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• W2091966984 abstract "Calcium influx through voltage-dependent calcium channels (VDCCs) mediates a variety of functions in neurons and other excitable cells, but excessive calcium influx through these channels can contribute to neuronal death in pathological settings. Oxyradical production and membrane lipid peroxidation occur in neurons in response to normal activity in neuronal circuits, whereas excessive lipid peroxidation is implicated in the pathogenesis of of neurodegenerative disorders. We now report on a specific mechanism whereby lipid peroxidation can modulate the activity of VDCCs. The lipid peroxidation product 4-hydroxy-2,3-nonenal (4HN) enhances dihydropyridine-sensitive whole-cell Ca2+ currents and increases depolarization-induced increases of intracellular Ca2+levels in hippocampal neurons. Prolonged exposure to 4HN results in neuronal death, which is prevented by treatment with glutathione and attenuated by the L-type Ca2+ channel blocker nimodipine. Tyrosine phosphorylation of α1 VDCC subunits is increased in neurons exposed to 4HN, and studies using inhibitors of tyrosine kinases and phosphatases indicate a requirement for tyrosine phosphorylation in the enhancement of VDCC activity in response to 4HN. Phosphorylation-mediated modulation of Ca2+ channel activity in response to lipid peroxidation may play important roles in the responses of neurons to oxidative stress in both physiological and pathological settings. Calcium influx through voltage-dependent calcium channels (VDCCs) mediates a variety of functions in neurons and other excitable cells, but excessive calcium influx through these channels can contribute to neuronal death in pathological settings. Oxyradical production and membrane lipid peroxidation occur in neurons in response to normal activity in neuronal circuits, whereas excessive lipid peroxidation is implicated in the pathogenesis of of neurodegenerative disorders. We now report on a specific mechanism whereby lipid peroxidation can modulate the activity of VDCCs. The lipid peroxidation product 4-hydroxy-2,3-nonenal (4HN) enhances dihydropyridine-sensitive whole-cell Ca2+ currents and increases depolarization-induced increases of intracellular Ca2+levels in hippocampal neurons. Prolonged exposure to 4HN results in neuronal death, which is prevented by treatment with glutathione and attenuated by the L-type Ca2+ channel blocker nimodipine. Tyrosine phosphorylation of α1 VDCC subunits is increased in neurons exposed to 4HN, and studies using inhibitors of tyrosine kinases and phosphatases indicate a requirement for tyrosine phosphorylation in the enhancement of VDCC activity in response to 4HN. Phosphorylation-mediated modulation of Ca2+ channel activity in response to lipid peroxidation may play important roles in the responses of neurons to oxidative stress in both physiological and pathological settings. membrane lipid peroxidation voltage-dependent calcium channel 4-hydroxy-2,3-nonenal Neurons are subjected to considerable oxidative stress as the result of oxyradical production that occurs during mitochondrial respiration and activities of enzymes such as nitric oxide synthase and cyclooxygenases (1Dagani F. Marzatico F. Curti D. J. Neurochem. 1988; 50: 1233-1236Crossref PubMed Scopus (21) Google Scholar, 2Lipton S.A. Singel D.J. Stamler J.S. Prog. Brain Res. 1994; 103: 359-364Crossref PubMed Scopus (86) Google Scholar, 3Kaufmann W.E. Andreasson K.I. Isakson P.C. Worley P.F. Prostaglandins. 1997; 54: 601-624Crossref PubMed Scopus (244) Google Scholar). Membrane lipid peroxidation (MLP)1 is a common consequence of such oxidative stress (4Mattson M.P. Trends Neurosci. 