Anal. Chem. 1998, 70, 1677-1681
Articles
Simultaneous Detection of Catecholamine Exocytosis and Ca2+ Release from Single Bovine Chromaffin Cells Using a Dual Microsensor Quan Xin and R. Mark Wightman*
Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599-3290
A dual microsensor with a 5 µm radius was fabricated to detect simultaneously Ca2+ and catecholamines following their secretion from individual biological cells. Detection of Ca2+ was based on changes in fluorescence as a result of its binding with a surface-attached dye, and catecholamines were detected by amperometry. The fluorescent dye employed, calcium green-1 dextran, is a selective chelator for Ca2+. It was attached to the tip of a carbon fiber electrode by cross-linking with 5% glutaraldehyde. The dual microsensor has a subsecond response time for both Ca2+ and catecholamine concentration changes. Ca2+ concentrations of 100 nM can be detected, while the detection limit for catecholamine is in the micromolar range. The utility of the dual microsensor was evaluated at the surface of bovine adrenal medullary cells. Release of catecholamines by exocytosis was evoked by transient application of histamine. This was detected by amperometry, and it was found to be accompanied by Ca2+ release, as measured by fluorescence from the same sensor. Chemical microsensors provide the unique opportunity to probe chemical dynamics in microscopic environments. Both electrochemical and spectroscopy-based sensors have been widely employed in such investigations. An example of an electrochemical microsensor is the carbon fiber microelectrode.1 It was developed to detect easily oxidized compounds, such as catecholamines2 and 5-hydroxytryptamine.3 Its speed and sensitivity have enabled the rates of neurotransmitter transport into neurons to be measured inside the rat brain,4 as well as allowing the direct observation of individual exocytotic events from adrenal chroma* Corresponding author. Telephone: (919) 962-1472. Fax: (919) 962-2388. (1) Michael, A. C.; Wightman, R. M. Microelectrodes. In Laboratory Techniques in Electroanalytical Chemistry; Kissinger, P. T., Heinemann, W. R., Eds.; Marcel Dekker: New York, 1996; pp 367-402. (2) Wightman, R. M.; Jankowski, J. A.; Kennedy, R. T.; Kawagoe, K. T.; Schroeder, T. J.; Leszczyszyn, D. J.; Near, J. A.; Diliberto, J., E. J.; Viveros, O. H. Proc. Natl. Acad. Sci. 1991, 88, 10754-10758. (3) Jackson, B. P.; Dietz, S. M.; Wightman, R. M. Anal. Chem. 1995, 67, 11151120. (4) Giros, B.; Jaber, M.; Jones, S. R.; Wightman, R. M.; Caron, M. G. Nature 1996, 379, 606-612. S0003-2700(97)00746-4 CCC: $15.00 Published on Web 03/20/1998
© 1998 American Chemical Society
ffin cells.5 This sensor has also been coupled with enzymes to provide a route to compounds that are not electroactive, such as glucose, acetylcholine, and choline.6-8 In the area of optical microsensors, surface-modified fiber-optic microsensors predominate. The fibers guide excitation light to the location of interest and simultaneously collect the emission from photoexcited species. Reagents that undergo a change in spectroscopic properties in the presence of a specific analyte can be attached to the tip of the optical fiber. The most commonly used reagents are fluorescent dyes9 and enzymes.10,11 For example, submicrometer fiberoptic sensors have been developed by Kopelman and co-workers to detect pH and glucose.12,13 Recently, sensors have been developed that yield multidimensional information. Pantano and Walt have obtained simultaneously spatial and chemical information by fabricating fiber-optic chemical sensors with coherent imaging fibers.14 In the electrochemical sensor field, multidimensional chemical information has been generated by time-resolved detection of histamine and 5-hydroxytryptamine cosecretion from individual biological cells using carbon fiber microelectrodes with fast-scan cyclic voltammetry.15 In this paper, we describe a multidimensional microsensor based on both electrochemistry and fluorescence. The surface of a carbon fiber microelectrode has been modified with a fluorescent dye to allow simultaneous detection of Ca2+ and catecholamines at the same microlocation. The dual microsensor described herein was developed to allow simultaneous measurements of catecholamines and Ca2+ during (5) Wightman, R. M.; Schroeder, T. J.; Finnegan, J. F.; Ciolkowski, E. L.; Pihel, K. Biophys. J. 1995, 65, 383-390. (6) Ohara, T. J.; Rajagopalan, R.; Heller, A. Anal. Chem. 1994, 66, 2451-2457. (7) Garguilo, M. G.; Huynh, N.; Proctor, A.; Michael, A. C. Anal. Chem. 1993, 65, 523-528. (8) Huang, Z.; Villarta-Snow, R.; Lubrano, G. J.; Guilbault, G. G. Anal. Biochem. 