Depolarization and Increased Conductance Precede Superoxide

Proc. NatL Acad. Sci. USA Vol. 80, pp. 3726-3728, June 1983 Cell Biology

Depolarization and increased conductance precede superoxide release by concanavalin A-stimulated rat alveolar macrophages (respiratory burst/membrane potential) ALAN R. CAMERON, JUNE NELSON, AND HENRY JAY FORMAN Department of Physiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104 Communicated by Irwin Fridovich, March 24, 1983 ABSTRACT Rat alveolar macrophages release superoxide into see ref. 15) and in the relative responses to different stimuli the extracellular medium when stimulated by concanavalin A. This caution against extrapolation between the different cell types. process, the respiratory burst, is characterized by a delay be- In this study of rat alveolar macrophages, we used micro- tween binding of the stimulus and release of superoxide. It has electrodes to obtain a direct measurement of the membrane been proposed that a key event that occurs during this delay pe- potential at rest and after addition of concanavalin A, a stim- riod is the alteration of membrane electrical potential. Microelec- ulant of superoxide release, along with measurements of the trode impalement was used to directly measure electrical prop- membrane input resistance under the same conditions. The erties of the plasma membrane. Upon addition of concanavalin A, length of time for the maximal changes in membrane electrical the membrane potential depolarized 21%, and membrane elec- the lag time for superoxide re- trical resistance decreased 16%. Parallel chemical measurement properties was compared with of superoxide release indicated that these changes in electrical lease. properties precede the release of superoxide. METHODS AND MATERIALS Generation of superoxide radical is an integral part of the an- timicrobial activity of phagocytic cells (1). The process in which Preparation of Alveolar Macrophages. Alveolar macro- superoxide is released by these cells is characterized by a lag phages were obtained from male Sprague-Dawley specific- between recognition of a stimulus and activation of a plasma pathogen-free rats [Crl: CDR (SD) BR; Charles River Breeding membrane NADPH oxidase, which catalyzes the reduction of Laboratories, Wilmington, MA] by lung lavage (16) as modified oxygen to superoxide. Based on studies of altered transmem- (17). The yield of cells was -1 x 107 from each rat and was brane distribution of various lipid-soluble cationic probes, an >95% alveolar macrophages; >98% of the cells excluded essential step in the process whereby the NADPH oxidase is erythrosin B. activated appears to be a change in the plasma membrane po- Electrophysiologic Measurements. For electrophysiologic tential. However, there is controversy as to whether the re- measurements, cells were centrifuged onto high-density oil quired change is a depolarization, hyperpolarization, or a se- (Fluorinert FC-40; 3M, Minneapolis, MN) and covered with quence of potential fluctuations (2-11). Among the reasons for Krebs-Ringer bicarbonate buffer (pH 7.4). Microelectrodes were the uncertainty in these measurements are (i) the time nec- pulled with final resistances of between 35 MfQ and 55 MfQ and essary for movement of the cations after perturbation; (ii) ac- were filled with 3 M KCI, and these were maneuvered with a cumulation or redistribution (or both) of the cations across the Leitz micromanipulator. Cells were impaled, and membrane inner mitochondrial membrane, which has a much greater po- potential was recorded through a high-impedance electrome- tential difference (negative inside) than that of the plasma ter. Results were displayed on a dual-beam oscilloscope and membrane (12); and (iii) metabolic alterations of the probes. recorded on a oscillographic recorder. Input resistance was For example, Whitin et al. (11) have shown that one of the flu- measured by passing 500-msec square-wave pulses of hyper- orescent cationic dyes used in these studies, 3,3'-dipropyl- polarizing current through the recording electrode. Pulses were thiodicarbocyanine, is oxidized by the myeloperoxidase system generated through the electrometer by using a Grass Instru- associated with the respiratory burst, resulting in decreased ments type 88 stimulator. Input resistance was measured con- fluorescence that has been misinterpreted as hyperpolariza- tinuously with pulses of 1 Hz for 20 sec before addition and for tion. several minutes after addition of concanavalin A (250 ,ug/ml). Studies with microelectrodes also have been made of the To avoid exciting the cells by mechanical agitation, we added plasma membrane potential of cultured peritoneal macro- the concanavalin A solution just above the well in which the phages (13, 14). Although the addition of chemotactic factors punctured cell was