©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Simultaneous Detection of Free Radical Release and Membrane Current during Phagocytosis (*)

(Received for publication, October 20, 1994; and in revised form, January 16, 1995)

Kathleen O. Holevinsky Deborah J. Nelson (§)

From the Departments of Neurology, Medicine, and Pharmacological and Physiological Sciences, University of Chicago, Chicago, Illinois 60637

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Stimulation of macrophages induces the ``respiratory burst'' response which is associated with the generation of superoxide (O(2)), a drop in cytoplasmic pH, and a pronounced depolarization of the membrane potential. The purpose of the present studies was to determine whether an increase in O(2) was temporally related to changes in membrane potential and transmembrane current. Release of O(2) at the single cell level was photometrically monitored during phagocytosis of immune complexes while simultaneously measuring whole-cell current. Membrane depolarization and the generation of a non-selective current followed an increase in O(2) production with a variable lag time which was correlated with the state of cellular maturation in culture. In the absence of phagocytosis, the exposure of macrophages to O(2) generated by a xanthine-xanthine oxidase reaction activated a non-selective current similar to that seen after phagocytosis. These results provide the first demonstration of the relationship between free radical release and the ensuing electrophysiological signaling events which are linked to particle engulfment in phagocytic cells.


INTRODUCTION

The binding of particulate as well as soluble ligands to phagocytic membranes may result in particle phagocytosis, lysosomal enzyme release, and generation of a respiratory burst response. The ability to initiate a respiratory burst and thereby to produce reactive oxygen species, e.g. superoxide anion (O(2)), (^1)hydrogen peroxide, and singlet oxygen is central to the cytotoxic properties of phagocytes. Superoxide produced following the phagocytic event is concentrated within the phagocytic vacuole and is only produced in the region of the plasma membrane in direct contact with the particle (see review by Segal and Abo(1993)).

Activation of the respiratory burst in phagocytic cells has been shown to be closely associated with stimulus-induced changes in membrane potential. Studies with HL-60 cells, for example, have demonstrated that differentiating granulocytes gain the capacity for depolarization concomitantly with O(2) production (Kitagawa et al., 1984). Moreover, when activated macrophages are cultured in vitro, the heightened membrane depolarization and respiratory burst reactivity are coincidentally lost (Kitagawa and Johnston, 1986). A close correlation between the development of depolarization and cytotoxic activities in normal myeloid cells further emphasizes that membrane potential changes and functional reactivity are coupled (Newburger et al., 1979). Stimulus-induced changes in membrane potential have been reported to antecede changes in cellular oxidative metabolism in granulocytes (Jones et al., 1981; Korchak and Weissman, 1978) suggesting that depolarization may be an important element in the signal transduction cascade leading from membrane receptor ligation to an increase in NADPH oxidase activity.

In an attempt to elucidate the molecular basis of the stimulus-induced changes in membrane potential that are associated with the initiation of the respiratory burst, we examined the relationship between the free radical release, transmembrane current, and membrane potential changes following receptor-mediated phagocytosis in peripheral mononuclear cells maintained in culture. We provide evidence that superoxide release subsequent to the phagocytosis of immune complexes is associated with activation of a calcium-dependent, non-selective current, and membrane depolarization. The apparent superoxide-induced changes in membrane potential may contribute to the depression of macrophage respiratory burst capacity observed following FcR-mediated phagocytosis.


EXPERIMENTAL PROCEDURES

Materials

DCFH-IC was obtained from Molecular Probes (Eugene, OR) as Fc-Oxyburst®. Fibronectin was obtained from Collaborative Research Inc. (Bedford, MA). Diphenylene iodonium chloride was obtained from Toronto Research Chemicals, Inc. (Downsview, Ontario, Canada).

