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Am J Physiol Cell Physiol 294: C1552-C1565, 2008. First published April 16, 2008; doi:10.1152/ajpcell.00571.2007
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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Volume-sensitive NADPH oxidase activity and taurine efflux in NIH3T3 mouse fibroblasts

Martin Barfred Friis, Katrine Gribel Vorum, and Ian Henry Lambert

Department of Biology, University of Copenhagen, Copenhagen, Denmark

Submitted 3 December 2007 ; accepted in final form 9 April 2008


   ABSTRACT
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 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reactive oxygen species (ROS) are produced in NIH3T3 fibroblasts during hypotonic stress, and H2O2 potentiates the concomitant release of the organic osmolyte taurine (Lambert IH. J Membr Biol 192: 19–32, 2003). The increase in ROS production [5-(and-6)-carboxy-2', 7'-dichlorodihydrofluorescein diacetate fluorescence] is detectable after a reduction in the extracellular osmolarity from 335 mosM (isotonic) to 300 mosM and reaches a maximal value after a reduction to 260 mosM. The swelling-induced ROS production is reduced by the flavoprotein inhibitor diphenylene iodonium chloride (25 µM) but is unaffected by the nitric oxide synthase inhibitor N{omega}-nitro-L-arginine methyl ester, indicating that the volume-sensitive ROS production is NADPH oxidase dependent. NIH3T3 cells express the NADPH oxidase components: p22phox, a NOX4 isotype; p47phox; and p67phox (real-time PCR). Exposure to the Ca2+-mobilizing agonist ATP (10 µM) potentiates the release of taurine but has no effect on ROS production under hypotonic conditions. On the other hand, addition of the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA, 100 nM) or the lipid messenger lysophosphatidic acid (LPA, 10 nM) potentiates the swelling-induced taurine release as well as the ROS production. Overexpression of Rac1 or p47phox or p47phox knockdown [small interfering (si)RNA] had no effect on the swelling-induced ROS production or taurine release. NOX4 knockdown (siRNA) impairs the increase in the ROS production and the concomitant taurine release following osmotic exposure. It is suggested that a NOX4 isotype plus p22phox account for the swelling-induced increase in the ROS production in NIH3T3 cells and that the oxidase activity is potentiated by PKC and LPA but not by Ca2+.

organic osmolytes; NOX4; lysophospholipids; arachidonic acid mobilization; adenosine triphosphate; calcium

REACTIVE OXYGEN SPECIES (ROS), e.g., superoxides, hydroxyl radicals, and H2O2, are produced in a variety of cells upon ligand stimulation, during stress, cell proliferation, and apoptosis (9, 16, 59, 62, 63). Within recent years, it has been shown that ROS are produced also upon osmotic stress in various cell types (35, 51, 71) and that ROS are involved in the swelling-induced activation and inactivation of the volume-sensitive release pathway for the organic osmolyte taurine in, e.g., NIH3T3 fibroblasts (35, 38) and in the activity of the volume-sensitive, outwardly rectifying Cl– channel in liver cells (71). Activation of the volume-sensitive taurine release pathway in NIH3T3 fibroblasts involves specific phospholipase A2 (PLA2)(40, 41), and it appears that the swelling-induced ROS production involves a NADPH oxidase, which is activated at a step downstream to the PLA2 activation (35). Colston and coworkers (13) similarly demonstrated that a NADPH oxidase is activated downstream to PLA2 in cardiac fibroblasts following exposure to exogenous H2O2.

A major source of ROS in mammalian cells is the NADPH oxidase, which was originally identified in neutrophils. This membrane bound, multicomponent enzyme catalyzes the formation of superoxides, which are rapidly converted to H2O2 by a superoxide dismutase. The catalytic core of the NADPH oxidase in neutrophilic cells consists of the two membrane-spanning units, gp91phox plus p22phox, known as flavocytochrome b558. Additional NADPH oxidase components include the monomeric GTP-binding protein Rac plus the two cytosolic, regulatory units, p47phox and p67phox. Apart from neutrophilic cells, many nonphagocytic cells seem to have a basal level of ROS production, even though their ROS production is quantitatively lower compared with neutrophiles (19). This variation in ROS production seems to reflect a difference in the type of NADPH oxidase expressed, i.e., proteins have been identified in nonphagocytic cells that share sequence homology and are functionally related to the phagocytic NADPH oxidase components (reviewed in Refs. 66 and 67). Some of these proteins are known to be expressed in human cardiac fibroblasts (10) and adventitial fibroblasts (52) as well as mouse embryonic fibroblasts (5) and NIH3T3 fibroblasts (9). It is emphasized that fibroblasts in contrast to neutrophils release superoxide radicals mainly to the intracellular compartment (70).