1993, 215, 31-37. (9) Shortreed, M.; Kopelman, R.; Kuhn, M.; Hoyland, B. Anal. Chem. 1996, 68, 1414-1418. (10) Blair, T. L.; Yang, S. T.; Smith-Palmer, T.; Bachas, L. G. Anal. Chem. 1994, 66, 300-302. (11) Rosenzweig, Z.; Kopelman, R. Anal. Chem. 1996, 68, 1408-1413. (12) Tan, W.; Shi, Z.; Smith, S.; Birnbaum, D.; Kopelman, R. Science 1992, 258, 778-781. (13) Rosenzweig, Z.; Kopelman, R. Anal. Chem. 1995, 67, 2650-2654. (14) Pantano, P.; Walt, D. R. Anal. Chem. 1995, 67, 481A-487A. (15) Pihel, K.; Hsieh, S.; Jorgenson, J. M.; Wightman, R. M. Anal. Chem. 1995, 67, 4514-4521.
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secretion from individual biological cells. In this application, the cell characteristics dictate the sensor requirements with respect to spatial and temporal resolution as well as chemical sensitivity. The size of the sensor should match that of a single adrenal chromaffin cell, which is approximately 15 µm in diameter. The temporal resolution should be sufficient to detect exocytotic events that occur on the second time scale. It has already been demonstrated that carbon fiber microelectrodes have the requisite sensitivity to detect catecholamine secretion.2 The sensitivity for Ca2+ should be in the nanomolar range so that the anticipated low amounts (approximately 170 000 ions/vesicle16,17) can be detected. A variety of fluorescent molecules have been developed that have high specificity and sensitivity for Ca2+.18 These have been used to image intracellular Ca2+ in many cell types19,20 and, with lipophilic chains attached to the dyes, to detect membranelocalized Ca2+ changes.21 A conjugate of calcium green-1 dextran9 was used in this work. It was selected because its fluorescence linearly increases with bound Ca2+ and because its large size prevents overconcentration of the reagent at the sensor tip. Furthermore, the dextran portion forms a readily accessible hydrogel environment that should allow access of analytes to the tip. The importance of Ca2+ in endocrine cells is well established and has been reviewed.22,23 Elevation of intracellular calcium triggers exocytosis, a process in which the vesicles stored in a cell fuse with the cell membrane and release their components into the extracellular environment. Catecholamines are among the released components from adrenal medullary cells,24 and their exocytosis from individual vesicles has been directly measured with an amperometric carbon fiber microelectrode.2 Intracellular Ca2+ is then restored to its resting level through mechanisms that are not clearly understood. Although Ca2+ (another species that is stored in vesicles) release has been detected from populations of chromaffin cell cultures,25,26 it has not been examined at the single chromaffin cell level. In this work, calcium release was simultaneously observed with catecholamine exocytosis from single bovine adrenal chromaffin cells using the microsensors described in this paper. METHODS AND CHEMICALS Chemicals. Culture medium (Dulbecco’s modified Eagles medium with Ham’s F12, DMEM/F12) was obtained from Gibco Laboratories (St. Louis, MO). Calcium green-1 dextran (70 000 Da) was from Molecular Probes (Eugene, OR). Collagenase (Type I) was from Worthington Chemicals (Freehold, NJ), and (16) Ornberg, R. L.; Kuijpers, A. J.; Leapman, R. D. J. Biol. Chem. 1988, 263, 1488-1493. (17) Haigh, J. R.; Parris, R.; Phillips, J. H. Biochem. J. 1989, 259, 485-491. (18) Tsien, R. Methods Cell Biol. 1989, 30, 127-156. (19) Cheek, T. R.; Jackson, T. R.; O’Sullivan, A. J.; Moreton, R. B.; Berridge, M. J.; Burgoyne, R. D. J. Cell Biol. 1989, 109, 1219-1227. (20) Finnegan, J. F.; Borges, R.; Wightman, R. M. Neuroscience 1996, 71, 833843. (21) Lloyd, Q. P.; Kuhn, M. A.; Gay, C. V. J. Biol. Chem. 1995, 270, 2244522451. (22) Ghosh, A.; Greenberg, M. E. Science 1995, 268, 239-247. (23) Burgoyne, R. D. Biochim. Biophys. Acta 1991, 1071, 174-202. (24) Winkler, H.; Apps, D. K.; Fischer-Colbrie, R. Neuroscience 1986, 18, 261290. (25) Houchi, H.; Okuno, M.; Yoshizumi, M.; Oka, M. Neurosci. Lett. 1995, 198, 177-180. (26) Schneider, A. S.; Jan, C. Ann. N.Y. Acad. Sci. 1996, 779, 356-365.
