role of O2·¯ production in mediating AβP production, we incubated ρ0 cells in the O2·¯-generating X/XO system (33Valencia A. Moran J. Free Radic. Biol. Med. 2004; 36: 1112-1125Crossref PubMed Scopus (162) Google Scholar) and examined Ca2+ current amplitudes. The ability of this system to mimic CH by enhancing Ca2+ currents supports a role for O2·¯ in hypoxic signal transduction. Given that the effects of CH were ablated by anti-AβP antibodies and secretase inhibitors (4Scragg J.L. Fearon I.M. Boyle J. Ball S.G. Varadi G. Peers C. FASEB J. 2004; 19: 150-152Crossref PubMed Scopus (48) Google Scholar), taken together these data suggest a role for (O2·¯) in promoting AβP production. In support of this proposal, we found that the effects of O2·¯ production (via X/XO) on Ca2+ currents were abolished by selective inhibitors of the AβP-producing β- and γ-secretases. Furthermore X/XO increased the intensity of immunocytochemical staining of ρ0 HEK 293 cells when using the AβP-specific 3D6 primary antibody (27Johnson-Wood K. Lee M. Motter R. Hu K. Gordon G. Barbour R. Khan K. Gordon M. Tan H. Games D. Leiberburg I. Schenk D. Seubert P. McConlogue L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1550-1556Crossref PubMed Scopus (584) Google Scholar). In accordance with these findings, AβP accumulation in mice genetically engineered to remove their ability to dismutate O2·¯ (34Li F. Calingasan N.Y. Yu F. Mauck W.M. Toidze M. Almeida C.G. Takahashi R.H. Carlson G.A. Flint Beal M. Lin M.T. Gouras G.K. J. Neurochem. 2004; 289: 1308-1312Crossref Scopus (236) Google Scholar) supports this proposed role of O2·¯ in enhancing AβP production. In summary, our data support the proposal that during prolonged exposure to hypoxia, regulation of the mitochondrial ETC enhances O2·¯ production, which elevates AβP production and consequently alters cellular Ca2+ homeostasis by the trafficking/retention of voltage-gated Ca2+ channel α1C subunits. Interestingly the protein levels of two components of complex I have been reported to be reduced in the brains of Alzheimer patients (35Kim S.H. Vlkolinsky R. Cairns N. Fountoulakis M. Lubec G. Life Sci. 2001; 68: 2741-2750Crossref PubMed Scopus (127) Google Scholar), and thus ETC regulation may provide a causal link between hypoxic episodes such as those that occur during brain ischemia and the resultant Ca2+ dyshomeostasis and subsequent neurodegeneration, which are hallmarks of Alzheimer disease. We thank Dr. Gyula Varadi (University of Cincinnati) for providing us with the pCDNA3.1-α1C construct. The 3D6 antibody was gratefully received from Elan Pharmaceuticals." @default.
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