Acid-sensing Ion Channels in Malignant Gliomas
2003; Elsevier BV; Volume: 278; Issue: 17 Linguagem: Inglês
10.1074/jbc.m300991200
ISSN1083-351X
AutoresBakhrom K. Berdiev, Jiazeng Xia, Lee Anne McLean, James M. Markert, G. Yancey Gillespie, Timothy B. Mapstone, Anjaparavanda P. Naren, Biljana Jovov, James K. Bubien, Hong-Long Ji, Catherine M. Fuller, Kevin L. Kirk, Dale Benos,
Tópico(s)Pancreatic function and diabetes
ResumoHigh grade glioma cells derived from patient biopsies express an amiloride-sensitive sodium conductance that has properties attributed to the human brain sodium channel family, also known as acid-sensing ion channels (ASICs). This amiloride-sensitive conductance was not detected in cells obtained from normal brain tissue or low grade or benign tumors. Differential gene profiling data showed that ASIC1 and ASIC2 mRNA were present in normal and low grade tumor cells. Although ASIC1 was present in all of the high grade glial cells examined, ASIC2 mRNA was detected in less than half. The main purpose of our work was to examine the molecular mechanisms that may underlie the constitutively activated sodium currents present in high grade glioma cells. Our results show that 1) gain-of-function mutations of ASIC1 were not present in a number of freshly resected and cultured high grade gliomas, 2) syntaxin 1A inhibited ASIC currents only when ASIC1 and ASIC2 were co-expressed, and 3) the inhibition of ASIC currents by syntaxin 1A had an absolute requirement for either γ- or δ-hENaC. Transfection of cultured cells originally derived from high grade gliomas (U87-MG and SK-MG1) with ASIC2 abolished basal amiloride-sensitive sodium conductance; this inhibition was reversed by dialysis of the cell interior with Munc-18, a syntaxin-binding protein that typically blocks the interaction of syntaxin with other proteins. Thus, syntaxin 1A cannot inhibit Na+ permeability in the absence of adequate plasma membrane ASIC2 expression, accounting for the observed functional expression of amiloride-sensitive currents in high grade glioma cells. High grade glioma cells derived from patient biopsies express an amiloride-sensitive sodium conductance that has properties attributed to the human brain sodium channel family, also known as acid-sensing ion channels (ASICs). This amiloride-sensitive conductance was not detected in cells obtained from normal brain tissue or low grade or benign tumors. Differential gene profiling data showed that ASIC1 and ASIC2 mRNA were present in normal and low grade tumor cells. Although ASIC1 was present in all of the high grade glial cells examined, ASIC2 mRNA was detected in less than half. The main purpose of our work was to examine the molecular mechanisms that may underlie the constitutively activated sodium currents present in high grade glioma cells. Our results show that 1) gain-of-function mutations of ASIC1 were not present in a number of freshly resected and cultured high grade gliomas, 2) syntaxin 1A inhibited ASIC currents only when ASIC1 and ASIC2 were co-expressed, and 3) the inhibition of ASIC currents by syntaxin 1A had an absolute requirement for either γ- or δ-hENaC. Transfection of cultured cells originally derived from high grade gliomas (U87-MG and SK-MG1) with ASIC2 abolished basal amiloride-sensitive sodium conductance; this inhibition was reversed by dialysis of the cell interior with Munc-18, a syntaxin-binding protein that typically blocks the interaction of syntaxin with other proteins. Thus, syntaxin 1A cannot inhibit Na+ permeability in the absence of adequate plasma membrane ASIC2 expression, accounting for the observed functional expression of amiloride-sensitive currents in high grade glioma cells. acid-sensing ion channel cystic fibrosis transmembrane conductance regulator epithelial Na+ channel glioblastoma multiforme 4-morpholineethanesulfonic acid 4-morpholinepropanesulfonic acid glutathioneS-transferase reverse transcriptase solubleN-ethylmaleimide-sensitive factor attachment protein receptors protein kinase C Primary intracranial neoplasms remain a significant cause of mortality and morbidity in both children and adults. In patients with malignant gliomas of World Health Organization Grades III and IV, disease progression is uniformly rapid despite