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Renograffin-60 was from Squibb Diagnostics (New Brunswick, NJ). Glutaraldehyde (25% w/w), chelating resin (Chelex 100, iminodiacetic acid, sodium form), histamine, caffeine, and all other chemicals were from Sigma (St. Louis, MO). Solutions were prepared in doubly distilled water. The balanced salt solution (BSS) used with chromaffin cells contained a final concentration of 145 mM NaCl, 5 mM KCl, 1.3 mM MgCl2, 1.2 mM NaH2PO4, 10 mM glucose, and 20 mM HEPES. It was prepared from a 10× solution containing NaCl, KCl, and NaH2PO4. A 500-mL volume of this solution was stirred with 10 g of chelating resin overnight to remove calcium impurities, and the solution was then filtered into a plastic bottle. MgCl2, glucose, and HEPES were then added, and the resulting solution was stored at 4 °C. Before use, it was diluted 10-fold, and the pH was adjusted to 7.4 with concentrated sodium hydroxide. In most experiments, this buffer also contained 0.2 mM EGTA to lower free [Ca2+] that could saturate the sensor response. Cell Cultures. Primary cultures of bovine adrenal medullary cells were prepared from fresh tissue.27 A single-step Renografin gradient was performed to separate epinephrine-enriched cells. After being washed with a buffer containing Ca2+ and magnesium, cells were diluted to 3.0 × 105/mL in growth medium, placed in 35-mm-diameter Petri dishes, and kept in an incubator. The medium was changed every other day starting on day 3. Singlecell experiments were done between days 3 and 8. Construction of Microsensors. A carbon fiber with a radius of 5 µm (Thornel P-55, Amoco Corp., Greenville, SC) was aspirated into a glass capillary (A-M Systems, Inc., Everett, WA). The tip was pulled to micrometer dimensions around the carbon fiber with a pipet puller (Narishige Scientific Instrument Lab, Tokyo, Japan). The protruding fiber was cut so that approximately 200 µm of carbon extended beyond the tip of glass. It was sealed in the capillary with epoxy (Epon 828 resin with 14% m-phenylenediamine hardener, Miller-Stephenson, Danbury, CT) and cured at two temperatures (100 and 150 °C, each for 2 h). The protruding portion of the fiber was completely insulated by electrodepositing an insulation layer from 25% (v/v) BASF ZQ84-3225 electrocoat solution at 4 V for 4 min, followed by curing at 200 °C for 5 min.28 The tip was exposed by polishing at a 45° angle on a diamondcoated wheel (Sutter Instrument Co., Novato, CA), followed by a 15-min soak in 2-propanol to remove particles attached to the surface. Before use, the tip was dipped for 10 s into a freshly prepared solution containing 0.5 mg/µL calcium green-1 dextran and 5% glutaraldehyde. The film was allowed to cross-link for 2 h in a dark box before use. Apparatus. An inverted microscope (Axiovert 35, Zeiss, Thornwood, NY) with a 40× oil immersion objective was used to visualize single cells and conduct fluorescence experiments (Figure 1). A xenon arc lamp was used to excite immobilized calcium green-1 dextran on the sensor’s surface. The excitation filter (490 nm, Omega Optical, Brattleboro, VT) was mounted between the arc lamp and the objective. The resulting emission passed through an emission filter (530 nm, Omega Optical, Brattleboro, VT) and a pinhole to a photomultiplier tube. The pinhole gave an effective viewing diameter from the sample of 27 (27) Wilson, S. P.; Viveros, O. H. Exp. Cell Res. 1981, 133, 159-169. (28) Bach, C. E.; Nichols, R. J.; Beckmann, W.; Meyer, H.; Schulte, A.; Besenhard, J. O.; Jannakoudakis, P. D. J. Electrochem. Soc. 1993, 140, 1281-1284.
