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J. Biol. Chem., Vol. 275, Issue 25, 18745-18750, June 23, 2000

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The Small G-protein Rac Mediates Depolarization-induced Superoxide Formation in Human Endothelial Cells^*

Hae-Young Sohn, Matthias Keller, Torsten Gloe, Henning Morawietz^§, Uwe Rueckschloss^§, and Ulrich Pohl

From the Institute of Physiology, Ludwig Maximilians University Munich, 80336 Munich, Germany and the ^§ Institute of Pathophysiology, University of Halle, 06097 Halle, Germany

Received for publication, January 5, 2000, and in revised form, March 2, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Superoxide anions impair nitric oxide-mediated responses and are involved in the development of hypertensive vascular hypertrophy. The regulation of their production in the vascular system is, however, poorly understood. We investigated whether changes in membrane potential that occur in hypertensive vessels modulate endothelial superoxide production. In cultured human umbilical vein endothelial cells, changes in membrane potential were induced by high potassium buffer, the non-selective potassium channel blocker tetrabutylammonium chloride (1 mM), and the non-selective cation ionophore gramicidin (1 µM). Superoxide formation was significantly elevated to a similar degree by all three treatments (by ~60%, n = 23, p < 0.01), whereas hyperpolarization by the K_ATP channel activator Hoe234 (1 µM) significantly decreased superoxide formation. Depolarization also induced an increased tyrosine phosphorylation of several not yet identified proteins (90-110 kDa) and resulted in a significant increase in membrane association of the small G-protein Rac. Accordingly, the Rac inhibitor Clostridium difficile toxin B blocked the effects of depolarization on superoxide formation. The tyrosine kinase inhibitor genistein (30 µM, n = 15) abolished depolarization-induced superoxide formation and also prevented depolarization-induced Rac translocation associated with it. It is concluded that depolarization is an important stimulus of endothelial superoxide production, which involves a tyrosine phosphorylation-dependent translocation of the small G-protein Rac.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

An increased production of vascular superoxide anions causes an impairment of NO-dependent vasodilation and plays an important pathophysiologic role for the initiation and progression of atherosclerosis and hypertension (1). Superoxide anions are also involved in promoting smooth muscle proliferation and vessel restenosis after angioplasty (2, 3). They might, however, also have a physiologic role termed "redox priming" in modulating cytokine receptors (4). Although vascular smooth muscle (5) and adventitial cells (6) are able to produce superoxides, endothelium-derived superoxides were shown to play the predominant pathophysiologic role in cardiovascular disorders such as hyperlipidemia (7).

Recent studies suggest that a neutrophil-type NAD(P)H oxidase is the main source of cellular superoxide generation in endothelial cells (8, 9). This multicomponent enzyme includes the membrane-bound cytochrome b₅₅₈ (p22^phox and gp91^phox) and cytosolic proteins (p47^phox, p67^phox, Rac1/2, and p40^phox) that translocate to the plasma membrane during stimulation to form a catalytically active oxidase (10, 11). The expression of these subunits in endothelial cells has been demonstrated (12, 13) although the regulation of the whole enzyme complex in endothelial cells is not clear. In leukocytes a number of signaling pathways have been shown to be involved in activation of the enzyme. Protein kinase C is known to phosphorylate p47^phox at several serine residues during NAD(P)H oxidase activation, thereby enabling interaction with cytochrome b₅₅₈ (10, 14). Alternatively, protein kinase C-independent activation of NAD(P)H oxidase involves tyrosine phosphorylation-dependent steps (15-18). In both pathways, the translocation of the small GTP-binding protein Rac1/2 could be an integral step because it is required in leukocytes for the full activation of the respiratory burst (19, 20).