1998; 21: 53-57Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar). Increased MLP occurs in paradigms of synaptic plasticity such as long-term potentiation, and antioxidants that suppress MLP can modify such processes (5Nilova N.S. Polezhaeva L.N. Neurosci. Behav. Physiol. 1996; 26: 23-26Crossref PubMed Scopus (3) Google Scholar, 6Murray C.A. Lynch M.A. J. Biol. Chem. 1998; 273: 12161-12168Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 7McGahon B.M. Murray C.A. Horrobin D.F. Lynch M.A. Neurobiol. Aging. 1999; 20: 643-653Crossref PubMed Scopus (60) Google Scholar), suggesting roles for MLP in neuronal plasticity (4Mattson M.P. Trends Neurosci. 1998; 21: 53-57Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar). However, studies of patients and experimental models suggest a major role for MLP in the pathogenesis of an array of neurodegenerative conditions including Alzheimer's and Parkinson's diseases, stroke, and traumatic brain and spinal cord injuries (for review, see Ref. 4Mattson M.P. Trends Neurosci. 1998; 21: 53-57Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar). MLP can promote neurodegeneration by impairing the function of membrane transport proteins including ion-motive ATPases and glucose and glutamate transporters (8Mark R.J. Hensley K. Butterfield D.A. Mattson M.P. J. Neurosci. 1995; 15: 6239-6249Crossref PubMed Google Scholar, 9Mark R.J. Lovell M.A. Markesbery W.R. Uchida K. Mattson M.P. J. Neurochem. 1997; 68: 255-264Crossref PubMed Scopus (708) Google Scholar, 10Mark R.J. Pang Z. Geddes J.W. Uchida K. Mattson M.P. J. Neurosci. 1997; 17: 1046-1054Crossref PubMed Google Scholar, 11Kruman I. Bruce-Keller A.J. Bredesen D.E. Waeg G. Mattson M.P. J. Neurosci. 1997; 17: 5089-5100Crossref PubMed Google Scholar, 12Blanc E.M. Keller J.N. Fernandez S. Mattson M.P. Glia. 1998; 22: 149-160Crossref PubMed Scopus (132) Google Scholar). The mechanism whereby MLP damages neurons involves an aldehyde called 4-hydroxy-2,3-nonenal (4HN), which is liberated from peroxidized fatty acids, particularly arachidonic acid, and can covalently modify proteins on cysteine, lysine, and histidine residues (10Mark R.J. Pang Z. Geddes J.W. Uchida K. Mattson M.P. J. Neurosci. 1997; 17: 1046-1054Crossref PubMed Google Scholar, 12Blanc E.M. Keller J.N. Fernandez S. Mattson M.P. Glia. 1998; 22: 149-160Crossref PubMed Scopus (132) Google Scholar, 13Esterbauer H. Schaur R.J. Zollner H. Free Radic. Biol. Med. 1991; 11: 81-128Crossref PubMed Scopus (5936) Google Scholar, 14Pedersen W.A. Chan S.L. Mattson M.P. J. Neurochem. 2000; 74: 1426-1433Crossref PubMed Scopus (117) Google Scholar). When neurons are exposed to agents that induce MLP, 4HN accumulates in the cells at concentrations of 1–10 μm (9Mark R.J. Lovell M.A. Markesbery W.R. Uchida K. Mattson M.P. J. Neurochem. 1997; 68: 255-264Crossref PubMed Scopus (708) Google Scholar). Similar concentrations of 4HN can impair synaptic function and facilitate neuronal apoptosis and excitotoxicity (9Mark R.J. Lovell M.A. Markesbery W.R. Uchida K. Mattson M.P. J. Neurochem. 1997; 68: 255-264Crossref PubMed Scopus (708) Google Scholar, 10Mark R.J. Pang Z. Geddes J.W. Uchida K. Mattson M.P. J. Neurosci. 1997; 17: 1046-1054Crossref PubMed Google Scholar, 11Kruman I. Bruce-Keller A.J. Bredesen D.E. Waeg G. Mattson M.P. J. Neurosci. 1997; 17: 5089-5100Crossref PubMed Google Scholar,15Keller J.N. Pang Z. Geddes J.W. Begley J.G. Germeyer A. Waeg G. Mattson M.P. J. Neurochem. 1997; 69: 273-284Crossref PubMed Scopus (414) Google Scholar). It was recently reported that 4HN can alter signal transduction pathways by modifying the activities of kinases and transcription factors (16Liu W. Akhand A.A. Kato M. Yokoyama I. Miyata T. Kurokawa K. Uchida K. Nakashima I. J. Cell Sci. 1999; 112: 2409-2417Crossref PubMed Google Scholar, 17Camandola S. Poli G. Mattson M.P. Mol. Brain Res. 2000; 85: 53-60Crossref PubMed Scopus (70) Google Scholar, 18Camandola S. Poli G. Mattson M.P. J. Neurochem. 