aggressive surgical and adjunctive therapies. Median survival of optimally treated individuals with the most aggressive of these tumors, glioblastoma multiforme, is 12 months, and this statistic has not varied for more than 30 years. The ability to treat these tumors successfully has been hampered by a fundamental lack of understanding of the control of the growth and differentiation of glial cells, how the transformed phenotype evolves, and how glioma cells modify their environment to support their increased energy demands. We have identified a novel amiloride-sensitive inward Na+ current that appears to be constitutively activated in malignant gliomas but not in low grade or normal astrocytes (1Bubien J.K. Keeton D.A. Fuller C.M. Gillespie G.Y. Reddy A.T. Mapstone T.B. Benos D.J. Am. J. Physiol. 1999; 276: C1405-C1410Crossref PubMed Google Scholar). In addition, glioma cells display up-regulation of Cl− and K+ channels not found, at least functionally, in normal glia (2Brismar T. Collins V.P. Brain Res. 1989; 480: 259-267Crossref PubMed Scopus (28) Google Scholar, 3Ullrich N. Sontheimer H. Am. J. Physiol. 1996; 270: C1511-C1521Crossref PubMed Google Scholar). Thus, it is reasonable to hypothesize that ion transport systems specifically expressed by glioma cells are intimately related to and indeed may define the unique growth and migratory ability of these cells. The main objective of the present study was to explore the molecular mechanisms that underlie the constitutively activated Na+currents present in high grade glioma cells. We assume, based on our previous electrophysiological, pharmacological, and molecular biological studies, that the brain Na+ channels, also known as acid-sensing ion channels (ASICs),1 may comprise the core conduction element of these channels. To date, six members of the ASIC family have been cloned in mammals (4Bassler E.L. Ngo-Anh T.J. Geisler H.S. Ruppersberg J.P Grunder S. J. Biol. Chem. 2001; 276: 33782-33787Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). These channels share the common property of generating excitatory currents in response to acidic pH when studied in heterologous expression systems, except for ASIC2b that, at least in its homomeric form, does not appear to respond to low pH (5Lingueglia E. de Weille J.R. Bassilana F. Heurteaux C. Sakai H. Waldmann R. Lazdunski M. J. Biol. Chem. 1997; 272: 29778-29783Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar). Moreover, ASIC4 is inactive by itself and hence is not thought to encode a proton-gated ion channel (6Akopian A.N. Chen C.C. Ding Y. Cesare P. Wood J.N. Neuroreport. 2000; 11: 2217-2222Crossref PubMed Scopus (188) Google Scholar, 7Grunder S. Geisler H.S. Rainier S. Fink J.K. Eur. J. Hum. Genet. 2001; 9: 672-676Crossref PubMed Scopus (15) Google Scholar). Although the subunit composition of brain Na+ channels in native tissues is unknown, evidence for heteromultimeric channel formation with distinctive functional characteristics has been obtained (7Grunder S. Geisler H.S. Rainier S. Fink J.K. Eur. J. Hum. Genet. 2001; 9: 672-676Crossref PubMed Scopus (15) Google Scholar, 8Bassilana F. Champigny G. Waldmann R. de Weille J.R. Heurteaux C. Lazdunski M. J. Biol. Chem. 1997; 272: 28819-28822Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 9Babkinski K. Catarsi S. Biagini G. Seguela P. J. Biol. Chem. 2000; 275: 28519-28525Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 10Zhang P. Canessa C.M. J. Gen. Physiol. 2001; 117: 563-572Crossref PubMed Scopus (28) Google Scholar). These brain Na+channels, like their epithelial counterparts, can be inhibited by amiloride and its analogs, although with a much lower affinity (11Waldmann R. Champigny G. Lingueglia E. De Weille J.R. Heurteaux C. Lazdunski M. Ann. N. Y. Acad. Sci. 1999; 868: 67-76Crossref PubMed Scopus (183) Google Scholar). A role for chemical pain sensation has been proposed for these channels in sensory neurons (12Waldmann R. Lazdunski M. Curr. Opin. Neurobiol. 1998; 8: 418-424Crossref PubMed Scopus (450) Google Scholar, 13Babinski K. Le K.T. Seguela P. J. Neurochem. 1999; 72: 51-57Crossref PubMed Scopus (159) Google Scholar), but their role in the brain is obscure. Proton-activated neuronal currents have been identified in different brain regions (13Babinski K. Le K.T. Seguela P. J. Neurochem. 