Anal. Chem. 1995, 67, 2599-2605
Simultaneous Amperometric Measurement of Ascorbate and Catecholamine Secretion from Individual Bovine Adrenal Medullary Cells Paula S. Cahill and R. Mark Wightman* Department of Chemistty, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3290
Secretion of ascorbate and catecholamines from single bovine adrenal medullary cells has been detected with amperometryat carbon-fibermicroelectrodes. kcarbonfiber electrodes were employed. One was beveled, and voltammograms at this electrode showed overlap of the responses for ascorbate and the catecholamines. The other was beveled and electrochemically oxidized to shift the ascorbate oxidation wave to potentials more negative than that of the catecholamines. Thus, at an applied potential of 0.05 V vs SSCE, ascorbate was selectively oxidized at the treated electrode and both catecholamines and ascorbate were oxidized at an applied potential of 0.65 V at the untreated electrode. Exocytotic release from the cell was stimulated with K4, nicotine, and digitonin. Nicotine and K- depolarize the cell membrane and elicit vesicular release. Digitonin is a detergent that reacts with cholesterol in the plasma membrane and causes the formation of pores. Ascorbate d u x from individual cells could be induced by digitonin but not by K+ or nicotine and was observed as a single peak with a full width at halfmaximum of 4 s. In contrast, catecholamine release was observed as many rapid, sequential current spikes when the cell was exposed to either digitonin, K+, or nicotine. The two different types of release show that ascorbate and catecholamines are being released from two different cellular compartments. The calcium independence of the digitonin-induced ascorbate release provides additional evidence that ascorbate is released by a nonexocytotic process. Chemical analysis at the level of single biological cells presents several challenges. The small size of a single cell means that small volumes, picoliters or less, and trace concentrations are present. Additionally, the biological complexity and heterogeneity of cell populations require chemical methods with high selectivity and sensitivity. The contents of single cells have been successfully characterized at invertebrate bovine adrenal medullary cells,j and human Capillary zone electrophoresis and open tubular liquid chromatography with sensitive detectors M. D.; Cooper, B. R.; Nickerson, B.; Jorgenson, J. W. Science 1989,246,57-63. (2) Kennedy, R. T.; Jorgenson, J. W. Anal. Chem. 1989,61,436-441. (3) Ewing, A G.; Mesaros, J. M.; Gavin, P. F. Anal. Chem. 1994,66,527A(1) Kennedy, R. T.; Oates,
537A. (4) Olefirowicz, T. M.; Ewing, A. G. Anal. Chem. 1990,62,1872-1876. (5) Cooper, B. R.; Jankowski, J. A; Leszczyszyn, D. J.; Wightman, R M.; Jorgenson, J. W. Anal. Chem. 1992,64,691-694. (6) Lee, T. T.; Yeung, E. S. Anal. Chem. 1992,64, 3045-3051. (7) Hogan, B. L.; Yeung, E. S. Anal. Chem. 1992,64,2841-2845.
0003-2700/95/0367-2599$9.00/0 0 1995 American Chemical Society
have been very useful in this regard because of their low volumes, which reduce sample dil~tion.',~The detectors include laserinduced fluorescence for the detection of and indirect detection of cations7 and electrochemical detection for neurotransmitters.'-j Analytical techniques have also been shown to be useful for monitoring dynamic chemical changes, i.e., making measurements in real time as the cell interacts with its environment. Dynamic cell experiments can be used to probe cellular processes such as secretion, metabolism, and ion r e g ~ l a t i o n . ~Intracellular -~~ fluorescent probes have been developed to measure levels of calcium and other ions inside single cells.10J2Microelectrodespositioned outside the cell have measured the release of easily oxidized compound^.^^^^-^^ Dynamic measurements should also be useful in revealing cell compartments. Cytoplasmic and vesicular dopamines have been distinguished in the giant dopamine cell with voltammetric microelectrodes.16 A concern with dynamic measurements at single cells is the trade-off that must be made between chemical identification and temporal resolution. For example, the release of catecholamines from single bovine adrenal medullary cells can be measured with amperometry with high temporal resolution.llJi However, with amperometry, the compounds detected cannot be identified and this limits measurement and interpretation to one compound or class of compounds per cell. Identiiication is improved when fastscan cyclic voltammetry is used, as demonstrated by the selective determination of epinephrine (E) and norepinephrine (NE), which are both released from single medullary cells, but the time resolution is decrea~ed.'~"~ Another approach is to use sensors that exhibit more selectivity for the molecules of interest. For (8) Yeung, E. S. Acc. Chem. Res. 1994,27,409-414. (9) Alvarez de Toledo, G.; Femandez-Chacon,R; Femandez, J. M. Nature 1993, 363,554-558. (10) Gryznkiewicz, G.; Poenie, M.; Tsien, R. Y.J. Biol. Chem. 1985,260, 34403450. (11) Wightman, R M.; Jankowski, J. A.; Kennedy, R. T.; Kawagoe, K. T.;
Schroeder, T. J.; Leszczyszyn, D. J.; Near, J. A,; Diliberto, E. J., Jr.; Viveros, 0. H. PYOC.Ndl. A d . S C ~U.S.A. . 1991,88, 10754-10758. (12) Tsien, R Y. Chem. Eng. News 1994,72 (29), 34-44. (13) Leszczyszyn, D. J.; Jankowski, J. A; Viveros, O.-H.; Diliberto, E. J., Jr.; Near, J. A.; Wightman, R M. J. Biol. Chem. 1990,265,14736-14737. (14) Chow, R. H.; yon Ruden, L.; Neher, E. Nature 1992,356,60-63. (15) Kennedy, R. T.; Huang, L.; Atkinson, M. A,; Dush. P. Anal. Chem. 1993, 65,1882-1887. (16) Chien, J. B.; Wallingford, R A; Ewing, A. G.J. Neurochem. 1990,54,633638. (17) Leszczyszyn,D.J.; Jankowski, J. A; Viveros, 0. H.; Diliberto, E. J., Jr.; Near, .J. A; Wightman, R M.J Neurochem. 1991,56,185551862, (18) Ciolkowski, E. L.; Cooper, B. R.; Jankowski, J. A; Jorgenson, J. W.; Wightman, R. M.J Am. Chem. SOC.1992,114,2815-2821. (19) Pihel, K.; Schroeder. T. J.; Wightman, R. M. Anal. Chem. 1994,66,45324537.
Analytical Chemistry, Vol. 67, No. 15,August 1, 1995 2599
example, microelectrodes modified with ruthenium oxide/cyanoruthenate films have been used to monitor secretion of insulin from single pancreatic P - ~ e l l s .Selectivity ~~ is achieved because the ruthenium film catalyzes the oxidation of a disulfide bond in insulin. In the present work, we have developed a strategy to simultaneously monitor the secretion of ascorbate and catecholamines from single bovine adrenal medullary cells. Previous work has shown that the release of the catecholamines contained in membrane-bound vesicles inside the cell appears as oxidation spikes with millisecond width at an amperometric electrode.11,20 Vesicular catecholaminescan be released by nicotine stimulation, membrane depolarization by K+, or exposure to digitonin. Digitonin is a detergent that reacts with cholesterol in the plasma membranes of medullary cells to produce pores with 50-300-nm diameters as measured by scanning electron microscopy.21,22 Although the medullary vesicle membranes also contain cholesterol, they are known to be stable in 20 pM digitonin for at least 15 min.23-25In contrast, the plasma membrane becomes permeable to calcium ions within a few secondsz0rz6and to soluble cytosolic proteins such as lactate dehydrogenase within a few minute~.~~