2000; 74: 159-168Crossref PubMed Scopus (153) Google Scholar, 19Rinaldi M. Barrera G. Aquino A. Spinsanti P. Pizzimenti S. Farace M.G. Dianzani M.U. Fazio V.M. Biochem. Biophys. Res. Commun. 2000; 272: 75-80Crossref PubMed Scopus (32) Google Scholar). Voltage-dependent Ca2+ channels (VDCCs) play critical roles in the regulation of levels of intracellular Ca2+ and thereby control an array of physiological processes in neurons including neurotransmitter release, synaptic plasticity, and gene expression (for review, see Refs. 20Berridge M.J. Lipp P. Bootman M.D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 11-21Crossref PubMed Scopus (4493) Google Scholar and 21Mattson M.P. LaFerla F.M. Chan S.L. Leissring M. Shepel P.N. Geiger J.D. Trends Neurosci. 2000; 23: 222-229Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar). Overactivation of VDCCs, particularly under conditions of reduced energy availability or increased oxidative stress, contributes to neuronal death in experimental models of stroke, trauma, and Alzheimer's disease (22Miljanich G.P. Ramachandran J. Annu. Rev. Pharmacol. Toxicol. 1995; 35: 707-734Crossref PubMed Scopus (288) Google Scholar, 23Alkon D.L. Nelson T.J. Zhao W. Cavallaro S. Trends Neurosci. 1998; 21: 529-537Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). VDCCs are heteromeric complexes of α, β, and sometimes γ subunits. The activity of VDCCs is modulated by phosphorylation such that channel activity is increased in response to phosphorylation of one or more subunits on tyrosine or serine residues (24Sculptoreanu A. Scheuer T. Catterall W.A. Nature. 1993; 364: 240-243Crossref PubMed Scopus (213) Google Scholar, 25Haby C. Larsson O. Islam M.S. Aunis D. Berggren P.O. Zwiller J. Biochem. J. 1994; 298: 341-346Crossref PubMed Scopus (42) Google Scholar, 26Rane S.G. Pollock J.D. J. Neurosci. Res. 1994; 38: 590-598Crossref PubMed Scopus (28) Google Scholar, 27Kavalali E.T. Hwang K.S. Plummer M.R. J. Neurosci. 1997; 17: 5334-5348Crossref PubMed Google Scholar, 28Evans G.J. Pocock J.M. Eur. J. Neurosci. 1999; 11: 279-292Crossref PubMed Scopus (39) Google Scholar). Despite the evidence that MLP can alter synaptic function and promote neuronal degeneration, the effects of MLP on VDCCs are unknown. In the present study, we employed whole-cell patch clamp, Ca2+ imaging, and immunochemical methods to examine the effects of 4HN on VDCCs in hippocampal neurons. Our data demonstrate that 4HN can increase the activity of VDCCs by a mechanism involving increased tyrosine phosphorylation of channel subunits. Primary hippocampal cell cultures were established from embryonic rats (day 18 of gestation) as detailed elsewhere (29Mattson M.P. Lovell M.A. Furukawa K. Markesbery W.R. J. Neurochem. 1995; 65: 1740-1751Crossref PubMed Scopus (595) Google Scholar). Cells were plated into polyethyleneimine-coated plastic culture dishes or 22-mm2 glass coverslips at a density of 80–100 cells/mm2. The cultures were maintained in Neurobasal Medium with B27 supplements (Invitrogen). The atmosphere consisted of 6% CO2/94% room air and was maintained near saturation with water. Experiments were performed in cultures that had been maintained for 8–10 days. Using these culture conditions, ∼95% of the cells are neurons, and the remaining cells are astrocytes. Immediately before experimental treatment, the culture maintenance medium was replaced with Locke's buffer (154 mm NaCl, 5.6 mm KCl, 2.3 mm CaCl2, 1.0 mm MgCl2, 3.6 mmNaHCO3, 10 mm glucose, and 5 mmHepes buffer, pH 7.2). 