In endothelial cells, the mechanisms involved in activation of NAD(P)H oxidase have not yet been studied and may depend on the endothelial stimulus. Recently, Al-Mehdi et al. (21) reported that in isolated perfused lungs the production of reactive oxygen species was significantly increased in the presence of membrane-depolarizing potassium buffer. This may also hold true for other (patho-)physiologic conditions because stimulation of vascular smooth muscle cells frequently goes along with acute changes in membrane potential. Moreover, vascular smooth muscle of hypertensive rats has been shown to be chronically depolarized (22). These acute and chronic changes of smooth muscle membrane potential can be conducted to the endothelial layer via heterologous gap junctions (23, 24). Consistent with the hypothesis of a pressure-induced depolarization, isolated arterioles exposed to high intravascular pressure for 30 min exhibited increased vascular superoxide formation (25).

Little is known, however, about the cellular mechanisms that could lead to superoxide generation after depolarization of the cell membrane. In particular it is not clear whether the above mentioned signaling pathways are activated or whether there is a direct voltage-sensitive activation mechanism of the enzyme itself as suggested by the structural homology between NAD(P)H-dependent oxidoreductases and a shaker potassium channel (26). We therefore first studied whether depolarization induces an activation of endothelial superoxide formation. Second, we analyzed whether this effect is mediated by protein kinase C or tyrosine phosphorylation-dependent pathways. Moreover, we tested whether depolarization would lead to an increased membrane translocation of Rac, which would make a direct voltage-dependent activation of the enzyme unlikely.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Culture-- Human umbilical vein endothelial cells were isolated from freshly obtained human umbilical veins as described (27). The cells were maintained in Medium 199 supplemented with 16% fetal calf serum and 20% endothelial growth medium (Promocell, Heidelberg, Germany). The cells used for experiments were in the 1-3 subpassage. The protein content of the samples was measured by the method of Bradford (28).

Membrane Potential-- For the measurements of the membrane potential the fluorescent dye bis-[1,3-dibutylbarbituric acid]trimethineoxonol (bis-oxonol)¹ was used as an indicator of changes of membrane potential. This dye has been used previously for determination of changes in membrane potential of endothelial cells (29). Human umbilical vein endothelial cells cultured on coverslips were preincubated with bis-oxonol (100 nM) in modified Tyrode's buffer (135 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl₂, 0.49 mM MgCl₂, 0.28 mM NaH₂PO₄, 5.5 mM glucose, 20 mM HEPES) for 30 min to equilibrate the cells with the dye and transferred into a perfusion chamber. The cells were continuously superfused with modified Tyrode's buffer containing bis-oxonol (0.5 ml/min). The fluorescence intensity (excitation, 488 nm; emission, >515 nm) was recorded every 20 s using a confocal microscope (Zeiss LSM 410). The fluorescence intensities were calibrated in terms of changes in membrane potential using the cationic ionophore gramicidin in the presence of different potassium concentrations according to Langheinrich and Daut (30).

Superoxide Production-- Superoxide formation was determined by the cytochrome c assay. Endothelial cells were incubated in a modified Tyrode's buffer containing 40 µM cytochrome c with or without superoxide dismutase (200 units/ml). After 30-60 min the supernatant was removed, and the reduction of cytochrome c was measured at 550 nm (Ultrospec 2000, Amersham Pharmacia Biotech). The superoxide-dependent part of cytochrome c reduction was calculated from the difference in absorbance between samples incubated with or without superoxide dismutase (₅₅₀ nmol/liter = 21.1 mM¹ cm¹). All measurements of superoxide formation were performed in the presence of the NO synthase inhibitor N^G-nitro-L-arginine (30 µM) to prevent modulating effects of NO. In separate experiments, the NADH-induced superoxide production in cell lysates was measured. The cells were suspended in lysis buffer (20 mM potassium phosphate buffer containing 1 mM EDTA, 5 µg/ml aprotinin, 2 µg/ml pepstatin, 2 µg/ml leupeptin, pH 7.0). In 400 µl of phosphate-buffered saline (160 mM NaCl, 2.7 mM KCl, 8 mM Na₂HPO₄, 1.5 mM KH₂PO₄, pH 7.40) containing aliquots of the samples (5 µg protein content) with or without superoxide dismutase (200 units/ml), NADH (100 µM) was added, and the superoxide dismutase-dependent reduction of cytochrome c (40 µM) was determined for 15 min as described.

