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Views the Authors Declare That They Have No Competing Interests Journal of Biomedical Science BioMed Central Research Open Access Menthol increases human glioblastoma intracellular Ca2+, BK channel activity and cell migration Robert Wondergem* and Jeremy W Bartley Address: Department of Physiology, James H Quillen College of Medicine, East Tennessee State University, PO Box 70,576, Johnson City, Tennessee 37614-1708, USA Email: Robert Wondergem* - [email protected]; Jeremy W Bartley - [email protected] * Corresponding author Published: 24 September 2009 Received: 13 July 2009 Accepted: 24 September 2009 Journal of Biomedical Science 2009, 16:90 doi:10.1186/1423-0127-16-90 This article is available from: http://www.jbiomedsci.com/content/16/1/90 © 2009 Wondergem and Bartley; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract This study examined the effect of menthol, an agonist for transient receptor potential melastatin 8 2+ 2+ (TRPM8) ion channels, to increase intracellular Ca concentration, [Ca ]i, in human glioblastoma cells (DBTRG cells), which resulted in activation of the large-conductance Ca2+-activated K+ membrane ion channels (BK channels). Voltage ramps applied over 300 ms from -100 to 100 mV resulted in membrane currents with marked inwardly- and outwardly-rectifying components. Paxilline (2 PM) abolished the outwardly-rectifying current. Outwardly-rectifying on-cell patch currents were increased markedly by menthol (100 PM) added to the bath. The estimated on-cell conductance of these channels was 253 pS. Kinetic analysis showed that added menthol increased channel open probability and mean open frequency after 5 min. In a similar time course menthol 2+ increased [Ca ]i, and this increase was abolished either by added paxilline, tetraethylammonium ion or by Ca2+-free external solution. Finally, menthol stimulated the rate of DBTRG cell migration into scratch wounds made in confluent cells, and this also was inhibited by paxilline or by tetraethylammonium ion. We conclude that menthol, a TRPM8 agonist, increases DBTRG cell 2+ [Ca ]i that in turn activates membrane BK ion channels. Inhibition of BK channels by paxilline 2+ reverses menthol-stimulated increase of [Ca ]i and of cell migration. Thus, BK channels function 2+ to maintain elevations in [Ca ]i needed to sustain increases in DBTRG cell migration. Background We have shown recently that a transient receptor potential Glioblastoma multiforme (GBM) has a particularly grim melastatin 8 ion channel, TRPM8, is expressed in a human prognosis with a median survival time of 15 months [1]. glioblastoma cell line (DBTRG), and its function contrib- The extreme invasive property of this tumor, along with utes to increasing the intracellular Ca2+ concentration, 2+ propensity for phenotypic changes to occur within the [Ca ]i, that is necessary for cell migration and, presuma- population of invading cells compared with the primary bly, tumor invasion [2]. TRPM8 is highly expressed in tumor mass, present unyielding dilemmas for effective prostate and other cancer cells [3], but, apart from its prin- surgical, chemo- or radiation therapy. Thus, efforts to cipal function in neurons as a detector of environmental understand the mechanisms by which tumor cells migrate cold [4], its physiological and pathological function in and invade will provide useful information to alleviate epithelia and cancer cells is unknown [5]. the progression of this disease. The cost of publication in Journal of Biomedical Science Page 1 of 7 is bourne by the National Science Council, Taiwan. (page number not for citation purposes) Journal of Biomedical Science 2009, 16:90 http://www.jbiomedsci.com/content/16/1/90 Others have shown that the large-conductance Ca2+-acti- Inc, Cincinnati). [Ca2+] was determined by interpolation vated K+ ion channels (ie. maxi-K or BK) are over- from a standard curve generated from a Ca2+ calibration expressed in human glioma cells [6]. These BK channels buffer kit #2 (Molecular Probes) and Fura2/K5-salt. are the product of spliced-variant hslo gene and have 2+ enhanced sensitivity to [Ca ]i [7,8]. They function to Cell migration assay enhance the migration and invasive property of gliomas DBTRG cells were plated into 12-well tissue culture plates presumably by contributing to the plasma membrane and grown until confluent. A disposable pipette tip (250 ionic fluxes that underscore cell volume regulation, partic- PL) was used to scratch a wound on midline of the culture ularly as these cells alter their volume through the well. Photomicrographs were taken at various times, and restricted intercellular spaces available to tumor cells wound width was measured and recorded using Meta- invading the brain parenchyma [9]. Morph software (Molecular Devices), which was