1999; 72: 51-57Crossref PubMed Scopus (159) Google Scholar, 14Chesler M. Kaila K. Trends Neurosci. 1992; 15: 396-402Abstract Full Text PDF PubMed Scopus (483) Google Scholar). Hence, these channels may function as acid pH sensors in normal brain and in pathophysiological states such as ischemia or epilepsy where tissue acidification occurs (15Biagini G. Babinski K. Avoli M. Marcinkiewicz M. Seguela P. Neurobiol Dis. 2001; 8: 45-58Crossref PubMed Scopus (68) Google Scholar, 16Johnson M.B. Kin K. Minami M. Chen D. Simon R.P. J. Cereb. Blood Flow Metab. 2001; 21: 734-740Crossref PubMed Scopus (80) Google Scholar). Gain-of-function mutations of ASIC1 and ASIC2 have also been detected and have been proposed to participate in neurodegenerative disease (7Grunder S. Geisler H.S. Rainier S. Fink J.K. Eur. J. Hum. Genet. 2001; 9: 672-676Crossref PubMed Scopus (15) Google Scholar,17Waldmann R. Champigny G. Voilley N. Lauritzen I. Lazdunski M. J. Biol. Chem. 1996; 271: 10433-10436Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, 18Adams C.M. Snyder P.M. Price M.P. Welsh M.J. J. Biol. Chem. 1998; 273: 30204-30207Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The function of these channels in glia remains a mystery, yet functional amiloride-sensitive Na+ current expression selectively characterizes high grade gliomas. There are a number of potential mechanisms that may produce a constitutive activation of this class of channel. We tested two hypotheses. First, as a consequence of oncogenic transformation, gain-of-function mutations of ASIC1 may occur. Second, we tested the hypothesis that syntaxin 1A may down-regulate ASIC1 activity by analogy with the effects of syntaxin 1A on other ion channel activities, such as voltage-dependent Ca2+ channels, CFTR, and ENaC. Our results show that gain-of-function mutations of ASIC1 are not present in a multitude of freshly resected and cultured high grade gliomas (glioblastoma multiforme or GBM, World Health Organization classification Grade IV). However, syntaxin 1A inhibits ASIC currents, but only when both ASIC1 and ASIC2 are co-expressed. Moreover, the inhibition of ASIC currents by syntaxin 1A requires either γ- or δ-hENaC. Furthermore, message for ASIC2 could be detected in 30–40% of high grade gliomas. We suggest that it is the failure of syntaxin 1A to inhibit ASIC1 activity in the absence of plasma membrane-localized ASIC2 that accounts for the functional expression of amiloride-sensitive Na+ currents in astrocytoma cells. The cDNAs encoding full-length γ-ENaC subunits, ASIC1, and ASIC2 are described elsewhere (19McDonald F.J. Snyder P.M. McCray Jr., P.B. Welsh M.J. Am. J. Physiol. 1994; 266: L728-L734Crossref PubMed Google Scholar, 20McDonald F.J. Price M.P. Snyder P.M. Welsh M.J. Am. J. Physiol. 1995; 268: C1157-C1163Crossref PubMed Google Scholar, 21Garcia-Añovers J. Derfler B. Neville-Golden J. Hyman B.T. Corey D.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1459-1464Crossref PubMed Scopus (297) Google Scholar). The human ASIC 1a (ASIC1) and ASIC2a (ASIC2) cDNAs were a gift from Drs. David Corey and Jaime Garcia-Añoveros of the Harvard Medical School. The human ENaC subunits were given to us by Dr. Michael J. Welsh of the University of Iowa. cDNAs were transcribed and translated in vitro using the TNT transcription/translation system (Promega) as previously described (22Jovov B. Tousson A. Ji H.L. Keeton D. Shlyonsky V. Ripoll P.J. Fuller C.M. Benos D.J. J. Biol. Chem. 1999; 274: 37845-37854Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). To test for protein-protein interaction between different ENaC subunits and syntaxin 1A or between ASIC1 and ASIC2, we translated these constructs either with radioactive or nonradioactive methionine, immunopurified them, and reconstituted them in different combinations in proteoliposomes as previously described (22Jovov B. Tousson A. Ji H.L. Keeton D. Shlyonsky V. Ripoll P.J. Fuller C.M. Benos D.J. J. Biol. Chem. 1999; 274: 37845-37854Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Proteoliposomes were solubilized in precipitation buffer that contained 50 mmTris, pH 7.5, 150 mm NaCl, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS. All of the precipitation