Tyrosine Phosphorylation-- Endothelial cells were suspended in ice-cold lysis buffer (phosphate-buffered saline, 1% Triton X-100, 1 mM EDTA, 10 µg/ml leupeptin, 10 µg/ml pepstatin, 10 µM phenylmethylsulfonyl fluoride, 2 mM orthovanadate, 10 mM NaF, pH 7.0) and further disrupted by passing them five times through a 29-gauge needle. The lysates were centrifuged (10,000 × g for 5 min), and the supernatants were used for experiments. Proteins were separated via SDS-polyacrylamide gel electrophoresis following standard procedures and transferred onto a nitrocellulose membrane. After incubation with the phosphotyrosine antibody (Upstate Biotechnology, Lake Placid, NY) and a secondary antibody linked to alkaline phosphatase, tyrosine phosphorylation was detected with the nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate system.

Rac Translocation-- Endothelial cells were lysed as described above in ice-cold lysed buffer and centrifuged (500 × g for 5 min) to separate unbroken cells and nuclei. After centrifugation of the supernatant (22,000 × g for 90 min, 4 °C) the membrane fraction was used. After SDS-polyacrylamide gel electrophoresis the proteins were transferred onto polyvinylidene difluoride membranes (Schleicher & Schüll). Equal protein loading was confirmed by reprobing the membrane with anti-actin (data not shown). The amount of membrane-located Rac was determined using a specific antibody against Rac (Upstate Biotechnology). It recognizes human Rac1 and Rac2. The antibody was used at a concentration of 1 µg/ml dissolved in the blocking buffer (20% horse serum, 3% albumin). The secondary antibody (Calbiochem) was linked to horseradish peroxidase. The band intensities for Rac were determined using a videodensitometric system (Bio-Rad).

p22^phox/gp91^phox Expression-- The expression of the NAD(P)H oxidase subunits p22^phox and gp91^phox was determined either by semiquantitative (p22^phox) or standard calibrated competitive (gp91^phox) reverse transcription-polymerase chain reaction. Total RNA was isolated using the single step TRI reagent as described in the product protocol (Life Technologies, Inc.). 1 µg of the total RNA was used for the reverse transcription-polymerase chain reaction, which was performed with the Titan reverse transcription-polymerase chain reaction system (Roche Molecular Biochemicals). The primer sequence used in this study for the p22^phox is described by Jones et al. (13) (forward, GTT TGT GTG CCT GCT GGA GT; reverse, TGG GCG GCT GCT TGA TGG T). As internal control, the expression of glyceraldehyde-3-phosphate dehydrogenase was used. For gp91^phox mRNA expression equal amounts of total RNA were incubated in separate reactions with defined amounts of gp91^phox standard cRNA molecules and subsequently reverse transcribed into cDNA using random hexamer primers and SuperScript II RNase H reverse transcriptase (Life Technologies, Inc.). Afterward, 25% of each reverse transcription reaction was amplified in separate reactions with gp91^phox primers (sense, 5'-GCT GTT CAA TGC TTG TGG CT-3'; antisense, 5'-TCT CCT CAT CAT GGT GCA CA-3').

Materials-- Hoe234 was a gift from Dr. Hansen (Aventis, Frankfurt, Germany). Clostridium difficile toxin B was provided by Dr. M. Essler, University of Munich. Superoxide dismutase was from Roche Molecular Biochemicals. Endothelial growth medium was purchased from PromoCell, sodium orthovanadate was purchased from Alexis Biochemicals, and bis-oxonol was purchased from Molecular Probes. All other substances were from Sigma.