placed. induced hyperpolarization of the plasma membrane of cultured Superoxide Generation. The assay for extracellular release human neutrophils (13), measurements of superoxide release of superoxide of Cohen and Chovaniec (18) was used with the by these cells were not made. The activation process for al- following modifications: the reaction was monitored with a dual- veolar macrophages, peritoneal macrophages, and polymor- wavelength spectrophotometer at 540-550 nm in Krebs-Ringer phonuclear leukocytes may be similar; however, differences in phosphate buffer (pH 7.4) containing 2 x 10-5 M ferricyto- metabolic requirements for activation of superoxide release (e.g., chrome c. Approximately 1 X 106 cells were incubated for 15 versus generation of the required ATP; min at 25°C, and then concanavalin A (250 ,ug/ml) was added. mitochondrial glycolytic All rates were expressed as the superoxide produced in 4 min after the end of the lag phase, as defined by Cohen and Chova- The publication costs of this article were defrayed in part by page charge of payment. This article must therefore be hereby marked "advertise- niec (18). To assess the superoxide dependence cytochrome ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact. c reduction, we added superoxide dismutase. 3726 Downloaded by guest on September 28, 2021 Cell Biology: Cameron et aL Proc. Natl. Acad. Sci. USA 80 (1983) 3727 0.21 O.'%

V. - 3E 0.4- E0.4 5 x 106 L.. O0 OC0 >0 ,vS0.3C < a. E _100~~~~~~a 0 0I~~~~~~~~ L)-o+ 3106 0. O 1-l2- 3 0.1 0. MINUTES C,) FIG.. Time course for the electrical changes and production of su- 0 60 120 180 peroxide stimulated by the addition of concanavalin A to rat alveolar SECONDS macrophages. Results are indicated for one typical experiment. Con- canavalin A was added at 0 min. Curves were smoothed to more clearly FIG. 2. The effect of cell concentration on the lag time. Alveolar illustrate the timing of events. macrophages were incubated at the indicated number in a 1-ml assay system as described. Actual tracings are shown. RESULTS Addition of concanavalin A to rat alveolar macrophages pro- able. Regardless of this variability, both the input resistance duced a small but significant change in both the membrane po- and potential changes were complete in =30-35 sec. Once these tential (16.8 ± 4.0%) and input resistance (15.9 ±- 8.3%) of the changes were complete, the membrane potential and input re- plasma membrane. The resting membrane potential of these sistance remained constant for at least 10 min. freshly isolated cells was -20.8 ± 1.9 mV at 250C. Because The timing for the changes in electrical properties contrasted concanavalin A could not be removed from the cells by wash- markedly with the timing for superoxide release. The lag in su- ing, we controlled for nonspecific effects by varying the inter- peroxide production was =61 sec, and superoxide production val of recording prior to the addition of concanavalin A. Both continued for more than 5 min after the lag. Thus, production the input resistance and membrane potential were found to be of superoxide follows the changes in electrical properties and stable in the absence of concanavalin A. After initial penetra- does not contribute to them. To test whether the limit of de- tion, the electrical properties stabilized within 5 sec and re- tection of superoxide production (-0.02 nmol) could account mained stable for upmyto 30 in the absence of concanavalin for the lag, the cell number was varied in the assay. Increasing A. Any cell that did not remain stable for at least 15 min was the number of cells in the assay to 5 x 106 caused a 5-fold in- not used for further measurements. Fig. 1 illustrates tracings crease in the amount of superoxide detected but did not alter from one combined electrophysiologic-biochemical experi- the lag time (Fig. 2). When the concentration of cytochrome ment, and Table 1 shows the results of the accumulated mea- c was increased 3-fold, neither the lag time nor the total measurements. Changes in electrical properties began within 0-18 sured superoxide production was affected. sec after the addition of the stimulus. We assume that the delay in the change in electrical properties in some experiments was, DISCUSSION in part, due to the manner in which concanavalin A had to be These experiments have demonstrated directly that both de- added in the electrophysiologic measurements. The time for polarization and increased membrane conductance (the recip- the full changes in electrical properties to occur was quite van- rocal of the input resistance) precede the production of su- Table 1. Membrane electrical properties and superoxide release peroxide. Whether the electrical changes must be complete in of rat alveolar macrophages any particular cell before superoxide production by that cell occurs can not be