4-Hydroxy-2,3-nonenal (Cayman Chemical) was prepared as a 1000× stock in ethanol. Nimodipine, genistein, and okadaic acid (Sigma) were prepared as 500× stocks in dimethyl sulfoxide. ω-Conotoxin-GVIA and ω-agatoxin-TK (Research Biochemicals International) were dissolved in saline. Sodium vanadate (Sigma) was prepared as 500× stock, and glutathione ethyl ester (Sigma) was prepared as a 50× stock in saline. In all experiments, an equivalent volume of vehicle was added to control cultures. Neuron survival was quantified by counting the number of viable neurons in premarked microscope fields before and at indicated time points after exposure to experimental treatments as described previously (29Mattson M.P. Lovell M.A. Furukawa K. Markesbery W.R. J. Neurochem. 1995; 65: 1740-1751Crossref PubMed Scopus (595) Google Scholar). These methods were similar to those used previously (30Furukawa K., Fu, W., Li, Y. Witke W. Kwiatkowski D.J. Mattson M.P. J. Neurosci. 1997; 17: 8178-8186Crossref PubMed Google Scholar, 31Furukawa K. Mattson M.P. J. Neurochem. 1998; 70: 1876-1886Crossref PubMed Scopus (224) Google Scholar). Briefly, responses were recorded at room temperature using a whole-cell recording configuration with a patch clamp amplifier (Axopatch-1D) and data acquisition and analysis software (pCLAMP-8) with filtering at 2 kHz. Glass pipettes were pulled with a Flaming-Brown horizontal puller (Sutter Instruments, Novato, CA). Electrodes were coated with Sylgard (Dow Corning, Midland, MI) and had an average resistance of 2 megaohms. For recording whole-cell currents through VDCCs, the ionic composition of the external solution was 145 mm NaCl, 5 mm CsCl, 8 mm CaCl2, 10 mm glucose, 0.3 μm tetrodotoxin, and 10 mm Hepes (the pH was adjusted to 7.35 with NaOH, and the osmolarity was adjusted to 330 mosmol with sucrose). The internal solution contained 145 mm methanesulfonic acid, 10 mm Hepes, 11 mm EGTA, 2 mm MgCl2, 1 mm CaCl2, 5 mm Mg-ATP, 13 mm tetramethylammonium chloride, 0.1 mmleupeptin, and 10 mm Hepes. Whole-cell current amplitude was normalized by dividing by whole-cell capacitance to yield current density. Cell capacitance and series resistance were measured at the start of each recording. Cells were constantly perfused with external solution, and test agents were applied to neurons via rapid switch of solutions using a six-channel valve controller apparatus (Warner Instrument Corp.). Intracellular free Ca2+ levels ([Ca2+] i ) were quantified by fluorescence imaging of the calcium indicator dye Fura-2 as described previously (29Mattson M.P. Lovell M.A. Furukawa K. Markesbery W.R. J. Neurochem. 1995; 65: 1740-1751Crossref PubMed Scopus (595) Google Scholar). Briefly, cells were incubated for 30 min in the presence of 2 μm acetoxymethylester form of Fura-2 (Molecular Probes, Eugene, OR), washed with Locke's buffer, and incubated for 40 min before imaging. Cells were imaged on a Zeiss Axiovert microscope (×40 oil immersion objective) coupled to an Attofluor imaging system. The [Ca2+] i in 12–20 neuronal cell bodies/microscope field was monitored before and after exposure of cells to KCl, which was added to the bathing medium by dilution from a 4× stock. Immunoprecipitations were performed as described previously (32Chan S.L. Mayne M. Holden C.P. Geiger J.D. Mattson M.P. J. Biol. Chem. 2000; 275: 18195-18200Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). Briefly, after exposure to 4HN or vehicle, cultures were lysed in radioimmune precipitation buffer (10% glycerol, 1% Triton X-100, 150 mm NaCl, 100 mm NaF, 5 mm EDTA, 2 mm phenylmethylsulfonyl fluoride, 1 mm sodium orthovanadate, 50 mm Tris-HCl (pH 7.5), and 1 μg/ml leupeptin). Channel subunits were immunoprecipitated from 200 μg of cell lysate using rabbit polyclonal antibodies that recognize either all isoforms of the α subunit of VDCCs or only the α1c subunit. 