Statistical Analysis-- Statistical comparisons with and without treatment within the same experimental group were performed using the Wilcoxon signed rank test for paired observations. Differences were considered as significant at an error probability of p < 0.05. For descriptive means all results are expressed as means ± S.E.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Measurements of Changes in Endothelial Membrane Potential-- The resting membrane potential of human umbilical vein endothelial cells was found to be 47.1 ± 1.8 mV (n = 23). Treatment by 90 mM potassium buffer, the non-selective potassium channel blocker tetrabutylammonium chloride (TBA, 1 mM), and the non-selective cation ionophore gramicidin (1 µM) elicited nearly the same magnitude of depolarization, whereas the K_ATP channel opener Hoe234 (1 µM) significantly hyperpolarized the cells (Fig. 1). The compounds immediately changed the membrane potential, but a new steady state potential was achieved only after 7-10 min. The latter result was due to the low flow rate in the perfusion chamber; the low flow rate was used to avoid flow- or pressure-dependent changes of the membrane potential. The new steady state potential remained constant over the whole perfusion time with a depolarizing agent.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Measurements of endothelial membrane potential. Endothelial cells were continuously superfused with modified Tyrode's buffer containing the potential-sensitive dye bis-oxonol (100 nM). The changes in fluorescence intensities were recorded after addition of compounds that modulate the membrane potential (n = 3). The fluorescence intensities were calibrated in the presence of different potassium concentrations as described under "Experimental Procedures."

Effects of Changes in Membrane Potential on Endothelial Superoxide Generation-- The effect of membrane depolarization on endothelial superoxide generation is shown in Fig. 2A. Superoxide formation was significantly elevated by high potassium buffer (90 mM), gramicidin (1 µM), and TBA (1 mM) to a similar degree (0.17 ± 0.03 versus 2.9 ± 0.03, 0.27 ± 0.4, and 0.28 ± 0.4 nmol of superoxide/min/mg of protein, respectively; n = 23, p < 0.01). In contrast, hyperpolarization by Hoe234 (1 µM) led to a significant decrease in superoxide formation (Fig. 2B, n = 11, p < 0.01). This decrease was prevented by preincubation of the cells with the K_ATP channel blocker glibenclamide (10 µM, n = 7, p < 0.05).

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Depolarization increases endothelial superoxide formation (A), and hyperpolarization attenuates endothelial superoxide formation (B). A, endothelial cells were depolarized by high potassium buffer (90 mM), the cation ionophore gramicidin (1 µM), and the potassium channel blocker TBA (1 mM), and superoxide formation was measured using the cytochrome c assay (n = 23; **, p < 0.01 versus control). B, hyperpolarization-dependent superoxide formation was determined after addition of the KATP channel opener Hoe234 (1 µM; n = 11; **, p < 0.01 versus control). In some experiments the cells were preincubated with the KATP channel blocker glibenclamide (Glibenclam.; 10 µM; n = 7; #, p < 0.05 versus Hoe234).

Effect of Depolarization on NAD(P)H-dependent Superoxide Formation-- To examine whether a neutrophil-type NAD(P)H oxidase is involved in depolarization-induced superoxide formation, in a separate series of experiments, NADH-induced superoxide formation was examined in lysates of endothelial cells. This method allows the assessment of the amount of assembled and activated enzyme complexes. A depolarization for 1 h by high potassium buffer (90 mM), gramicidin (1 µM), and TBA (1 mM) resulted in a significant increase in NADH-dependent superoxide formation (Fig. 3A; 1.68 ± 0.2 versus 3.22 ± 0.36, 3.46 ± 0.54, and 2.87 ± 0.41 nmol of superoxide/min/mg of protein, respectively; n = 18). Accordingly, in intact cells the NAD(P)H oxidase blocker diphenyleniodonium chloride (DPI, 30 µM) significantly attenuated depolarization-induced superoxide formation by 76.2% (n = 10, p < 0.05, Fig. 3B).