reactions were carried out in this buffer. As a rule, antibodies directed against nonlabeled proteins were used, and the presence of co-precipitated radioactively labeled proteins were detected using SDS-PAGE and autoradiography. DNA samples werein vitro transcribed using the SP6 or T3mMessage Machine kits (Ambion, Austin, TX). The integrity of the cRNA was assessed by running the samples under denaturing conditions on a formaldehyde-agarose gel. RNA concentration and purity were determined by UV spectrophotometry at 260 nm. The oocytes were removed from appropriately anesthetized adult female Xenopus laevis (Xenopus Express, Beverly Hills, FL) using standard techniques (23Ji H.L. Fuller C.M. Benos D.J. J. Biol. Chem. 1999; 274: 37693-37704Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Follicular cells were removed by the addition of collagenase to calcium-free medium as described (23Ji H.L. Fuller C.M. Benos D.J. J. Biol. Chem. 1999; 274: 37693-37704Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Defolliculated oocytes were washed in OR-2 medium (82.5 mm NaCl, 2.4 mm KCl, 1.0 mm MgCl2, 1.8 mm CaCl2, and 5.0 mm HEPES, pH 7.4) and allowed to recover overnight in half-strength Liebovitz medium at 18 °C. Groups of stage VI oocytes were injected with cRNA in a 50-nl volume containing 12.5 ng of ASIC and/or ENaC subunits depending upon the experiments. Standard two-electrode voltage clamp procedures were performed at room temperature on the oocytes 24–72 h post-injection. The oocytes were impaled with two 3 m KCl-filled electrodes, each having resistances of 0.5–2 MΩ. A Dagan TEV-200 voltage clamp amplifier was used. Two Ag-AgCl reference electrodes were connected to the bath by 3 m KCl, 3% agar bridges. The regular perfused solution was ND96 (96 mm NaCl, 1 mm MgCl2, 1.8 mm CaCl2, 2 mm KCl, and 5 mm HEPES, pH 7.4). To prepare ND96 having pH values below 6.0, MES replaced HEPES. To permit ASIC to recover completely from desensitization following acidification, at least 45 s elapsed prior to a subsequent challenge of the oocyte with another acidic pH solution. The experiments were controlled by pCLAMP 8.0 software (Axon Instruments, Burlingame, CA), and the proton-sensitive currents measured at −60 mV were digitized. The current-voltage (I-V) relationships were determined by changing the clamp potential in 20-mV increments from −100 to +100 mV from a holding potential of 0 mV. All of the animal care and experimental protocols were approved by the University of Alabama at Birmingham Institutional Animal Care and Use Committee (protocol number APN 020506241). Planar lipid bilayers were formed from a solution of a 2:1 diphytanoyl-phosphatidyl-ethanolamine:diphytanoyl-phosphatidylserine dissolved in n-octanol (total concentration, 25 mg/ml). The membranes were spread onto a 200-μm diameter hole drilled in a polystyrene cup. Membrane capacitance averaged 250–350 picofarads. The standard bathing solution was 100 mmNaCl plus 10 mm MOPS buffer, pH 7.4. The lipids were obtained from Avanti Polar Lipids (Alabaster, AL). All of the solutions were filtered sterilized using 0.22-μm Sterivex-GS filters (Millipore, Bedford, MA). Electrical connections and current measurements were made as previously described (24Ismailov I.I. Shlyonsky V.G. Alvarez O. Benos D.J. J. Physiol. 1997; 504: 287-300Crossref PubMed Scopus (15) Google Scholar). The voltage was applied to the cis chamber, and the trans chamber was held at virtual ground. Oocyte membrane vesicles containing the channels of interest were applied to a preformed bilayer with a glass rod from the trans compartment with the potential held at −40 mV. Only membranes containing a single ion channel were used for experiments. The data were analyzed as before (24Ismailov I.I. Shlyonsky V.G. Alvarez O. Benos D.J. J. Physiol. 