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Electrochemical Analysis of Cell Plasma Membrane Cholesterol at the Airway Surface of Mouse Trachea Dechen Jiang,† Danjun Fang,† Thomas J. Kelley,‡ and James. D. Burgess*,†
Departments of Chemistry and Pediatrics and Pharmacology, Case Western Reserve University and Rainbow Babies and Children’s Hospital, Cleveland, Ohio 44106
Electrochemical detection of plasma membrane cholesterol at the surface of excised mouse trachea tissue is reported. Cholesterol oxidase is covalently linked to an 11-mercaptoundecanoic acid submonolayer on the platinum electrode surface. The cholesterol oxidase-modified electrodes show steady-state responses for cholesterol in solution at physiological temperatures. Experiments for direct contact between the cholesterol oxidase-modified electrode and the surface of excised trachea tissue at 37 °C indicate steady-state responses that are largely independent of the position of contact on the tissue surface. Tissue samples are mounted on a quartz crystal microbalance electrode to gauge contact force between the electrode and the tissue surface, and the steady-state electrode response for tissue cholesterol is shown to be largely independent of the contact force. Trachea tissue excised from a mouse model of cystic fibrosis, which is known to exhibit evaluated cholesterol in airway cells, shows an electrode response that is ∼40% larger than the response observed at wild-type mouse trachea tissue. This group recently reported a microelectrode strategy to track plasma membrane cholesterol in single living mammalian cells at 37 °C.1 The measurements implement Pt electrodes modified with a submonolayer of covalently attached cholesterol oxidase. The submonolayer functionalization allows the Pt electrode surface to be placed directly at the membrane/solution interface so that measurements of cholesterol as a component of the plasma membrane can be resolved. Because the rate of cholesterol consumption at the electrode is low, analysis can be performed without significant depletion as mass transfer of cholesterol in living cells to the electrode contact site sustains the apparent steady-state amperometric response. The electrochemical measurements have proven useful in evaluating the effect of specific disease-state models1 and drug treatments on intracellular trafficking of cholesterol to the plasma membrane. Extension of cholesterol electroanalysis for evaluation of cell plasma membrane cholesterol at the surface of primary tissue will allow more * To whom correspondence should be addressed. E-mail:
[email protected]. † Department of Chemistry. ‡ Department of Pediatrics and Pharmacology. (1) Jiang, D.; Devadoss, A.; Palencsa´r, M. S.; Fang, D.; White, N.W.; Kelley, T. J.; Smith, J. D.; Burgess, J. D. J. Am. Chem. Soc. 2007, 129, 11352-11353. 10.1021/ac7019909 CCC: $40.75 Published on Web 01/16/2008
© 2008 American Chemical Society
clinically relevant studies of animal disease-state models and the efficacy of drug treatments. This paper reports an electrochemical method for gauging cholesterol at the airway surface of mouse trachea tissue. Cholesterol is an essential structural component of mammalian cell membranes. For example, the fibroblast plasma membrane is estimated to contain ∼60-80% of the total cellular cholesterol, and complex trafficking pathways exist within cells to maintain this distribution.2,3 Dysfunctional intracellular cholesterol transport can lead to accumulation of cellular cholesterol and is thus believed to be inherently involved in the onset of atherosclerosis and Niemann-Pick disease.4 Work by Kelley and co-workers has demonstrated elevated cellular cholesterol in mouse and cell line models of cystic fibrosis (CF).5,6 CF is a genetic disease caused by the lost function of the cAMP-dependent chloride channel cystic fibrosis transmembrane conductance regulator (CFTR). Although the mechanism leading to the aggressive inflammatory signaling in CF is still unclear, it is believed that improper cholesterol processing is a trigger for altered inflammatory signaling responses.5 Our recent electrochemical studies on plasma membrane cholesterol of cell line models of CF and of excised nasal tissue from CF mouse models indicate that plasma membrane cholesterol is elevated in the CF models relative to the basal physiological level observed in wild-type (WT) cells and nasal tissue.6 Here we demonstrate the methodology for electrochemical cholesterol analysis of animal tissue using excised mouse trachea, a tissue sample that provides a relatively large homogeneous and well-defined (airway) surface for study by direct contact with cholesterol oxidase-modified electrodes. Data are reported for contacting a single tissue sample at multiple positions on the airway surface to verify a cholesterol content that is spatially homogeneous. Tissue samples are mounted on quartz crystal microbalance (QCM) electrodes to qualitatively estimate the contact force between the electrode and the tissue surface. The elevated cholesterol content of CF airway epithelial cells, including those of the trachea, provides the ability to demonstrate electro(2) Liscum, L.; Munn, N. J. Biochim. Biophys. Acta 1999, 1438, 19-37. (3) Maxfield, F. R.; Tabas, I. Nature 2005, 438, 612-621. (4) Maxfield, F. R.; Wu ¨ stner, D. J. Clin. Invest. 2002, 110, 891-898. (5) White, N. M.; Corey, D. A.; Kelley, T. J. Am. J. Respir. Cell Mol. Biol. 2004, 31, 538-543. (6) White, N. W.; Jiang, D.; Burgess, J. D.; Bederman, I. R.; Previs, S. F.; Kelley, T. J. Am. J. Respir. Cell Mol. Biol. 2006, 292, L476-L486.