Electrophysiological Studies

Human monocyte-derived macrophages (HMDMs) isolated from peripheral blood were cultured in Teflon vials for up to 2 weeks as described previously (Nelson et al., 1990). Cells were plated on glass-bottom recording dishes which were coated with 2% gelatin followed by 10 µg/ml fibronectin. Whole-cell recordings from HMDM membranes were obtained using the techniques of Hamill et al.(1981). A dish containing cultured cells was placed in a chamber on the movable stage of an inverted microscope equipped with phase-contrast optics. Experiments involving the phagocytic uptake of the immune complex fluorescent dye conjugate (DCFH-IC) were performed at elevated temperature (33 °C) using a temperature controlled stage (Brook Industries, Lake Villa, IL). Recording pipettes were formed from soda lime glass (Blue-Dot Hematocrit Glass, Fisher) using a horizontal puller (model P-87, Sutter Instruments, San Rafael, CA). Whole-cell currents were obtained using a List EPC-7 (List Electronic, Darmstadt, West Germany) voltage clamp. The output of the current-to-voltage converter was filtered through a low pass filter at 500 Hz. The current signal was sampled at 1 kHz by a 12-bit A/D converter (Data Translation 2818, Marlborough, MA.) and written into data files using an IBM-AT.

Experimental Solutions

Whole-cell recordings were made in an external recording solution that contained 140 mM NaCl, 5.4 mM KCl, 2 mM CaCl(2), 2 mM MgCl(2), 10 mM HEPES buffered to pH 7.4. The pipette solution contained 144 mM KCl, 2 mM CaCl(2), 2 mM MgCl(2), 11 mM EGTA, 10 mM HEPES buffered to pH 7.2. Solution osmolarities were monitored using a vapor pressure osmometer (model 5500, Wesco, Logan, UT). For the selectivity studies, the pipette solution contained 40 mM NaCl, 100 mM NMDG-glutamate, 0.2 mM CaCl(2), 1 mM MgCl(2), 10 mM HEPES, 1 mM EGTA buffered to pH 7.2. The bath solution was the standard external recording solution. The equilibrium potentials for a perfectly cation- or anion-selective conductance under these ionic conditions were +31 mV and -31 mV, respectively. Experiments performed in the asymmetric salt solutions allowed us to separate leak current from current carried by monovalent cations. In studies examining the relative cation selectivity of the current, NaCl in the bathing solution was replaced with the chloride salts of either K, Cs, Li, or NMDG.

Microfluorimetric Measurement of Free Radical Release

Superoxide production following phagocytosis of the DCFH-labeled immune complexes was monitored directly using a PTI D104 (Photon Technologies International, South Brunswick, NJ). The PTI D104 is a filter-based illumination system consisting of a 75-watt xenon arc lamp and a variable speed reflective optical chopper under computer control. Excitation light at 490 nm was deflected with a 510-nm dichroic mirror. Emitted fluorescence filtered at 525 nm was collected by a Hammamatsu R928 photomultiplier tube and photon-counting photometer through a variable rectangular aperture roughly equal to the area of the single cell. Photomultiplier output was sampled at 20 Hz and processed using a PTI interface on a 386/20 personal computer.

Digital Fluorescence Imaging

Cells were illuminated at 490 nm using a slewing monochromator and a xenon-light source as described above. In the imaging experiments, a 40 times (Nikon Fluor, N.A. 1.3) oil immersion objective was used to gather fluorescence data from a number of cells in the field of view. Fluorescence emission at 525 nm was collected with a Hammamatsu c2400 SIT video camera connected to a PTI image processor. Individual frames were summed (number of frames summed depended on fluorescence intensity) and averaged. Frame averages were collected at 30-s intervals following exposure of the cell to the dye-coupled immune complexes.

Data Analysis

Summary data are expressed as means ± standard error of the mean with the number of experiments in parentheses.