assessed; however, measurable superoxide pro- Membrane potential, mV duction always considerably lagged behind the changes in elec- Resting -20.8 ± 1.9 trical properties. It is possible that the slight overlap in these Change with concanavalin A 4.4 ± 1.0* Time course, sec events reflects the variability of the timing of the electrical Start 12.3 ± 2.3 changes that were observed directly with single cells, whereas superoxide production was measured with 1 X 106 cells si- To maximum change 30.9 ± 11.8 multaneously. However, because neither increasing the num- Input resistance, Mfl ber of cells nor increasing the ferricytochrome c concentration Before concanavalin A 76.5 ± 22.8 affected the lag time for superoxide production, it is unlikely Change with concanavalin A -14.5 ± 8.2t that the measurement of superoxide generation contributed Time course, sec significantly to the lag time. Start 12.3 ± 2.3 The plasma membrane potential for resting rat alveolar mac- To maximum change 34.4 ± 11.5 rophages measured in this work is close to the Cl potential (-22 mV) reported for these cells by Castranova et al. (19), but Superoxide production, differs from the potential they measured with cationic probes. nmol/4 min/1 x 106 cells 0.17 ± 0.02 However, as mentioned above, accumulation of these probes Lag time, sec 60.6 ± 6.1 in intracellular compartments causes an overestimation of the The methods are described in the text; N = eight experiments. Each plasma membrane potential (12) that most likely produced this value shown is the mean ± SEM. discrepancy. *P < 0.002. We presume that the alterations in electrical properties of tp < 0.05. the plasma membrane that precede the release of superoxide Downloaded by guest on September 28, 2021 3728 Cell Biology: Cameron et al. Proc. Natl. Acad. Sci. USA 80 (1983)

are actually involved in the activation process. Although there 6. Seligmann, B. & Gallin, J. I. (1980) Mol. Immunol. 17, 191-200. is no direct evidence of that connection in the present studies, 7. Seligmann, B. & Gallin, J. I. (1980) J. Clin. Invest. 66, 493-503. it has been shown that neutrophils from patients with chronic 8. Jones, G. S., Van Dyke, K. & Castranova, V. (1981) J. Cell. Phy- granulomatous disease are apparently defective in superoxide siol. 106, 75-84. 9. Miles, P. R., Bowman, L. & Castranova, V. (1981)J. Cell. Physiol. production because they do not depolarize (7, 10). Influx of Na', 106, 109-117. Ca", or both into phagocytes also has been implicated in the 10. Whitin, J. C., Chapman, C. E., Simons, E. R., Chovaniec, M. activation of superoxide release (9, 20-24). The relative timing E. & Cohen, H. J. (1980) J. Biol. Chem. 255, 1874-1878. of the changes in electrical properties of the alveolar macro- 11. Whitin, J. C., Clark, R. A., Simons, E. R. & Cohen, H. J. (1981) phage plasma membrane and in production of superoxide sug- J. Biol. Chem. 256, 8904-8906. 12. Hoek, J. B., Nicholls, D. G. & Williamson, J. R. (1980)J. Biol. gests that the membrane depolarization may reflect the influx Chem. 255, 1458-1464. of those cations. 13. Gallin, E. K. & Gallin, J. I. (1977) J. Cell Biol. 75, 277-289. Finally, the stability of the membrane potential and con- 14. Dos Reis, G. A. & Oliveira-Castro, G. M. (1977) Biochim. Bio- ductance during most of the period of superoxide release pre- phys. Acta 469, 257-263. cludes the suggestion that superoxide release by stimulated al- 15. Cohen, H. J. & Chovaniec, M. E. (1978)J. Clin. Invest. 61, 1088- veolar macrophages is electrogenic. 1096. 16. Myrvik, Q. N., Leake, E. S. & Fariss, B. (1961)J. Immunol. 86, 128-132. The authors thank Dr. Aron Fisher and Dr. Michael Seltzer for their 17. Forman, H. J., Nelson, J. & Fisher, A. B. (1980)J. Biol. Chem. valuable comments. This work was supported by National Institutes of 255, 9879-9883. Health Grants HL 23790 and HL 19737. 18. Cohen, H. J. & Chovaniec, M. E. (1978)J. Clin. Invest. 61, 1081- 1087. 1. Babior, B. M. (1978) N. Engl. J. Med. 298, 659-668; 721-725. 19. Castranova, V., Bowman, L. & Miles, P. R. (1979) J. Cell. Physiol. 2. Utsumi, K. K., Sugiyama, M., Miyahara, M., Naito, M., Awai, 101, 471-480. M. & Inoue, M. (1977) Cell Struct. Func. 2, 203-209. 20. Lew, P. D. & Stossel, T. P. (1981)J. Clin. Invest. 67, 1-9. 3. Korchak, H. M. & Weissmann, G. (1978) Proc. NatL Acad. Sci. USA 21. Simchowitz, L. & Spilberg, I. (1979) J. Lab. Clin. Med. 93, 583- 75,3818-3822. 593. 4. Korchak, H. M. & Weissmann, G. (1980) Biochim. Biophys. Acta 22. Simchowitz, L. & Spilberg, I. (1979)J. Immunol. 123, 2428-2435. 601, 180-194. 23. Sweeney, T. D., Castranova, V., Bowman, L. & Miles, P. R. (1981) 5. Seligmann, B., Gallin, E. K., Martin, D. L., Shain, W. & Gallin, Exp. Lung Res. 2, 85-96. J. I. (1980) . Membr. Biol. 52, 257-272. 24. Forman, H. J. & Nelson, J. J. Appl. Physiol., in press. Downloaded by guest on September 28, 2021