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chemical evaluation of plasma membrane cholesterol in cells at the natural surface of tissue samples. The data indicate that the CF trachea surface exhibits increased plasma membrane cholesterol. This result further verifies that transport of cholesterol to the plasma membrane from stores inside the cell is upregulated in CF airway epithelial cells. EXPERIMENTAL SECTION Mice. Mice lacking CFTR expression (CFTRtm1Unc) are obtained from Jackson Laboratories (Bar Harbor, MA). CFTR WT mice are siblings of cftr-/- mice. All mice are used between 6 and 8 weeks of age and are back-crossed over 10 generations onto a C57Bl/6 background. CF mice are fed a liquid diet as described by Eckman et al.7 Mice are cared for in accordance with the Case Western Reserve University Institutional Animal Care and Use Committee guidelines by the CF Animal Core Facility. Tracheas of mouse epithelium are obtained from WT and CF animals. The Animal Care and Use Committee of Case Western Reserve University approved all animal procedures. Trachea tissue samples are surgically excised using established procedures. Electrode Modification. Platinum disk electrodes (100-µm diameter) are fabricated in-house as described ealier.1,8 The platinum disk electrodes are immersed in a 5 mM hexane solution of 11-mercaptoundecanoic acid (Aldrich Chemical Co.) for 2 h. This reaction condition produces a submonolayer coverage of 11mercaptoundecanoic acid on the platinum electrode surface. The submonolayer-modified platinum surface is exposed to a 100 mM sodium phosphate aqueous solution (pH 7.4) containing 2 mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (Sigma Chemical Co.) and 5 mM N-hydroxysulfosuccinimide (NHS) (Fisher) for 30 min for conversion of the carboxylic acid end groups of the submonolayer to an NHS ester. This activated surface is immersed in a 1 mg/mL solution of recombinant cholesterol oxidase (Wako Chemicals USA, Inc., ∼33.0 units/mg) for 2 h for covalent attachment of the enzyme to the electrode surface. The NHS esters of the submonolayer serve as leaving groups when amide bonds are formed with lysine residues on the surface of the enzyme.9 Preparation of Cholesterol Solutions. Cholesterol is dissolved in chloroform and dried under nitrogen before preparing the solutions of required concentration. The dried cholesterol is dissolved in an aliquot of PBS containing 1% (v/v) Triton X-100. Solution flow injection analysis-type experiments are conducted using a flow cell constructed in-house. Briefly, the electrochemical flow cell is a microscope glass slide with a circular silicone barrier in the middle to hold a buffer volume of ∼200 µL. The flow rate is 500 µL/min. The flow is controlled using a six-way valve (Rheodyne model 5020, Supelco Corp.) and two syringe pumps (Harvard Apparatus) to direct buffered Triton X-100 or buffered Triton X-100 containing cholesterol over the electrode. Injection times are controlled manually. Electrode responses to cholesterol solution at 37 °C are collected in quiet buffer after spiking with aliquots of cholesterol solution. The spiked aliquots are preheated to 37 °C, and the solution is kept at 37 °C during the experiments. (7) Perez, A.; Risma, K. A.; Eckman, E. A.; Davis, P. B. Am. J. Physiol. Lung Cel.l Mol. Physiol. 1996, 271, L85-L92. (8) Devadoss, A.; Palencsar, M. S.; Jiang, D.; Honkonen, M. L.; Burgess, J. D. Anal.Chem. 2005, 77, 7393-7398. (9) Fung, Y. S.; Wong, Y. Y. Anal. Chem. 2001, 73, 5302-5309.