RESULTS AND DISCUSSION

Respiratory burst activation in HMDMs was triggered and monitored photometrically through detection of superoxide using the reduced fluorochrome DCFH covalently linked to bovine serum albumin (BSA)-anti-BSA immunoglobulin immune complexes. The Fc receptor-mediated binding and phagocytosis of the fluorochrome-coupled derivative (DCFH-IC) transfers the conjugate to the phagocytic vacuole. The presence of oxygen metabolites released into the phagocytic vacuole following NADPH-oxidative activation oxidizes DCFH to fluorescent dichlorofluorescein. Flow cytometric studies on peripheral blood polymorphonuclear leukocytes have previously shown that the DCFH-IC conjugate is capable of monitoring the kinetics of the production of activated oxygen species within the phagocytic vacuoles of neutrophils (Ryan et al., 1990). To gain insight into the interrelationship between free radical release and ion channel activation in mononuclear cells during activation, whole-cell voltage clamp experiments were carried out in physiological solutions during phagocytosis of DCFH-ICs.

Fig. 1shows the time-dependent increase in membrane current following exposure of a representative cell to the fluorochrome-coupled immune complexes. Current activation was linear throughout the voltage range and was accompanied by a depolarizing shift in the zero current potential. Fig. 2shows examples of simultaneous current and microfluorimetry recordings from three macrophages at different stages of development in culture. The membrane potential was held at -60 mV and voltage ramps from -100 to 100 mV, 300 ms in duration were repetitively applied every 3 s throughout the experiment. Exposure of the cells to DCFH-ICs induced an increase in fluorescence, which represented an increase in free radical production and was associated with the generation of a current with a reversal potential of -0.9 ± 0.8 mV (n = 9). The lag time preceding the onset of the current response appeared to be correlated with the length of time cells were maintained in culture. Older cells displayed an increase in the time required to observe both free radical release as well as current activation and membrane depolarization, data which is summarized in Table 1. In the absence of stimulus, cells exhibited stable whole-cell currents and no change in fluorescence.


Figure 1: Phagocytosis of fluorescent-labeled immune complexes (DCFH-IC) induces current activation in macrophages. A, whole-cell current following step depolarizations between -110 and 100 mV at 30 mV intervals from a holding potential of -60 mV under control conditions. B, current following 5 min of exposure of the cell to a solution of solubilized DCFH-IC (15 µg/ml) used to stimulate the oxidative burst activation following phagocytosis. C, maximal current activation observed at approximately 10 min following exposure of the cell to DCFH-IC. D, associated current-voltage relationship for the currents in A-C .




Figure 2: Response time for superoxide release as well as membrane depolarization following phagocytosis of DCFH-IC becomes slower the longer cells are maintained in culture. Coupled voltage-clamp and microfluorimetry experiments showing simultaneous changes in fluorescence, membrane potential, and membrane current in three representative cells following exposure to 15 µg/ml DCFH-IC. After uptake of immune complexes, reactive oxygen products are generated, as indicated by changes in fluorescence (top panel in A-C). The accumulation of intracellular oxidants was associated with the activation of a non-selective current and with an accompanying membrane potential depolarization. The fluorescent response was normalized to the peak response for each for each cell. Variability in cell size and differences in the rate of particle uptake precluded comparative analysis of free radial release among cells examined. Membrane currents measured in response to 300 ms voltage ramps from -100 to 100 mV every 3 s from a holding potential of -60 mV are seen in the second panel in A-C. Illustrated membrane currents were obtained from voltage ramps at six different membrane potentials between 100 and -100 mV and shown at 40-mV intervals. Membrane potential was determined as the zero current potential for each of the voltage ramps over the duration of the experiment as shown in the third panel from each of the representative cells. In some cells, a transient hyperpolarization was seen prior to depolarization. The time before fluorescent response and the lag time between initial increase in fluorescence and membrane depolarization was dependent on the age of the cell in culture. A, single cell microfluorimetric measurement of O(2), membrane current, and membrane potential in a macrophage after 1 day in culture. B, recording from a cell after 6 days in culture. C, recording from a cell after 13 days in culture.