4HN-modified proteins were immunoprecipitated using a mouse monoclonal antibody that specifically recognizes 4HN-modified proteins (14Pedersen W.A. Chan S.L. Mattson M.P. J. Neurochem. 2000; 74: 1426-1433Crossref PubMed Scopus (117) Google Scholar, 33Waeg G. Mimsity G. Esterbauer H. Free Radic. Res. 1996; 25: 149-159Crossref PubMed Scopus (162) Google Scholar). The antibody-lysate solution was left overnight at 4 °C on a rotary shaker, and the antibody-antigen complex was then pelleted using protein A linked to acrylic beads (M01; Sigma). The pellet was washed three times with ice-cold radioimmune precipitation buffer, and the final pellet was suspended in 2× Laemmli sample buffer. Samples were boiled for 10 min and centrifuged at 3000 rpm for 30 s, and the supernatant was loaded on a 7.5% SDS-PAGE gel. Protein was transferred to nitrocellulose, and the blot was incubated with a primary antibody. The blot was further processed using horseradish peroxidase-conjugated secondary antibody and a chemiluminescence detection method (AmershamBiosciences). Primary antibodies used included an antibody that recognizes all isoforms of the α subunit of VDCCs (anti-panα, mouse monoclonal antibody from Alomone Laboratories; 1:50 dilution for immunoprecipitation and 1:1000 dilution for immunoblots), antibodies specific for either the α1c or α1d subunits of VDCCs (anti-α1c, mouse monoclonal antibody from Alomone Laboratories; 1:50 dilution for immunoprecipitation and 1:1000 dilution for immunoblots), anti-phosphotyrosine (mouse monoclonal antibody from Transduction Laboratories; 1:1000 dilution), anti-phosphoserine (mouse monoclonal antibody from Sigma; 1:500 dilution), and anti-α-tubulin (rabbit polyclonal antibody from Sigma; 1:1000 dilution). Previous studies have shown that concentrations of 4HN in the range of 1–10 μm accumulate in cultured hippocampal neurons exposed to agents that induce MLP and that exposure of hippocampal neurons to similar concentrations of 4HN can alter synaptic function and promote excitotoxicity (9Mark R.J. Lovell M.A. Markesbery W.R. Uchida K. Mattson M.P. J. Neurochem. 1997; 68: 255-264Crossref PubMed Scopus (708) Google Scholar, 15Keller J.N. Pang Z. Geddes J.W. Begley J.G. Germeyer A. Waeg G. Mattson M.P. J. Neurochem. 1997; 69: 273-284Crossref PubMed Scopus (414) Google Scholar). In preliminary studies, we determined that concentrations of 1–10 μm4HN were not acutely toxic to neurons but did induce delayed death of neurons and increase their vulnerability to excitotoxicity (9Mark R.J. Lovell M.A. Markesbery W.R. Uchida K. Mattson M.P. J. Neurochem. 1997; 68: 255-264Crossref PubMed Scopus (708) Google Scholar, 11Kruman I. Bruce-Keller A.J. Bredesen D.E. Waeg G. Mattson M.P. J. Neurosci. 1997; 17: 5089-5100Crossref PubMed Google Scholar). Whole-cell Ca2+ currents were recorded under basal conditions and at increasing time points after exposure to 4HN. Pretreatment of hippocampal neurons with 1 μm 4HN for 2 h resulted in a significant 40% increase in the amplitude of the Ca2+ current, and the current amplitude was increased by an additional 20% in neurons exposed to 10 μm 4HN (Fig. 1, A and C). Analysis of the current-voltage relationships of the currents recorded in the absence or presence of 4HN revealed that the voltage dependence and the voltage generating the maximum inward current (approximately 10 mV) remained unchanged in neurons exposed to 4HN (Fig. 1 B). The enhancement of the Ca2+ current by 4HN was time-dependent such that exposure times of 10 min or less had no significant effect on the current, whereas longer exposure periods resulted in progressive increases in the amplitude of the Ca2+ current, which reached a peak level at 2 h and remained elevated through 4 h of exposure (Fig. 1 D). The effect of 4HN on the Ca2+ current was specific for this aldehyde in that exposure of neurons to malondialdehyde (a major product of MLP) for 2 h at concentrations of up to 100 μm had no effect on the amplitude of the Ca2+current (data not shown), consistent with our previous data showing that among aldehydes produced as a result of MLP, 4HN is uniquely toxic to neurons (9Mark R.J. Lovell M.A. Markesbery W.R. Uchida K. Mattson M.P. J. Neurochem. 