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Data Acquisition. Amperometric measurements are conducted using a two-electrode cell and a voltammeter-amperometer (Chem-Clamp, Dagan corp.). The three-pole Bessel filter of the Chem-Clamp is set to 100 Hz. The signal is further processed using a noise-rejecting voltmeter (model 7310 DSP, Signal Recovery Inc.) to digitally filter 60-Hz noise and to provide a dc voltage output with a time constant of 100 ms. Collected data are smoothed using a moving boxcar average (10 data points). An Ag/AgCl (1 M KCl) reference electrode is used for all experiments, and the applied potential is 820 mV versus NHE for all experiments. For electrochemical evaluation of plasma membrane cholesterol, the excised tissue samples are rinsed with 37 °C Dulbecco’s phosphate-buffered saline (D-PBS) (Invitrogen) and kept in D-PBS at 37 °C during the experiment. Prior to the electrochemical experiments, the tissue sample is captured using a pulled glass capillary connected to a IM-6 microinjector and positioned ∼500 µm from the electrode to acquire baseline data. The electrode is repositioned so that it is in physical contact with the airway surface of the tissue sample for cholesterol analysis. Electrode contact is defined as the force required to physically alter the position of the tissue as gauged visually under optical magnification Contact Force Estimates. To investigate the dependence of contact force between the electrode and the tissue surface on electrode response, the trachea tissue is spread on a QCM electrode (10 MHz, International Crystal Manufacturers). Increased force applied between the tissue sample and the electrode produces a decrease in QCM resonant frequency providing a qualitative measure of contact force. No model is presented to quantify the absolute force applied. RESULTS AND DISCUSSIONS Microelectrode Detection of Cholesterol in Solution. The cholesterol oxidase-modified electrodes are characterized for detection of room-temperature cholesterol solution in flow injection-type experiments. Figure 1 shows typical amperometric responses for flow exposure of cholesterol solution (25 µM-1 mM) to an oxidase-modified electrode (Figure 1a). Plots of electrode response (oxidase electrodes and bare Pt control electrodes) versus cholesterol concentration are shown in Figure 1b. The control electrodes show no response for cholesterol solution, and the three oxidase electrodes show standard derivations in the range of 5-20% for all concentrations (see error bars in Figure 1b). These data indicate that cholesterol oxidase, covalently anchored to the electrode surface, retains activity for catalyzing the oxidation of cholesterol by molecular oxygen, and these data are consistent with a covalent scheme for immobilization of the enzyme on gold.10,11 The plateau behavior shown in Figure 1b could reflect enzyme saturation or molecular oxygen becoming a limiting reactant. The electron-transfer reaction scheme for cholesterol detection is shown in Scheme 1. The oxidase electrodes were characterized for detection of cholesterol solution at 37 °C (Figure 2). Amperometric data are collected for spiking a quiet solution with aliquots of cholesterol solution (Figure 2a). Plots of electrode response (three oxidase(10) Nakaminami, T.; Ito, S.; Kuwabata, S.; Yoneyama, H. Anal. Chem. 1999, 71, 1068-1076. (11) MacLachlan, J.; Wotherspoon, A. T. L.; Ansell, R. O.; Brooks, C. J. W. J. Steroid Biochem. Mol. Biol. 2000, 72, 169-195.
Figure 1. Characterization of cholesterol oxidase-modified electrodes for solution cholesterol at room temperature. (a) Flow injection analysis data for cholesterol solution at indicated concentrations. (v) indicates the times for injecting cholesterol solution and (V) indicates the times for reverting the flow to buffer. (b) Plot of steady-state current response vs cholesterol concentration for the oxidase-modified electrodes (squares) and bare Pt control electrodes (circles). Error bars are SD for three electrodes.
Figure 2. Characterization of cholesterol oxidase-modified electrodes for solution cholesterol at 37 °C. (a) Amperometric data collected at a cholesterol oxidase-modified electrode in quiet cholesterol solution as labeled. (v) indicates the times for injecting cholesterol solution. (b) Plot of steady-state current response vs cholesterol concentration for oxidase-modified electrodes (squares) and bare Pt control electrodes (circles). Error bars are SD for three electrodes.
Scheme 1. Enzyme Catalyzing the Oxidation of Cholesterol to Cholestenone, and the Reduction of Molecular Oxygen to Hydrogen Peroxidea
a At the electrode surface, hydrogen peroxide is electrochemically oxidized yielding oxygen and steady-state current responses.