In order to further confirm the observation that the depolarization and conductance changes following particle ingestion are indeed due to the presence of reactive oxygen intermediates, cellular responses were measured in the presence of the specific NADPH-oxidase inhibitor diphenylene iodonium (DPI). Addition of 50 µM DPI to phorbol myristate acetate (PMA)-stimulated intact neutrophils has been shown to cause marked inhibition of superoxide generation (Cross and Jones, 1986). Similar results have also been obtained in murine peritoneal macrophages (Hancock and Jones, 1987; Stuehr et al., 1991). Digital fluorescent imaging experiments were carried out on HMDMs in the presence and absence of DPI. Cells preincubated with 5 µM DPI prior to the addition of DCFH-IC failed to give rise to an increase in fluorescence as compared to control cells, as seen in Fig. 3. Similarly, we were unable to observe current activation in cells pre-treated with DPI (5 µM) 10 min prior to exposure to DCFH-IC (Fig. 4). Peak current amplitude at maximal depolarizations of 100 mV in the presence and absence of DPI is summarized in Table 2.


Figure 3: Oxidation of DCFH-IC requires NADPH oxidase activity. Fluorescence intensity (490 nm) was monitored by digital imaging, following addition of 15 µg/ml DCFH-IC. A heating stage was used to maintain temperature at 30 °C. A and B, fluorescent images of a field of HMDMs (times 400 magnification) 24 min following stimulation with DCFH-IC in standard 140 mM NaCl bath solution (A) and in 140 mM NaCl bath containing 5 µM DPI (B). An outline of cells in each field is overlaid on the fluorescent image. Boxes represent the areas of highest fluorescence, which were used for data in C and D. C, change in fluorescence intensity with time following stimulation with DCFH-IC in each of five representative cells as seen in A. D, change in fluorescence of five cells stimulated in the presence of DPI as seen in B. Addition of DCFH-IC is indicated by an arrow. At 15 min following exposure of the cells to the phagocytic stimulus, control cells exhibited a 34.2% increase in fluorescent intensity over that observed in DPI-treated cells. The average change in fluorescence intensity (in arbitrary units) for the five control cells in A at 15 min was 7.6 ± 0.7 compared to 5.0 ± 0.4 measured for the DPI-treated cells in B.




Figure 4: Current activation is dependent upon NADPH oxidase activity. HMDMs preincubated in DPI, a specific inhibitor of NADPH oxidase, do not exhibit current activation in response to DCFH-IC. Patch-clamp experiments were carried out at 30 °C in bath solutions containing 140 mM NaCl, 5.4 mM KCl, 2 mM Ca, 2 mM Mg, and 10 mM HEPES, with pipette solutions containing 140 mM KCl, 1 mM EGTA, 0.2 mM Ca, and 2 mM Mg. A and B, HMDMs which received no treatment prior to DCFH-IC activation. A, whole-cell current, following 100 mV pulse, before and 10 min following addition of 15 µg/ml DCFH-IC. B, current-voltage relationship for current shown in A. C and D, whole-cell current measured in cells preincubated for 10 min in 5 µM DPI prior to whole-cell formation. C, whole-cell current following a 100 mV pulse before and after addition of DCFH-IC. D, current-voltage relationship for current shown in C.





In order to demonstrate that particle uptake was necessary to elicit a fluorescent response, cells were preincubated in 5 µM cytochalasin B, an inhibitor of phagocytosis, prior to stimulation. A representative fluorescent response for a cytochalasin-treated cell is compared to that obtained to a control cell in Fig. 5. We were unable to observe an increase in fluorescence in response to DCFH-IC in a total of six cells following pretreatment with cytochalasin, indicating that oxidation of the dye was occurring intracellularly. A similar inhibition of the fluorescent response in cells pretreated with cytochalasin B was observed in the flow cytometric studies of Ryan et al.(1990).