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Depolarization increases NADH-dependent superoxide formation (A), and the NAD(P)H oxidase blocker DPI inhibits depolarization-induced superoxide formation (B). A, after depolarization of endothelial cells by high potassium buffer (90 mM), the cation ionophore gramicidin (1 µM), and the potassium channel blocker TBA (1 mM), the cells were lysed as described under "Experimental Procedures." In aliquots of the lysates (5 µg protein content), NADH (100 µM)-dependent superoxide production was measured using the cytochrome c method (n = 18; *, p < 0.05 versus control; **, p < 0.01 versus control). B, endothelial cells were depolarized (depol.) by high potassium buffer (90 mM), and superoxide formation was measured using the cytochrome c assay in the presence or absence of the flavoprotein inhibitor DPI (n = 10; *, p < 0.05 versus control, #, p < 0.05 versus high potassium buffer). DPI was preincubated for 30 min.

Effects of Depolarization on the Expression of the NAD(P)H Oxidase Subunits-- Fig. 4 demonstrates the expression of the NAD(P)H oxidase subunits p22^phox and gp91^phox in human umbilical vein endothelial cells. Depolarization for 1 and 6 h with high potassium buffer (90 mM) did not affect the expression of these two components of the endothelial NAD(P)H oxidase (n = 3).

View larger version (49K): [in this window] [in a new window]   Fig. 4.   Expression of NAD(P)H oxidase subunits in endothelial cells. Endothelial cells were depolarized by high potassium buffer (90 mM) for 1 or 6 h. The expression of the NAD(P)H oxidase subunits p22phox and gp91phox was analyzed by reverse transcription-polymerase chain reaction as described under "Experimental Procedures" (n = 3). The polymerase chain reaction products were assessed by ethidium bromide staining in 1% agarose gel.

Role of Tyrosine Phosphorylation in Depolarization-induced Superoxide Generation-- We examined whether depolarization-induced activation of endothelial superoxide formation involves tyrosine phosphorylation-dependent steps or activation of protein kinase C. Pretreatment of the cells with the protein kinase C inhibitor staurosporine (100 nM) did not significantly alter depolarization-induced superoxide production (n = 12, data not shown). In contrast, the tyrosine kinase blocker genistein (30 µM, n = 15) blocked the superoxide production elicited by high potassium buffer (90 mM), gramicidin (1 µM), and TBA (1 mM, Fig. 5) without altering the membrane potassium-induced depolarization (+11.4 ± 1.8 mV, n = 3). Daidzein, an inactive analog of genistein, had no effects on superoxide production. We also examined directly whether depolarization modulates cellular tyrosine phosphorylation. Membrane depolarization (30 min) induced an increased tyrosine phosphorylation of several not yet identified 90-110-kDa proteins (Fig. 6).

View larger version (33K): [in this window] [in a new window]   Fig. 5.   The tyrosine kinase blocker genistein inhibits depolarization-induced superoxide formation. Endothelial cells were depolarized by high potassium buffer (90 mM), the cation ionophore gramicidin (1 µM), and the potassium channel blocker TBA (1 mM), and superoxide formation was measured in the presence (n = 23; **, p < 0.01 versus control) or absence of the tyrosine kinase inhibitor genistein (30 µM, n = 15, not significant) using the cytochrome c assay.

View larger version (70K): [in this window] [in a new window]   Fig. 6.   Depolarization-induced tyrosine phosphorylation in endothelial cells. After depolarization by high potassium buffer (90 mM), the cation ionophore gramicidin (1 µM), and the potassium channel blocker TBA (1 mM), endothelial cells were lysed, separated using SDS-polyacrylamide gel electrophoresis, and blotted on nitrocellulose membranes. The membranes were probed with an antibody specific for tyrosine phosphorylation (n = 3). Membrane depolarization induced an increased tyrosine phosphorylation of several not yet identified 90-110-kDa proteins.