1997; 504: 287-300Crossref PubMed Scopus (15) Google Scholar). Micropipettes were constructed using a Narashigi pp-83 two-stage micropipette puller. The tips of these pipettes had internal diameters of ∼0.3–0.5 μm and outer diameters of 0.7–0.9 μm. When filled with an electrolyte solution containing 100 mm potassium gluconate, 30 mm KCl, 10 mm NaCl, 20 mm HEPES, 0.5 mm EGTA, <10 nm free Ca2+, 4 mm ATP at a pH of 7.2, the electrical resistance of the tip was 1–3 mΩ. The bath solution was serum-free RPMI 1640 cell culture medium. The solutions approximate the ionic gradients across the cell membranein vivo. The pipettes were mounted in a holder and connected to the head stage of an Axon 200A patch clamp amplifier affixed to a three-dimensional micromanipulator system attached to the microscope. The cells were viewed using a Nikon model TE200 inverted microscope fitted with an ultraviolet light source. Under fluorescence, transfected cells expressing jellyfish green fluorescence protein were identified for whole cell patch clamp analysis. The micropipettes were abutted to the cells, and slight suction was applied. Seal resistance was continuously monitored using pCLAMP 8. After the formation of seals with resistances in excess of 1 GΩ, another suction pulse was applied to form the whole cell configuration by rupturing the membrane within the seal but leaving the seal intact. Successful completion of this procedure produced a sudden increase in capacitance with no change in seal resistance, indicative of the whole cell configuration. The cells were then held at a membrane potential of 0 mV and clamped sequentially for 1600 ms each to membrane potentials of −100 mV to +100 mV in 20-mV increments, returning to the holding potential of 0 mV for 1 s between each test voltage. The currents were recorded digitally and filed in real time. The entire procedure was performed using a DOS Pentium computer modified for A/D signals with pCLAMP 8 software and with an A/D interface controlled by pCLAMP (Axon Instruments, Sunnyvale, CA). The anti-ASIC2a antibodies (Alamone, Jerusalem, Israel), anti-ASIC1 antibodies (Chemicon International, Temecula, CA), anti-γENaC antibodies (raised against peptide; CNTLRLDRAFSSQLTDTQLTNEL), and anti-syntaxin 1A antibodies (25Naren A.P. Nelson D.J. Xie W. Jovov B. Pevsner J. Bennett M.K. Benos D.J. Quick M.W. Kirk K.L. Nature. 1997; 390: 302-305Crossref PubMed Scopus (185) Google Scholar) were used for immunoprecipitation and Western blot detection. Crude membrane fractions from the glioblastoma cell line SK-MG1 were prepared as previously described (26Jovov B. Ismailov I.I. Berdiev B.K. Fuller C.M. Sorscher E.J. Dedman J.R. Kaetzel M.A. Benos D.J. J. Biol. Chem. 1995; 270: 29194-29200Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Immunoprecipitation and co-precipitation from SK-MG1 cell lysate were performed using the size X protein A immunoprecipitation kit from Pierce according to the manufacturer's instructions. Briefly, affinity-purified ASIC2 antibody (280 μg) was bound and cross-linked to protein A beads. After extensive washing, the beads were added to the SK-MG1 cell lysates (1.5 ml) and incubated overnight at 4 °C. The cells were lysed with 1.5 ml of 1% Triton X-100, 150 mm NaCl, 5 mm EDTA, 50 mm Tris, pH 7.5, containing protease inhibitors. Immunoprecipitated proteins were eluted and analyzed by electrophoresis and Western blotting. Standard electrophoresis and blotting protocols were followed. Briefly, the protein was run on 8% SDS-PAGE mini gels with 4% stacking gels for about 1 h at 200 V in a Bio-Rad Minisubcell apparatus. The gels were transferred onto polyvinylidene fluoride, treated with 5% nonfat dry milk/Tris-buffered saline/Tween, and probed with the appropriate antibodies. Secondary antibody was conjugated to horseradish peroxidase, and visualization was performed with chemiluminescent reagents (Amersham Biosciences). Controls included substitution of nonimmune rabbit IgG for primary antibodies. To determine whether expression of hASIC2 could restore the normal astrocyte Na+ current phenotype to astrocytoma cells, we transfected hASIC2 contained in a eukaryotic expression vector (GW1-CMV, the kind gift of Dr. David Corey, Massachusetts General Hospital) into the SK-MG1 (gift of Dr. Gregory Cairncross, University of Calgary) or U87-MG (purchased from ATCC) astrocytoma cell lines using LipofectAMINE (Invitrogen) for whole cell patch-clamp analysis. Briefly, 5–10 μg of hASIC2 was mixed with 100 μl of serum-free Optimem (Invitrogen). In most experiments, hASIC2 was co-transfected with 3 μg of a green fluorescence protein reporter vector. Control cells were either untransfected or transfected with GW1-CMV vector from