Figure 5: Changes in fluorescence seen during the respiratory burst are dependent on uptake of DCFH-IC particles and are due to intracellular oxidation of DCFH. Pretreatment of granulocytes with the microtubule-disrupting agent cytochalasin B has been shown to effectively inhibit phagocytosis. Two cells from the same HMDM isolation, each approximately 80 pF, in standard external solution were stimulated with 15 µg/ml DCFH-IC; fluorescence was monitored at 490 nm. Temperature was maintained at 30 °C. The trace labeled Cytochalasin B was recorded from a cell preincubated with 5 µM cytochalasin B for 25 min prior to exposure to DCFH-IC. The trace labeled Control was obtained from a stimulated cell that received no pretreatment. The decrease in fluorescence in the control cell over time may be a consequence of the pH sensitivity of the dye and attributable to the decrease in cytoplasmic pH which accompanies the oxidative burst. Upon acidification, the emission spectra of all the fluorescein dyes decrease in amplitude without a significant shift in wavelength (Tsien, 1989).



In order to determine whether the reactive oxygen species generated during the phagocytic response were directly responsible for current generation, we investigated the effect of O(2) on HMDMs in the absence of phagocytosis. Exposure of cells to O(2) generated in the presence of xanthine and xanthine oxidase gave rise to changes in current and shifts in zero current potential resembling those following phagocytosis of DCFH-ICs as seen in Fig. 6. This result indicates that the presence of oxygen metabolites alone is sufficient to induce current activation. Currents induced under either condition were completely inhibited in the presence of 1 mM La indicating that the non-selective currents were not simply the result of instabilities in the electrophysiological recording or free radical-induced membrane breakdown.


Figure 6: Membrane current activation following exposure to both DCFH-IC and the O(2) generating system, xanthine-xanthine oxidase. A: upper panel, changes in membrane potential after addition of 15 µg/ml DCFH-IC and in the presence of 1 mM La. Membrane potential was determined as the zero current potential during voltage ramps. Addition of DCFH-IC and 1 mM La is indicated by arrows. Lower panel, whole-cell currents elicited by 300-ms voltage ramps from -100 to 100 mV prior to addition to DCFH-IC, 8 min after addition of DCFH-IC, and 10 s after addition of 1 mM La to the cell following maximal current activation. Capacitive current was subtracted from the records shown. B, changes in membrane potential and current following exposure of cells to the O(2) generating system, xanthine-xanthine oxidase. Upper panel, changes in membrane potential prior to and following exposure of the cell to xanthine oxidase in the presence of 0.1 mM xanthine and in the presence of La. Addition of 0.05 unit/ml xanthine oxidase and 1 mM La is indicated by the arrows. Lower panel, whole-cell currents prior to activation in the presence of xanthine; maximal current activation 4 min after addition of xanthine oxidase, and current inhibition 4 min after addition of La.



The ionic selectivity of the free radical-induced current was determined in experiments in which Na and Cl were the major permeant species and the cells stimulated in the presence of DCFH-ICs. The cation to anion selectivity was determined from current reversal potentials obtained when 100 mM of the NaCl in the intracellular solution was substituted with NMDG-glutamate. Under these conditions, the cation to anion permeability ratio (P/P) was calculated to be 1.0. This value may reflect the fact that 1) the conductance is not selective for cations over anions, i.e. the Na/Cl permeability ratio is low, or 2) the conductance is partially permeant to glutamate, i.e. the Cl/glutamate permeability ratio is low, or 3) there are a number of conductances which are simultaneously activated during the response to free radical release.

The relative permeability of the DCFH-IC activated conductance to several monovalent cations was determined in experiments in which the Na in the bath solution was replaced with chloride salts of either K, Cs, Li, or NMDG. The ionic selectivity among cations was determined from shifts in the reversal potential as determined in three cells, each of which was exposed sequentially to the five cations and is summarized in Table 3. The relative cation permeability (P/P) of the DCFH-IC-induced current as determined in three cells was K (1.37) > Cs (1.28) > Na = Li (1.00) > NMDG (0.69) which corresponds to the Eisenman sequence IV indicating that permeation is likely to be weakly dominated by the dehydration energy of the permeating species and that with the exception of NMDG, the DCFH-IC-activated conductance showed poor selectivity among the monovalent cations examined. While our results clearly demonstrate that monovalent cations contribute to the free radical-induced current, these experiments did not determine whether current flow was through single or multiple channel types.