Role of Rac in Depolarization-induced Superoxide Production-- Because the membrane association of Rac is necessary for a full activation of NAD(P)H oxidase in leukocytes, we studied whether depolarization leads to an enhanced membrane translocation of Rac. Depolarization for 30 min resulted in a significant increase in membrane-associated Rac (Fig. 7, n = 11). Preincubation with genistein (30 µM) prevented depolarization-induced Rac translocation as well as superoxide formation. Genistein-treated cells did not exhibit in the resting state a significantly reduced membrane association of Rac (Fig. 7). Treatment with the Rac inhibitor C. difficile toxin B (0.5 ng/ml) also completely blocked the increase of endothelial superoxide formation following depolarization (Fig. 8, n = 7; *, p < 0.05 versus control; #, p < 0.05 versus depolarization). C. difficile toxin B did not affect the potassium-induced membrane depolarization (+13.4 ± 5.4 mV, n = 3).

View larger version (29K): [in this window] [in a new window]   Fig. 7.   Depolarization-induced membrane translocation of Rac. Top panel, after membrane depolarization endothelial cells were lysed, and the plasma membrane fraction was separated via SDS-polyacrylamide gel electrophoresis. After blotting on polyvinylidene difluoride membranes, the plasma membrane-located Rac was determined using an antibody specific for Rac (n = 11). Genistein alone did not affect the amount of the membrane-bound Rac (n = 3). The bottom panel shows the densitometric analysis of the Rac staining (*, p < 0.05 versus control; #, < 0.05 versus depolarization (depol.)).

View larger version (34K): [in this window] [in a new window]   Fig. 8.   Inactivation of Rac abolishes depolarization-induced superoxide formation. After depolarization with the cation ionophore gramicidin (1 µM) or the potassium channel blocker TBA (1 mM), superoxide formation was determined using the cytochrome c assay. In separate experiments superoxide production was measured in the presence of the Rac inhibitor C. difficile toxin B (0.5 ng/ml; n = 7; *, p < 0.05 versus control; #, p < 0.05 versus depolarization).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The results show that membrane depolarization induces an enhanced generation of superoxide in endothelial cells. This is because of the activation of a neutrophil-type NAD(P)H oxidase that involves a tyrosine phosphorylation-dependent membrane translocation of the small G-protein Rac. At the same time, these results make a conceivable direct voltage-dependent activation of this enzyme unlikely.

In neutrophils, the membrane translocation of the small G-protein Rac is known to be closely related to an activation of NAD(P)H oxidase and to be essential for full activation of the respiratory burst in phagocytes (19, 20, 31, 32). The translocation occurs in response to different stimuli such as protein kinase C activators or N-formyl-methionyl-leucyl-phenylalanine (15, 32). In vascular cells, Rac also seems to be of crucial importance for the production of reactive oxygen species induced by hypoxia/re-oxygenation or by elevated shear stress (33, 34). In support of the concept of a central role for Rac, depolarization-induced superoxide formation was also abolished following Rac inactivation by toxin B in this study. It is still not clear how Rac affects the activity of NAD(P)H oxidase. Dorseuil et al. (35) recently reported that the translocation of Rac2 but not of the cytosolic NADPH oxidase subunits p47^phox or p67^phox could be inhibited by genistein in chemoattractant-stimulated human neutrophils, which suggests that Rac may not control the assembly of the enzyme but rather a distal step. This remains to be determined in endothelial cells as well.