modified electrodes and three bare Pt control electrodes) versus cholesterol concentration are shown in Figure 2b. The standard derivations for electrode response to solution cholesterol at 37 °C are in the range of 8-20%. These data demonstrate that the surface structure is stable at physiological temperature. Tissue Cholesterol Analysis. Experiments for detection of plasma membrane cholesterol at the surface of excised trachea are conducted with the tissue sample submerged in buffer so that the electrode response can be resolved by comparing the baseline current (no contact) to the steady-state response observed with the electrode in direct physical contact with the airway tissue surface. Contact is defined as the applied force that slightly alters the position of the tissue (gauged visibly under optical magnification). Figure 3a shows the steady-state current responses for duplicate contacts at the same position on the tissue surface. About five sequential contact experiments can be conducted with a given oxidase electrode before response is diminished. Diminished electrode response is likely due to the adsorption of cellular material to the electrode surface that blocks hydrogen peroxide oxidation or that causes loss of enzyme activity. Control experiments (Figure 3b) using bare Pt electrodes do not show oxidative responses. These data suggest that the current observed at the
Figure 3. (a) Amperometric responses of a cholesterol oxidasemodified electrode for two consecutive contact experiments at the same position on trachea tissue surface. (v) indicates the time of contact, and (V) indicates the times of withdrawal. (b) Control experiment conducted using a bare Pt electrode to contact the same position on the trachea tissue surface.
oxidase-modified electrodes is a response to plasma membrane cholesterol in cells at the surface of the tissue. The average response for six replicate experiments conducted using different electrodes and different tissue samples is 27 ( 4.3 pA (relative error 16%), similar to the variation between electrodes in solution characterizations (Figures 1 and 2). Our mechanistic model for detection of plasma membrane cholesterol is that previously reported for detection of cholesterol at the surface of single macrophage cells, i.e., the aqueous Analytical Chemistry, Vol. 80, No. 4, February 15, 2008
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Figure 5. (a) QCM data for increase in the force applied between the cholesterol oxidase-modified electrode and the tissue surface. (b) Amperometric response of the cholesterol oxidase-modified electrode with increased contact force. (v) indicates the time of contact, and (v) indicates the time increasing applied force.
Figure 4. Data from two cholesterol oxidase-modified electrodes (a, b) for contacting the trachea tissue at different locations (1, 2, and 3) on the airway surface. (v) indicates the time of contact.
diffusion model. The aqueous diffusion mechanism is based on studies that show a maximum plateau in the rate of cellular cholesterol efflux to solution-phase acceptors with increasing acceptor concentration.12,13 The zero-order dependence of efflux on acceptor concentration (above 1.6 × 1016 phosphatidylcholine vesicles/mL) indicates that the rate of cholesterol removal from the plasma membrane is independent of collision frequency between acceptor and the plasma membrane. Thus, movement of cholesterol out of the plasma membrane to the aqueous phase is proposed as the initial step in efflux of cellular cholesterol to solution-phase acceptors. The physical model for contact of the tissue surface with the electrode assumes an aqueous hydration layer between the electrode surface and the plasma membrane tissue surface. Cholesterol in the aqueous phase between the electrode and tissue surface is consumed at the electrode surface resulting in mass transfer of cholesterol from the plasma membrane to the aqueous phase. Based on the electrode response at the tissue samples, cholesterol is estimated to be consumed at a rate of ∼2 pmol/s cm2. This rate of cholesterol extraction is in agreement with reported rates of cholesterol removal from a lipid monolayer at the air/water interface using cyclodextrin (a welldocumented aqueous-phase cholesterol acceptor of cellular efflux) as the solution-phase acceptor (1.3-6.6 pmol/s cm2 for various mole fractions of cholesterol in the lipid monolayer).14 Based on this model, cholesterol detected by the electrode is aqueous-phase cholesterol originating as cholesterol efflux from the cell plasma membrane. It is proposed that the rate of cholesterol efflux from the plasma membrane to the aqueous layer between the electrode and tissue surface influences the magnitude of the steady-state response. The efflux rate is dependent on the cholesterol content of the plasma membrane.14 The number of cholesterol molecules initially present in the plasma membrane (12) Rothblat, G. H.; Phillips, M. C. J. Biol. Chem. 1982, 257, 4775-4782. (13) Phillips, M. C.; Johnson, W. J.; Rothblat, G. H. Biochim. Biophys. Acta 1987, 906, 223-276. (14) Radhakrishnan, A.; McConnell, H. M. Biochemistry 2000, 39, 8119-8124.