The sensitivity of the DCFH-IC activated current to external Ca was examined in experiments conducted in Ca-free bath solutions buffered with 1 mM EGTA. In these experiments the pipette solution contained 10 mM BAPTA without additional Ca, with one exception where the standard Ca buffer was used. These experiments indicated that DCFH-IC-induced current activation was significantly attenuated under Ca-free conditions. Peak current at 100 mV in response to DCFH-IC in Ca-free solutions was 248 ± 52 pA (n = 3) as compared to 4804 ± 1240 pA (n = 3) obtained in Ca-containing solutions. The mean change in current after addition of DCFH-IC was -183 ± 33 pA in Ca-free solutions and 4475 ± 1428 pA in Ca containing solutions (p < 0.08). Normalized to capacitance, this corresponds to a decrease of 1.7 pA/pF in Ca-free solutions and an increase of 50.8 pA/pF in Ca-containing solutions. Capacitance values for these cells were estimated using data in which the capacitance for HMDMs relative to age in culture was previously determined (Nelson, 1990).

The decrease in current amplitude under Ca-free conditions could reflect the fact that the free radical sensitive-channels are Ca activated or alternatively that the activity of NADPH-oxidase is Ca-dependent. To differentiate between the two possibilities, fluorescence imaging experiments were carried out on cells in the presence and absence of external Ca. We were unable to observe a significant difference in the fluorescence response to DCFH-IC as a function of the external Ca concentration. The average increase in fluorescence intensity in Ca-containing solutions was 176.8 ± 20% (n = 5) as compared to an increase of 201.4 ± 49.8% observed in nominally Ca-free solutions. These results are consistent with the conclusion that the conductance increase and not the oxidase activity is Ca sensitive.

Data supporting a link between changes in membrane potential and cellular oxidative metabolism comes from population studies showing that conditions which inhibit membrane depolarization also prevent generation of respiratory burst activity (Simchowitz and Spilberg, 1979). The importance of depolarization in the generation of a respiratory burst response has also been emphasized in studies involving neutrophils from chronic granulomatous disease patients. These cells lack the ability to produce O(2), and they additionally fail to undergo significant depolarization upon surface stimulation (Seligmann and Gallin, 1980; Whitin et al., 1980). Furthermore, studies on lipopolysaccharide-activated macrophages (Kitagawa and Johnston, 1985) revealed that these cells concomitantly develop an increased capacity for depolarization and respiratory burst responsivity, suggesting that cell depolarization does not occur artifactually as a result of membrane perturbation, but may be an important mechanism of signal transduction.

Superoxide generation is not limited to phagocytes and bactericidal activity. The consequences of free radical generation might be generalized and expected to regulate variety of cellular functions. Transformed as well as normal human B lymphocytes efficiently release O(2) when stimulated by surface immunoglobulin cross-linking or phorbol esters (Dorseuil et al., 1992; Maly et al., 1988; Volkman et al., 1984). Moreover, it has been suggested that reactive oxygen intermediates may regulate genes involved in inflammatory and immune responses serving as signaling molecules in T lymphocytes controlling activation of the transcription factor NFkappaB (Schreck et al., 1991).