1997; 68: 255-264Crossref PubMed Scopus (708) Google Scholar). We next measured intracellular free Ca2+ levels by imaging of the Ca2+ indicator dye Fura-2 in control and 4HN-treated hippocampal neurons under basal conditions and after membrane depolarization. The basal concentration of Ca2+ was similar (70–110 nm) in neurons in control cultures and in cultures that had been exposed for 2 h to 1 μm 4HN (Fig.1 E). In control cultures, exposure to KCl resulted in a rapid rise of the intracellular Ca2+ concentration to a peak level of ∼280 nm, followed by a decrease to a sustained level of ∼200 nm 2 min later. In 4HN-treated cultures, both the peak and sustained depolarization-induced increase of Ca2+ levels were significantly increased to ∼400 nm compared with the responses in neurons in control cultures (Fig. 1, E and F). Cultured embryonic hippocampal neurons express VDCCs of the L, N, and P/Q types, with the majority of whole-cell current being contributed by L-type channels (31Furukawa K. Mattson M.P. J. Neurochem. 1998; 70: 1876-1886Crossref PubMed Scopus (224) Google Scholar, 34Blalock E.M. Porter N.M. Landfield P.W. J. Neurosci. 1999; 19: 8674-8684Crossref PubMed Google Scholar). To determine which of these types of channels were affected by 4HN, we employed pharmacological agents that selectively block each channel type. Neurons were sequentially exposed to blockers of three different classes of VDCCs, and the Ca2+ currents were recorded. In control cultures, addition of ω-conotoxin-GVIA (a blocker of N-type channels) resulted in a 34% decrease in the Ca2+ current, addition of nimodipine (a blocker of L-type channels) resulted in an additional 32% decrease, and addition of ω-agatoxin-TK (a blocker of P/Q channels; Refs. 35Wheeler D.B. Randall A. Tsien R.W. Science. 1994; 264: 107-111Crossref PubMed Scopus (842) Google Scholarand 36Sather W.A. Tanabe T. Zhang J.-F. Mori Y. Adams M.E. Tsien R.W. Neuron. 1993; 11: 291-303Abstract Full Text PDF PubMed Scopus (382) Google Scholar) caused an additional 30% decrease in the amplitude of the Ca2+ current (Fig. 2). In 4HN-treated neurons, ω-conotoxin-GVIA caused a 24% decrease in the Ca2+ current amplitude, nimodipine caused an additional 47% decrease, and ω-agatoxin-TK caused an additional 28% decrease (Fig. 2). Because the total increase in Ca2+ current induced by 4HN is ∼50%, these results suggest that the increase is due mainly to increases in current carried by L-type and P/Q-type channels. The increase in whole-cell Ca2+ current induced by 4HN might result from an increase in the number of channels present or from increased activity of a fixed number of channels. To discriminate between these two possibilities, we performed immunoblot analyses to assess relative levels of VDCC proteins in control and 4HN-treated hippocampal neurons. Similar levels of the α1 subunit of VDCC were present in homogenates from control cultures and cultures that had been exposed for 2 h to 10 μm 4HN, as determined in immunoblots probed with either an antibody that recognizes all isoforms of the α1 subunit of VDCCs or an antibody specific for the α1c isoform (Fig.3). Because proteins can be covalently modified by 4HN, we determined whether this was the case with VDCC subunits. Proteins in lysates of control and 4HN-treated cultures were immunoprecipitated using an antibody against 4HN-modified proteins (10Mark R.J. Pang Z. Geddes J.W. Uchida K. Mattson M.P. J. Neurosci. 