which the hASIC2 insert had been removed by cutting with EcoRI and religating the cut ends. The DNA/Optimem mixture was mixed with 100 μl of Optimem plus 7.5 μl of lipid and incubated at room temperature for 45 min, after which time a further 800 μl of Optimem was added. Tumor cells plated onto glass coverslips at 60–80% confluency were rinsed twice in Optimem and then incubated for 6 h in the DNA/lipid Optimem mixture. At the end of this incubation period, the medium was aspirated and replaced with fresh Dulbecco's modified Eagle's medium plus 10% fetal bovine serum. The cells were allowed to recover overnight and were used for patch-clamp experiments over the course of the next 48 h. Defined regions of syntaxin 1A and syntaxin 3 (amino acids 1–266; referred to as ΔC as they lack the C-terminal membrane anchor) were generated by polymerase chain reaction. Restriction sites (EcoRI in forward and XhoI in reverse), 5′overhangs (TATA), and a stop codon were introduced into the primers. The PCR product was cloned into pGEX5X-1 vector (Pharmacia Corp.) and transformed in a protease deficient Escherichia coli strain (BL21-DE3). The GST fusion protein (GST-Syn1AΔC and GST-Syn3ΔC) was purified on glutathione-Sepharose beads (Pharmacia Corp.). The protein was eluted using 20 mm reduced glutathione in phosphate-buffered saline, pH 7.4, and dialyzed extensively at 4 °C with at least four changes of cold phosphate-buffered saline. The protein was concentrated using a centrifugal filter device (Centriprep; 10,000 Dalton cut-off; Millipore). The protein was estimated using the BCA kit (Pierce) and stored in small aliquots at −80 °C. Freshly excised human brain tissue or primary cultured brain tumor cells (GBM or normal brain from temporal lobe) were frozen and stored in liquid nitrogen by the University of Alabama at Birmingham Neurosurgery Brain Tissue Bank under Institutional Review Board approval X9804090300. The frozen tissue was ground into a fine powder using a mortar and pestle, under liquid nitrogen, after which 1 ml of Trizol (Invitrogen) containing 250 μg of glycogen was added. The Trizol/powder mixture was transferred to a chilled glass/Teflon homogenizer and ground for 10 strokes while on ice. The homogenate was sequentially passed through 25- and 26-gauge needles to further reduce cellular debris and then transferred to a 1.5-ml microcentrifuge tube. 200 μl of chloroform was added to the homogenate, vortexed for 30 s to mix, and centrifuged at maximum speed (14,000 rpm) for 5 min using a tabletop centrifuge at room temperature. The aqueous phase was transferred to a fresh 1.5-ml tube, 500 μl of ice-cold isopropanol were added, and the RNA was allowed to precipitate overnight at −20 °C. The precipitated RNA was pelleted by centrifugation at maximal speed for 15 min at room temperature, washed with 1 ml of 70% ethanol, and centrifuged for 5 min. The pellet was dissolved in 100 μl of RNase-free H2O, and 100 μl of phenol:chloroform (1:1) was added and mixed by vortexing. After centrifugation, the aqueous phase was transferred to a fresh 1.5-ml tube, and 100 μl of chloroform was added, mixed by vortexing, and centrifuged. After transferring the aqueous phase to a fresh 1.5-ml tube, 10 μl of 7.5 mg/ml ammonium acetate and 250 μl of 100% ethanol were added and mixed well, and the RNA was allowed to precipitate overnight at −20 °C. The precipitated RNA was pelleted by centrifugation, washed twice with 70% ethanol, and resuspended in RNase-free H2O. The integrity of the RNA was verified following electrophoresis through 1% agarose-formaldehyde gels. All of the equipment (e.g.homogenizers, mortar, pestle, etc.) were pretreated with RNase-Zap (Ambion) and rinsed with diethyl pyrocarbonate-treated H2O prior to use. All of the human tissue acquisition and protocols were reviewed and approved by the University of Alabama at Birmingham Institutional Review Board (protocol numbers X021015002 and X980409003). RT-PCR was performed using a OneStep RT-PCR kit (Qiagen) according to the manufacturer's instructions, using 0.1–1 μg of total RNA as template. Custom primers specific to ASIC1 were synthesized by Invitrogen and used at a final concentration of 0.6 μm. For example, the forward and reverse primers 5′-CCCGCATGGCAAAGAG-3′ and 