Figure 2. Temporal response of a calcium green-1 dextran-modified microsensor during repeated 0.5-s exposures to a solution containing 1.4 µM Ca2+ introduced from a micropipet. I is the fluorescence intensity.
Figure 1. (Upper panel) Schematic of the experimental arrangement for single-cell measurements with the dual microsensor. The cylindrical microsensor was positioned touching a chromaffin cell that is attached to the floor of a Petri dish. A double-barrel microinjector was placed approximately 50 µm from the cell to introduce reagents onto it. (Lower panel) A bright-field photograph of cell experiments.
µm. Light from a red LED indicated the viewing location. Two piezoelectric drivers (PCS-1000 Patch Clamp Manipulator, Burleigh Instruments, Fishers, NY) mounted on the microscope stage were used to position the microsensors and a microinjector with a resolution of 1 µm. A potential of +650 mV vs SSCE was applied to the microsensor, and the electrochemical current was measured with a picoammeter (gain ) 100 pA/V, AI 403, Axon Instrumentation, Foster City, CA). The fluorescence from the sensor surface was simultaneously measured with the PMT. Both signals were filtered at 10 Hz with a programmable signal conditioner (Axon Instrumentation) and digitized with an interface (TL-1, Axon Instrumentation) for computer storage. The fluorescent baselines were adjusted with computer software with multiple-point baseline fitting and subtraction to remove the effects of photobleaching of the dye. Multibarrel pipets were used to deliver small amounts of solution to stimulate the cells and calibrate the microsensors. They were prepared by twisting and pulling attached glass capillaries with the pipet puller, and the tips were cut to a diameter of 15 µm under a microscope. A pressure injection device (Picospritzer, General Valve Co., Fairfield, NJ) operated at a pressure of 3 psi was used to force solution through the tips at a flow rate of approximately 1 nL/s.29 The sequence and duration of microinjections were triggered with a computer-generated TTL pulse. Single-Cell Procedure. A Petri dish containing cells was removed from the incubator and equilibrated to room temperature for 15 min. The dish was washed and filled with BSS containing (29) Leszczyszyn, D. J.; Jankowski, J. A.; Viveros, O. H.; Diliberto, E. J., Jr.; Near, J. A.; Wightman, R. M. J. Neurochem. 1991, 56, 1855-1862.
0.2 mM EGTA and mounted on the microscope stage. Because EGTA can deplete stored Ca2+ from cells,30 the concentration of EGTA used was relatively low (0.2 mM), and the experimental time with each dish was kept as short as possible (20 min). For measurements, the microsensor was positioned against the cell membrane while the microinjector was placed 50 µm away. The double-barrel microinjector contained a solution to stimulate the cell (50 µM histamine) and a postcalibration solution (1 mM CaEGTA (containing approximately 100 nM free Ca2+) and 50 µM epinephrine). It was raised quickly before stimulation, and both barrels were pressure injected at 40 psi twice to remove diluted solutions from the tips of both barrels. RESULTS AND DISCUSSION Immobilization of Calcium Green-1 Dextran. Calcium green-1 is a dye that complexes Ca2+ with a high affinity and whose fluorescence increases with increasing bound Ca2+. Its dextran conjugate, calcium green-1 dextran (CGD) also has a high affinity for Ca2+ (Kd ) 190 nM).31 It is excited with visible light (490 nm) and exhibits a single fluorescence maximum at 530 nm with a quantum yield of 0.75. For its immobilization on the sensor tip, we took advantage of the fact that, during synthesis, the dextran portion of CGD was derivatized with amine groups to enable attachment to calcium green-1. Sufficient amine groups remain that the probe can be cross-linked with glutaraldehyde. In Figure 2, the fluorescent response of a CGD modified sensor to pressure injection of a solution containing 1.4 µM Ca2+ is shown. Due to the Kd value, this concentration represents the upper limit of detection. To determine the optimum concentration of glutaraldehyde for cross-linking, solutions containing 0.5 mg/mL CGD were evaluated. Glutaraldehyde was added and the fluorescence of the uncomplexed form was monitored after cross-linking had occurred (Figure 3). The fluorescence decreased dramatically when the concentration of glutaraldehyde was 6% (v/v%) or greater. We attribute this to a decreased distance between fluorophores leading to quenching of fluorescence. For this reason, 5% glutaraldehyde was chosen for immobilization of CGD on microsensors. The intensity of the excitation light source was found to affect the lifetime of the microsensors. The excitation can photobleach the dye molecules, causing baseline drift and diminishing the response to Ca2+. The excitation intensity was adjusted using different neutral density filters, and a 32% transmission filter was (30) Guo, X.; Przywara, D. A.; Wakade, T. D.; Wakade, A. R. J. Neurochem. 1996, 67, 155-162. (31) Haugland, R. Handbook of Fluorescent Probes and Research Chemicals, 6th ed.; Molecular Probes Inc.: Eugene, OR, 1996.