1997; 17: 1046-1054Crossref PubMed Google Scholar,33Waeg G. Mimsity G. Esterbauer H. Free Radic. Res. 1996; 25: 149-159Crossref PubMed Scopus (162) Google Scholar), and the precipitated proteins were subjected to immunoblot analysis using the pan-α1 or α1c-specific antibodies. No band could be detected with the αl subunit antibodies, suggesting that these proteins are not modified by 4HN (Fig. 3). Because currents through VDCCs are known to be increased by phosphorylation of channel α subunit proteins (25Haby C. Larsson O. Islam M.S. Aunis D. Berggren P.O. Zwiller J. Biochem. J. 1994; 298: 341-346Crossref PubMed Scopus (42) Google Scholar, 26Rane S.G. Pollock J.D. J. Neurosci. Res. 1994; 38: 590-598Crossref PubMed Scopus (28) Google Scholar, 27Kavalali E.T. Hwang K.S. Plummer M.R. J. Neurosci. 1997; 17: 5334-5348Crossref PubMed Google Scholar), we performed additional experiments to determine whether 4HN might modify VDCC protein phosphorylation in a manner that enhances Ca2+currents. To this end, we exposed neurons to 4HN for increasing time periods, immunoprecipitated the α1 subunits of VDCCs from equal amounts of cell homogenates (200 μg of protein), and performed an immunoblot analysis using an antibody that selectively binds to proteins phosphorylated on tyrosine residues. The results indicated that levels of tyrosine phosphorylation of α1 proteins were increased within 30 min of exposure to 4HN, continued to increase through 90 min, and then decreased at 3 h (Fig. 4). Immunoprecipitation-immunoblot analysis using a phosphoserine antibody indicated that no significant increase in the level of serine phosphorylation of α1 proteins occurs in neurons exposed to 4HN, whereas okadaic acid (1 μm), an inhibitor of serine phosphatases, causes a large increase in the level of immunoreactivity of α1 subunits with the anti-phosphoserine antibody (Fig.5 A). The increase in tyrosine phosphorylation of α1 proteins in response to 4HN was comparable to that observed in neurons exposed to the tyrosine phosphatase inhibitor vanadate (Fig. 5 B). Immunoprecipitation-immunoblot analysis using an antibody specific for αlc and α1d revealed significant increases in tyrosine phosphorylation of these L-type VDCC subunits in neurons exposed to 4HN (Fig. 5,C and D).Figure 54HN increases tyrosine phosphorylation of the α1 VDCC subunit in hippocampal neurons. Hippocampal neurons were treated for 2 h with 1 μm 4HN, 10 μm okadaic acid, 1 mm vanadate, or vehicle. A−D, immunoblots of proteins (50 μg) immunoprecipitated using either the pan-α1 or αlc-specific antibodies were then performed using antibodies against phosphotyrosine (p-tyro) and phosphoserine (p-ser) as indicated. E, samples of cell lysates (50 μg of protein) were subjected to immunoblot analysis using an antibody against α-tubulin. Note the large increase in the level of phosphotyrosine immunoreactivity of the α1 subunit in neurons exposed to 4HN. ∗, p < 0.05; ∗∗,p < 0.01 compared with control value.IP, immunoprecipitation; WB, Western blot.View Large Image Figure ViewerDownload Hi-res image Download (PPT) If tyrosine phosphorylation of VDCC proteins was responsible for the increased Ca2+ current induced by 4HN, then the effect of 4HN should be mimicked by selective inhibition of tyrosine phosphatases and should be suppressed by inhibition of tyrosine kinases. Exposure of neurons to vanadate, an inhibitor of tyrosine phosphatases (37Gordon J.A. Methods Enzymol. 