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of cells directly adjacent to the 100-µm-diameter electrode (e.g., electrode footprint) is estimated to be 2 × 1010, which gives an equivalent of ∼6.7 nC (assuming 0.33 mole fraction cholesterol in the plasma membrane15 and 100% oxidation of the generated hydrogen peroxide). This calculation suggests that ∼10% of the available cholesterol is consumed over 20 s. Lateral diffusion of cholesterol in the plasma membrane, transbilayer movement (flipflop) of cholesterol in the plasma membrane, and delivery of cholesterol to the plasma membrane from stores inside the cell all likely contribute to replenishing cholesterol to the electrode contact site.16 The mass-transfer rate of cholesterol to the electrode contact site, caused by the local depletion, likely also contributes to the steady-state electrode response. Other factors possibly affecting electrode response are ion transport and enzyme kinetics. No speculation is provided here regarding these uncertainties. The objective is to establish the tissue cholesterol measurements as a diagnostic tool. Thus, experiments have been conducted to determine the dependence of electrode response on contact position at the tissue airway surface. The responses of an individual oxidase electrode collected at three different positions on the same trachea tissue sample are shown in Figure 4a. The standard derivation of the three responses is less than 10% for two replicate experiments (two electrodes and two tissue samples; Figure 4a and b). These data show that the cholesterol content of the tissue sample is homogeneous across the surface of the tissue, suggesting that a single oxidase electrode can be used to compare the plasma membrane cholesterol content at the surface of two different tissue samples. We speculate that the trachea airway surface has some topography that could change upon contact by the electrode. The nature of the electrode tissue contact (e.g., average thickness of the aqueous layer and area of plasma membrane under the electrode footprint) could feasibly be altered under varied contact forces. To verify that such behavior does not significantly affect the electrode response, trachea tissue samples were placed on a QCM electrode surface so that contact force applied between the cholesterol oxidase-modified electrode and the tissue surface could (15) Smaby, J. M.; Momsen, M.; Kulkarni, V. S.; Brown, R. E. Biochemistry 1996, 35, 5696-570419. (16) Oradd, G.; Lindblom, G. Magn. Reson. Chem. 2004, 42,123-131.
other experiments, the contact force is indicated and limited by physical movement of the tissue sample. These data demonstrate the ability to reliably assay tissue surface cholesterol without strictly controlling the applied force of electrode contact. Comparing Different Tissue Samples. As a means of demonstrating the ability to evaluate the relative plasma membrane cholesterol content of different tissue samples using a single cholesterol oxidase-modified electrode, data are presented for comparing electrode response at trachea tissue excised from a WT mouse to that excised from a CF mouse model. Figure 6 shows two independent experiments (a and b) comparing WT and CF tissue samples. While there is variation in response between different electrodes, the individual electrodes show nearly the same relative increase in response for the CF samples. An increased current response of more than 40% is observed at the CF trachea tissue, and these data are consistent with our earlier work comparing WT and CF cells and WT and CF nasal tissue.6
Figure 6. Two independent electrochemical experiments (a, b) using two oxidase-modified microelectrodes to compare CF and WT tissue samples. (v) indicates the time of contact.
be qualitatively tracked. Applying mechanical force to the QCM electrode causes a decrease in resonant frequency providing a real-time estimate of the contact force. While no model is given to quantify the QCM response to altered electrode contact force, the experiments do provide a relative measure of contact force with respect to the amperometric response of the oxidase electrode. Figure 5 shows the QCM resonant frequency and the amperometric oxidase electrode response for sequentially increasing the applied contact force in step functions with the first step being initial contact. Steady-state responses are shown for three different contract forces as verified by the decrease in QCM resonant frequency. When the force is manually increased by moving the electrode in the direction of the tissue (Figure 5a), no resolvable change in steady-state current is observed (Figure 5b). This indicates that complications from changes in tissue surface topography upon contact by the electrode do not significantly affect the measured steady-state response. No speculation is given regarding the current transients during and immediately after increasing the contact force. It is noted, however, that the electrode forces applied in these experiments are greater than those applied in all other experiments shown in this paper. In all
CONCLUSION Electrochemical analysis of plasma membrane cholesterol at the airway surface of mouse trachea is reported. Experiments for the exposures of cholesterol oxidase-modified electrodes to 37 °C cholesterol solution show steady-state responses. The data show that electrode response correlates with plasma membrane cholesterol content at the trachea surface and that the steadystate measurements are not significantly dependent on contact position or contact force. Cholesterol is elevated in the plasma membrane of cells at the airway surface of CF mouse trachea. This work rationalizes our goal of implementing electrochemical analysis of tissue surface cholesterol as a clinical tool for CF diagnostics and management. Sensor optimization studies where electrode sizes and amount of enzyme immobilized on the electrode surface are underway. ACKNOWLEDGMENT This work was supported by the National Institutes of Health (5 R21 EB003925) and the Department of Chemistry, Case Western Reserve University. Helpful discussions with Professosr Dan Scherson and Barry Miller are also acknowledged.
Received for review September 21, 2007. Accepted October 28, 2007. AC7019909
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