Whitin et al.(1980) have reported that phorbol ester-stimulated granulocytes undergo membrane depolarization under anaerobic conditions where no O(2) is produced, suggesting that membrane potential and superoxide production are not coupled. Phorbol esters known to activate protein kinase C, as well as purified protein kinase C have been shown to regulate the activation of both anion and cation channels (Berger et al., 1993; Charpentier et al., 1993; Fournier et al., 1993; Li et al., 1993; Oike et al., 1993; Tabcharani et al., 1991). Therefore, in phorbol ester-stimulated cells, it is not unreasonable to expect that changes in membrane potential might be observed as a consequence of protein kinase C-mediated ion channel phosphorylation independent of O(2) generation. The electrophysiological response of the cells undergoing phagocytosis in our studies might well be different from the response in cells stimulated with phorbol esters.

The time course of phorbol ester-induced depolarization has been shown to be a sensitive function of the fluorescent probe used to measure membrane potential in flow cytometric studies of phorbol ester-stimulated neutrophils (Seligmann and Gallin, 1983). Seligmann and Gallin(1983) were able to demonstrate that the fluorescence of the carbocyanine dye di-S-C(3)(5) used as the membrane potential probe is destroyed by reactive oxygen products produced by neutrophils following stimulation, giving rise to an apparent insensitivity of the cellular response to the effects of scavengers or inhibitors of various reactive oxygen species. This fact coupled with the toxicity of the carbocyanine dyes at high concentrations (Seligmann and Gallin, 1983) would lead to the erroneous conclusion in phorbol ester-stimulated cells monitored with carbocyanine dyes that membrane potential and O(2) are not coupled. In our studies, we have determined membrane potential directly, thereby circumventing the problems encountered with dye toxicity and sensitivity to free radical destruction.

The ability of mononuclear phagocytes to respond to respiratory burst activators is also related to the stage of cellular differentiation. Resident tissue macrophages produce only low levels of superoxide anion (Biggar et al., 1976; DeChatelet et al., 1975). Monocytes allowed to differentiate in culture resemble tissue macrophages as evidenced by a progressive loss in respiratory burst activity (Musson et al., 1982; Seim, 1982). Our observation that the time course of current activation following the generation of free radicals in HMDMs is dependent upon age in culture may be the consequence of the reduction in the O(2) generating capacity of the cells with differentiation and suggests that a critical concentration of free radical is needed to induce an electrophysiological response. The large lag time between oxidant production and current change observed in the older cells may alternatively be due to 1) a decrease in the oxidant sensitivity of the ionic channel(s) generating the current, 2) a decrease in the expression of the oxidant sensitive channel(s) or 3) a decrease in the expression and/or oxidant sensitivity of a regulatory protein which controls channel opening.

This study demonstrates that oxygen metabolites generated during the phagocytic response, as well as exogenous O(2), activate a non-selective membrane current which induces membrane depolarization and is inhibitable by La. Data from this investigation provide evidence that membrane depolarization does not signal activation of the superoxide generating system but rather is a consequence of its activation. It has been generally observed that following Fc receptor-mediated phagocytosis there is a general depression of macrophage respiratory burst capacity as well as tumoricidal activity (Commins et al., 1990; Loegering and Schwacha, 1991; Schwacha et al., 1993). Thus, membrane depolarization which accompanies the initial phagocytic event may be one of a number of signals which down-regulate the granulocyte response to subsequent stimuli.


FOOTNOTES

*
This work was supported by a grant from the National Institutes of Health RO1 GM36823. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Neurology, University of Chicago, MC2030, 5841 S. Maryland Ave., Chicago, IL 60637. Tel.: 312-702-0126; Fax: 312-702-9076.

(^1)
The abbreviations used are: O(2), superoxide anion; DCFH-IC, 2`,7`-dichlorodihydrofluorescein-labeled immune complex; BSA, bovine serum albumin; NMDG, N-methyl-D-glucamine; HMDM, human monocyte-derived macrophage; FcR, Fc receptor; DPI, diphenylene iodonium chloride; PMA, phorbol 12-myristate 13-acetate.


ACKNOWLEDGEMENTS

We thank Drs. E. R. Simons and V. Bindokas for many helpful discussions during this investigation.


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