1991; 201: 477-482Crossref PubMed Scopus (528) Google Scholar), significantly increased the amplitude of the whole-cell Ca2+ current (Fig. 6), suggesting that tyrosine phosphatases modulate basal VDCC activity by reducing channel phosphorylation. Combined treatment with 4HN and vanadate resulted in an increase in the amplitude of the Ca2+ current similar to that obtained with either agent alone, suggesting that they may act via a common mechanism. Exposure of neurons to the tyrosine kinase inhibitor genistein (30 μm) did not significantly affect the basal Ca2+ current but significantly attenuated the increase in current caused by 4HN (Fig. 6). Collectively, these data suggest that an increase in tyrosine phosphorylation of VDCC subunits is involved in enhancement of VDCC activity by 4HN. Prolonged exposure of hippocampal neurons to 4HN results in delayed cell death that is evident after 12–24 h (11Kruman I. Bruce-Keller A.J. Bredesen D.E. Waeg G. Mattson M.P. J. Neurosci. 1997; 17: 5089-5100Crossref PubMed Google Scholar). Previous studies have shown that glutathione is covalently modified by 4HN on the cysteine residue of the peptide and that glutathione can thereby detoxify 4HN and protect cells from injury induced by lipid peroxidation or direct exposure to 4HN (9Mark R.J. Lovell M.A. Markesbery W.R. Uchida K. Mattson M.P. J. Neurochem. 1997; 68: 255-264Crossref PubMed Scopus (708) Google Scholar, 11Kruman I. Bruce-Keller A.J. Bredesen D.E. Waeg G. Mattson M.P. J. Neurosci. 1997; 17: 5089-5100Crossref PubMed Google Scholar, 38Siems W.G. Pimenov A.M. Esterbauer H. Grune T. J. Biochem. 1998; 123: 534-539Crossref PubMed Scopus (29) Google Scholar). In the present study, we found that this delayed neuronal death was completely prevented in cultures cotreated with glutathione ethyl ester (Fig.7). When hippocampal cultures were cotreated with 500 μm glutathione ethyl ester and 10 μm 4HN, there was no increase in the amplitude of the Ca2+ current. Current amplitudes were as follows: control, 22 + 4.5 pA/picofarad; 4HN, 33 + 5.2 pA/picofarad; glutathione, 26 + 5.9 pA/picofarad; and glutathione + 4HN, 25 + 3.3 pA/picofarad (mean ± S.D.; n = 3). Treatment of neurons with nimodipine significantly attenuated 4HN-induced cell death (Fig. 7), demonstrating the involvement of Ca2+ influx through VDCCs in the neurotoxic action of 4HN. Neither ω-conotoxin-GVIA nor ω-agatoxin-TK affected 4HN-induced cell death (data not shown), consistent with our electrophysiological data showing that 4HN does not alter N- or P/Q-type VDCCs. The present findings are the first to document an effect of the MLP product 4HN on the channel activity of VDCCs. Several lines of evidence suggest that the enhancement of Ca2+ current in response to 4HN is an indirect effect mediated by increased phosphorylation of VDCC subunits on tyrosine residues. First, 4HN may not covalently modify VDCC α1 subunits because we could not detect association of 4HN with α1 subunits using an immunoprecipitation and immunoblot approach that we previously employed to demonstrate covalent modification by 4HN of the glucose transport protein GLUT3 (10Mark R.J. Pang Z. Geddes J.W. Uchida K. Mattson M.P. J. Neurosci. 1997; 17: 1046-1054Crossref PubMed Google Scholar), a glutamate transport protein (12Blanc E.M. Keller J.N. Fernandez S. Mattson M.P. Glia. 1998; 22: 149-160Crossref PubMed Scopus (132) Google Scholar), the GTP-binding protein Gq11 (39Blanc E.M. Kelly J.F. Mark R.J. Mattson M.P. J. Neurochem. 1997; 69: 570-580Crossref PubMed Sco" @default.
• W2091966984 created "2016-06-24" @default.
• W2091966984 creator A5024369563 @default.
• W2091966984 creator A5068840254 @default.
• W2091966984 creator A5080235402 @default.
• W2091966984 creator A5083040475 @default.
• W2091966984 date "2002-07-01" @default.
• W2091966984 modified "2023-10-16" @default.
• W2091966984 title "The Lipid Peroxidation Product 4-Hydroxynonenal Facilitates Opening of Voltage-dependent Ca2+ Channels in Neurons by Increasing Protein Tyrosine Phosphorylation" @default.
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