cali- brated at 1.04 Pm/pixel by the grid on a hemocytometer. In this study we report the integrative function of BK ion channels in relation to the increase in cell migration by Data analysis menthol, the putative agonist of TRPM8 ion channels. We Results are expressed as mean ± SEM. Differences among 2+ show that menthol increases [Ca ]i necessary for stimu- means were determined by Student's paired t-test, p < lating glioblastoma cell migration, and that blocking BK 0.05. Electrophysiological data were analyzed using channels abolishes the menthol-stimulated increase in WinASCD software (Guy Droogmans, Katholieke Univer- 2+ [Ca ]i as well as cell migration. siteit, Leuven, Belgium, ftp://ftp.cc.kuleuven.ac.be/pub/ droogmans/winascd.zip) Migration rates were deter- Materials and methods mined by regression analysis of wound width versus time Cell culture and materials (hrs), and treatments were deemed effective compared Human GBM cells (DBTRG cells) were a gift from G.F. with control if the null-hypothesis of common slopes (Ho: Vande Woude, Van Andel Research Institute, Grand Rap- b1-b2 = 0) was rejected at p < 0.05. ids, MI. Cells were grown in Dulbecco's MEM supple- mented with 10% FBS (Invitrogen) and 100 IU-100 Pg/ml Results penicillin-streptomycin (Sigma). DBTRG whole-cell membrane currents were recorded dur- ing voltage ramps (300 ms) from -100 to 100 mV (HP = - Whole-cell Voltage Clamp and On-cell Patch Clamp 30 mV), Figure 1. The control I-V plot displayed both Technique inwardly and outwardly rectifying currents; however, DBTRG cells were superfused on a microscope stage at added paxilline (2 PM) quickly reduced the outwardly rec- room temperature with a standard external salt solution tifying current, Figure 1A. The time course for this inhibi- containing (in mM): 150 NaCl, 6 KCl, 1 MgCl2, 1.5 CaCl2, tion by paxilline was rapid as is evident from the plot of 10 HEPES [N-(2-Hydroxethyl)piperazine-N'-(2- whole-cell current at -90 and 90 mV versus time, Figure ethanesulfonic acid)], 10 glucose, and pH 7.4 (1N HCl). 1B. Paxilline had similar effects on outward currents gen- In some instances KCl was increased from 6 mM to 60 erated by voltage steps from -30 mV (holding potential) to mM by isosmotic substitution for NaCl. Whole-cell volt- 90 mV, Figure 1C, and this inhibition was significant, Fig- age clamp pipettes were fabricated by means of a Brown/ ure 1D. Flaming micropipette puller (Sutter Instr. Co, Novato, 2+ CA) and were filled with (in mM): 140 KCl, 6 CaCl2 (302- Paxilline is known to inhibit the large-conductance Ca - nM free Ca2+; computed by WCaBuf software from activated K+ channels (BK) [12,13]. As a result, we per- [email protected]), 2 MgCl2, 11 formed on-cell patch-clamp recordings of DBTRG cells to EGTA, 50 HEPES, and pH 7.2 (1N KOH). The pipettes determine if the outwardly rectifying currents might be were 4-5 M: in the bath solution, and whole-cell voltage- attributable to BK ion channels. The inset in Figure 2A clamp measurements were performed by standard tech- shows the current-voltage (I/V) plot obtained from an on- nique [10]. On-cell patch clamp pipettes were filled with cell voltage ramp (-100 to 100 mV pipette potential; 300 (in mM): 20 CsCl, 100 aspartic acid, 0.1 CaCl2, 1 MgCl2, ms duration) applied to a DBTRG cell. Outward currents 5 EGTA 10 HEPES, and pH 7.4 (1N CsOH). (1-2 M: in were clearly evident in the pipette potential range of -100 bath solution). to -45 mV. Readout of channel currents recorded on-cell at a pipette potential of -100 mV is shown above the inset, Ca2+ Measurements by fluorescence imaging of Fura2 Figure 2A. A histogram of relative frequency versus these Cells on glass coverslips were loaded with Fura2 and channel currents shows three distinct peaks, with the 2+ [Ca ]i was measured by inverted microscopy (AccuScope smaller peaks at 34.6 and 50.6 pA, respectively, approxi- 3030) [11]. Filter-wheel and data acquisition were con- mating multiples of the unit current of 17.7 pA. Assuming trolled by the InCyte2 software (Intracellular Imaging, the membrane potential of the DBTRG cells at -30 mV, Page 2 of 7 (page number not for citation purposes) Journal of Biomedical Science 2009, 16:90 http://www.jbiomedsci.com/content/16/1/90 0.015 0.012 Control -90 mV A Paxilline (2PM B +90 mV 0.010 0.008 0.004 0.005 Paxilline (2 PM) Current (nA/pF) Current 0.000 Current (nA/pF) -100 -50 50 100 Membrane Potential (mV) -0.004 0 20406080100 -0.005 Time (s) CD Control 18 + Paxilline (2 PM) 16 (pA/pF) m 14 12 Differs from control 10 * p < 0.01, n = 3 8 * 0.005 nA/pF 6 50 mV 4 2 100 0 mV Outward Current at Vm = 90 V 0 Control Paxilline (2 PM) Whole-cellFigure 1 voltage clamp measurements of human glioblastoma cells Whole-cell voltage clamp measurements of human glioblastoma cells.
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