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Figure 5. Response of a dual microsensor following a 5-s exposure to 50 µM histamine (indicated by the long bar), followed by its response to the solution used for postcalibration (3-s injection of a solution containing 100 nM Ca2+ and 50 µM epinephrine indicated by the short bar). The upper panel is the Ca2+ signal, while the lower panel is the catecholamine signal. I is the fluorescence intensity. Figure 3. Effect of the glutaraldehyde concentration on the fluorescence of solutions containing CGD.
Figure 4. CGD-modified microsensor response to a solution containing 1.4 µM Ca2+ and 50 µM epinephrine. Upper trace, fluorescence; lower trace, amperometry. I is the fluorescence intensity.
chosen because it balanced the need for sensitivity and yet maintained the stability of the microsensors. Even so, the fluorescence of the CGD calcium microsensors was found to decrease 50% (n ) 3) after exposure to the excitation light source for 150 s. Similar photoinstability of calcium green has been noted before in sensor applications.9 Thus, while unsuitable for longterm use, this stability is sufficient for transient experiments at single cells that can be completed in 100 s. These measurements are necessarily qualitative, however, because the short lifetime of the sensor precludes accurate calibration. Rather, the Ca2+sensing portion of the dual microsensor can be viewed simply as an indicator that reports on an elevation in Ca2+ in the vicinity of the probe tip. Ca2+ and Catecholamine Dual Measurements. Carbon fiber-based microsensors also can detect catecholamines amperometrically with a sensitivity in the micromolar range and response times of less than 1 s. The CGD thin film on the surface of the microelectrode did not appear to block access of analytes to the carbon fiber. The amperometric response time of the microelectrodes increased by approximately 40 ms compared to that of an uncoated electrode, and the sensitivity decreased by 7% (n ) 3). Figure 4 shows a typical dual-channel trace from the microsensor when it was transiently exposed to solutions containing 1.4 µM Ca2+ and 50 µM epinephrine delivered from the microinjector. 1680 Analytical Chemistry, Vol. 70, No. 9, May 1, 1998
Both channels have a rise time of less than 1 s. However, the decay of the Ca2+ signal was much slower than that for the catecholamine. The catecholamine decay is due to combined effects of dilution into the bulk of solution and its consumption at the surface by the amperometric measurement. On the other hand, the Ca2+ decay is due to the combined effects of dilution and the competition for Ca2+ binding between CGD on the microsensor and the EGTA added to the solution. The kinetics of the binding reaction of EGTA with Ca2+ is known to be slower than that of BAPTA, a widely used calcium chelator,32 which is structurally similar to the chelating portion of CGD. This slow kinetic behavior is presumably reflected in the slow recovery of Ca2+ signals to baseline. Experimental Conditions for Single Cells. The measurement of Ca2+ efflux from single cells with the microsensors must be done in a Ca2+-free environment to minimize the background signal and maximize the dynamic range of the Ca2+-dye interaction. The extracellular buffer, BBS containing 0.2 mM EGTA, had [Ca2+] much below the detection limit of the sensors. The stimulation solution was prepared from the same solution. The only solution containing Ca2+ was the postcalibration solution in one of the microinjection barrels. This could provide interference if it diffused into the other barrel of the microinjector. This effect was minimized by flushing the tip contents of the microinjector barrels immediately before placement adjacent to the cell under investigation. Figure 5 shows a time trace in which the stimulation and postcalibration solution were alternately pressure-injected onto the sensor in the absence of a cell. During the histamine injection, neither the amperometric nor fluorescent signals changed from baseline values, indicating the effectiveness of the flushing process. Histamine as Secretagogue for Single Cells. Histamine interacts with H1 receptors on chromaffin cell membranes. This results in elevated intracellular Ca2+ levels by two mechanisms: entry from the extracellular fluid and mobilization of intracellular Ca2+ stores.23,33 The latter mechanism raises intracellular Ca2+ levels sufficiently that histamine can induce exocytosis from chromaffin cells even in Ca2+-free media.34 Histamine caused this effect in 50% of adrenal chromaffin cells in prior work.20 In this (32) Tsien, R. Biochemistry 1980, 19, 2396. (33) Zhang, L.; Del Castillo, A. R.; Trifaro, J. J. Neurochem. 1995, 65, 12971308. (34) Stauderman, K. A.; McKinney, R. A.; Murawsky, M. M. Biochem. J. 1991, 278, 643-650.