5′-GGCTCAGCAGGTAAAGTCC-3′ corresponded to bases 1109–1125 and 1572–1587 (plus 3 bases of the 3′-untranslated region) of ASIC1, respectively. Reverse transcription was performed using a single cycle of 50 °C for 30 min. This was followed by a single cycle of 95 °C for 15 min, which inactivates the reverse transcriptase while activating the HotStart Taq DNA polymerase, followed by 40 cycles of 94 °C for 1 min, 56 °C for 1 min, and 72 °C for 1 min, and finally a single 10-min cycle at 72 °C. Aliquots of each reaction mixture were electrophoresed on a 2% Nu-Sieve (FMC Corp.) agarose gel using PCR markers (Promega) to determine molecular size. Products of the correct molecular size were isolated from the gel using the QIAquick gel extraction kit (Qiagen) and subcloned into the pCR-2.1 vector using the TOPO-TA cloning kit (Invitrogen) following the manufacturer's instructions. Recombinants were selected by blue/white screening and restricted withEcoRI (Promega) to verify incorporation of insert of correct size. ASIC1 sequences were verified by further restriction enzyme digest analysis and automated DNA sequencing (DNA Sequencing Facility, Iowa State University). We previously reported that there was an amiloride-sensitive component to whole cell inward currents in high grade, highly invasive brain tumors (1Bubien J.K. Keeton D.A. Fuller C.M. Gillespie G.Y. Reddy A.T. Mapstone T.B. Benos D.J. Am. J. Physiol. 1999; 276: C1405-C1410Crossref PubMed Google Scholar). These currents were seen in primary cultures of tumors as well as in established glioma cell lines. This amiloride-sensitive current was not present in low grade brain tumors or normal brain tissue. To verify these initial findings and to extend these results to freshly resected GBM tumors, we performed whole cell patch-clamp experiments on freshly excised normal astrocytes and freshly resected and primary cultured brain tumor tissue samples (Fig.1). In the basal state, the current records for both freshly resected and primary cultures of World Health Organization Grade III and IV tumor cells were characterized by large inward currents (Fig. 1A), and these results were completely inhibited following superfusion with 100 μm amiloride (Fig. 1B). Fig. 1C shows the difference current (i.e. the amiloride-sensitive component). These results should be contrasted to the lack of effect of 100 μmamiloride in normal astrocytes and Grades I and II astrocytoma cells. The absolute magnitudes of the outward currents at +40 mV (Fig.2A) and inward currents at −60 mV (Fig.2B) in the absence and presence of amiloride for normal astrocytes, different grade gliomas, medulloblastoma, and two continuous GBM cell lines are summarized in Fig.2. Although there was no discernible pattern to the magnitudes of either the outward or inward currents, amiloride only blocked inward currents in the more aggressive, higher grade tumors (Grades III and IV and medulloblastoma). Amiloride likewise blocked inward currents in SK-MG and U87-MG cells, both originally derived from GBM. A summary of the current-voltage (I–V) characteristics of both freshly resected normal astrocytes and GBM cells is presented in Fig. 3. It is apparent that the GBM cells are depolarized by an average of 31 mV compared with normal astrocytes under these recording conditions. The depolarized zero current membrane potential is due to the presence of an enhanced Na+ conductance. These results confirm our previous findings and establish that this amiloride-inhibitable component to whole cell currents is present in freshly excised GBM cells.Figure 2Summary of absolute outward (+40 mV;A) and inward (−60 mV; B) currents obtained from a variety of gliomas and normal cells in the absence and presence of 100 μm amiloride, using whole cell patch clamp. At least four cells were measured from each preparation.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Summary I-V curves of freshly resected normal astrocytes and GBM cells. Inward currents (at −60 mV) were −7.5 + 1.2 pA (normal) and −43.8 + 14.5 (GBM). Outward currents (at +40 mV) averaged 42.2 + 2.4 and 47.2 + 12.5 pA for normal and GBMs, respectively, in this set of experiments.View Large Image Figure ViewerDownload Hi-res
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