The translocation of Rac following tumor necrosis factor- treatment in neutrophils was sensitive to the tyrosine kinase inhibitor genistein but not to protein kinase C blockade (15). Activation of the neutrophil NAD(P)H oxidase involves tyrosine phosphorylation although neither the phosphorylation site nor the involvement of tyrosine kinase has yet been identified (15-18). Thannickal et al. (36) reported recently that tyrosine phosphorylation of 103- and 115-kDa proteins mediates hydrogen peroxide production in lung fibroblasts by a membrane-bound NADH oxidase. In agreement with this observation in neutrophils, we found that depolarization-induced translocation of Rac (which was followed by enhanced superoxide formation) was inhibited by the tyrosine kinase inhibitor genistein but not by the protein kinase C blocker staurosporine. This indicates that Rac is activated in a similar way in both cell types. Actions of genistein not related to tyrosine phosphorylation, such as scavenging of superoxides (37), were excluded because daidzein, a functional inactive analog of genistein, had no effect, a result similar to that observed in our previous studies (27). We also found an enhanced tyrosine phosphorylation of several proteins (90-110 kDa) after depolarization, of which the functional significance remains to be elucidated.

In neutrophils, the respiratory burst is followed by a transient depolarization that is clearly a consequence of the electrical gradient induced by an outward flux of electrons (38, 39). Thus, this burst-induced depolarization could be prevented by the NAD(P)H oxidase inhibitor DPI, or this depolarization does not occur in neutrophils from patients suffering from chronic granulomatous disease (40, 41). In our experiments, however, depolarization preceded superoxide formation, suggesting that a change in membrane potential was the initial step in a signal cascade that involved tyrosine phosphorylation and Rac translocation. The identical effects of structurally and mechanistically different compounds used for cell depolarization on superoxide formation imply that our observations were in fact due to the modulating effects of these different compounds on the membrane potential and not due to unspecific side effects. This implication is further supported by the fact that membrane hyperpolarization by opening K_ATP channels attenuated superoxide production. This effect was not caused by an increased scavenging of superoxides by the enhanced production of NO due to hyperpolarization (42), because the effect was observed in the presence of a NO synthase inhibitor. Similar findings with K_ATP channel activation have also been described in neutrophils (43).

The structural homology between NAD(P)H-dependent oxidoreductases and a shaker potassium channel (26) suggests one mechanism by which depolarization could induce a direct voltage-sensitive activation of the enzyme itself. Our results do not support this mechanism because it should have bypassed Rac translocation and the preceding tyrosine phosphorylation. The stimulating effects of depolarization could also not be attributed to an alteration in gene transcription enzyme constituents because a long term depolarization (1 and 6 h) did not alter the expression of the NAD(P)H oxidase subunits p22^phox and gp91^phox. Other investigators, however, observed an enhanced expression of these components in aortas derived from angiotensin II-treated rats, suggesting that an activation of the renin angiotensin system has additional effects as compared with an alteration in membrane potential (44).

In conclusion, membrane depolarization results in increased endothelial superoxide formation, which is probably due to an activation of an NAD(P)H-dependent oxidase. A tyrosine phosphorylation-dependent translocation of the small G-protein Rac is involved in these processes, which may represent a new and interesting target for therapeutic control of endothelial superoxide production. Because many vasoactive compounds go along with transient changes of endothelial membrane potential (45), the mechanisms described here may represent a basic component in the control of vascular superoxide production.

	ACKNOWLEDGEMENTS

We thank D. Goessel and E. Musiol for excellent technical assistance and Paula H. Sohn for helpful discussions.

	FOOTNOTES

^* This study was supported by Deutsche Forschungsgemeinschaft (553/B2). This paper contains part of the doctoral thesis of Matthias Keller to be submitted to the medical faculty of the Ludwig-Maximilians-University Munich.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Institute of Physiology, Ludwig-Maximilians University Munich, Schillerstrasse 44, Munich 80336, Germany. Tel.: 49 89 5996384; Fax: 49 89 5996378; E-mail: sohn@lrz.uni-muenchen.de.

Published, JBC Papers in Press, April 10, 2000, DOI 10.1074/jbc.M000026200

	ABBREVIATIONS

The abbreviations used are: bis-oxonol, bis-[1,3-dibutylbarbituric acid]trimethineoxonol; TBA, tetrabutylammonium chloride; DPI, diphenyleniodonium chloride.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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