Figure 6. Experiments at a single cell exposed to 50 µM histamine for 5 s (indicated by the long bar). A 3-s exposure to a solution containing 100 nM Ca2+ and 50 µM epinephrine was used to postcalibrate the microsensor (shown by the short bar). In panel A, release from a single cell was evoked by histamine exposure and resulted in the amperometric spikes due to catecholamine oxidation (lower panel). At the same time Ca2+ release was detected. In panel B, neither catecholamine nor Ca2+ release was detected from a single cell after histamine exposure. I is the fluorescence intensity.
work, we took advantage of this observation because the sensor requires measurements in a Ca2+-free medium so that extruded Ca2+ can be observed. As shown in Figure 6A, both Ca2+ and catecholamine release could be simultaneously detected from a chromaffin cell after exposure to histamine (50 µM). Catecholamine secretion was observed as a series of sharp amperometric spikes arising from their electrooxidation. It has previously been shown that each spike corresponds to the catecholamine content of an individual vesicle.2 The fluorescence signal reveals that Ca2+ levels increase at the cell surface at the same time as catecholamine secretion occurs. The signal rose slowly to a plateau and then slowly returned to background. In the single cells that were successfully stimulated by 50 µM histamine (seven out of eight), no spikeshaped Ca2+ signals were found. The observed Ca2+ efflux lasted approximately 40 s, longer than the approximately 10 s for catecholamine signals. This was likely due to the slow disappearance of Ca2+ from the surface of the microsensors, as
described earlier. When histamine did not activate the exocytosis of catecholamine (one cell, Figure 6B), Ca2+ efflux was not detected from the cell. In all cases, the postcalibration of the Ca2+ response 50 s after the cell stimulation demonstrated that the microsensor retained sufficient sensitivity to detect approximately 100 nM Ca2+. There are two possible origins of the observed Ca2+ that was released from chromaffin cells. The observed Ca2+ release could originate from the same vesicles from which the catecholamines originate, because Ca2+ is costored there in relatively high (30 mM) concentration.24 Another source is the extrusion of the elevated cytoplasmic Ca2+ concentration that is triggered by the interaction of histamine with its receptor and that initiates exocytosis. Both the plasma membrane and the vesicular membrane contain a sodium-dependent Na+-Ca2+ exchanger that can accomplish this by pumping Ca2+ out of the cell and into the extracellular environment.26 These two mechanisms for increased extracellular Ca2+ levels would be expected to occur at similar times after stimulation of exocytotic events. Indeed, cytosolic Ca2+ is restored to baseline levels within 40 s after transient exposure to histamine. A faster responding sensor is required in order to see whether spike-shaped concentrations are observed, suggesting a vesicular origin. While the source of the released Ca2+ cannot be unequivocally assigned, these measurements show, for the first time at the single-cell level, that exocytosis of catecholamine is accompanied by Ca2+ efflux from bovine adrenal chromaffin cells. CONCLUSION Catecholamines and Ca2+ were simultaneously measured with a calcium green-1 dextran-modified carbon fiber microelectrode. Although the amperometric characteristics of the microelectrode were slightly changed by the fluorescent dye coating, the response ranges for both Ca2+ and catecholamine detection were satisfactory for measurements of dynamic events at single cells. The chief disadvantage of the approach described is the instability of the fluorescent signal. ACKNOWLEDGMENT This research was supported by NIH and ONR. Received for review July 14, 1997. Accepted February 6, 1998. AC970746O
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