The regulatory subunit of the NADPH oxidase p47phox is a 390 amino acid protein that consists of a NH2 terminal phox homology domain followed by a tandem Src homology domain 3, an arginine- and lysine -rich region (polybasic region), and finally a proline-rich region (1). In the inactive state, p47phox associates with p67phox through an autoinhibitory region (AIR) adjacent to the proline-rich region (1). This aggregation prevents p67phox from interaction with the flavocytochrome b558. Upon activation, several serine residues in the COOH-terminal part of p47phox become phosphorylated (21, 64), mainly by protein kinase C (PKC), protein kinase A (PKA), and, to a lesser extent, by the mitogen-activated protein kinase p42-ERK2 (21, 25). Phosphorylation of the COOH-terminal region of p47phox creates a conformational change in the protein relieving the inhibitory effect on p67phox, so that the entire complex is able to bind to and activate the catalytic flavocytochrome b558 (reviewed in Refs. 28 and 72). Conventional PKC isoforms are regulated by Ca2+ and calmodulin, and Ca2+ release can be triggered through activation of the G protein-coupled purinergic receptors P2Y by ATP or UTP [as reviewed by Erb et al. (22)] or by binding of lysophosphatidic acid (LPA) to the likewise G protein-coupled Edg receptors (Edg2, 4, 7, and LPA4) (4, 6, 30). Hence, NADPH oxidase could be activated by Ca2+ in a process that involves PKC and p47phox. However, some NADPH oxidase complexes seem to be constitutively active, i.e., the p47phox homolog NOXO1 lacks the AIR domain (43) relieving the p67phox homologue NOXA1 (66) so that NOXA1 may associate with the catalytic NOX1 [{approx}pg91phox (43)] and hence ensure the production of ROS in unpertubated cells (67). The pg91phox homolog NOX4 (43) seems to be constitutively active (13, 49, 65), and, for some cells, NOX4 seems not to be regulated by p47phox and p67phox in variance with the other catalytic NOX isoforms (49). Still, little is known about the regulation of NOX4 activity.

Mammalian cells swell as almost perfect osmometers following exposure to a hypotonic solution, whereafter they release KCl and organic osmolytes plus cell water to regain the original cell volume (see Ref. 37). H2O2 potentiates dramatically the release of taurine from NIH3T3 cells following hypotonic exposure, whereas the volume-sensitive taurine release is impaired in the presence of antioxidants or N-acetyl-cysteine, which stimulates the intracellular level of reduced glutathione and thus elimination of ROS via the glutathione peroxidase system (35). Furthermore, inhibition of the NADPH oxidase with diphenylene iodonium chloride (DPI) accelerates the inactivation of the volume-sensitive taurine release pathway in NIH3T3 and Ehrlich Lettre cells (35, 38). It has accordingly been suggested that the NADPH oxidase contributes to the ROS production in NIH3T3 cells under hypotonic conditions and that ROS modulate the volume-sensitive taurine efflux pathway when it is in its active state (35, 38). In a recent paper it was demonstrated that addition of the protein tyrosine phosphatase inhibitor vanadate is accompanied by a potentiation of the swelling-induced ROS production and the concomitant taurine release in NIH3T3 and Ehrlich Lettre cells (38). However, even though the inactivation of the volume-sensitive taurine efflux pathway was strongly impaired in NIH3T3 cells following inhibition of protein tyrosine phosphatase activity, it turned out that the vanadate-induced inactivation of the volume-sensitive taurine efflux pathway was completely lifted in the presence of DPI (38). It was proposed that an increased tyrosine phosphorylation of a NADPH oxidase component potentiated the ROS production and subsequently delayed the inactivation of the volume-sensitive taurine efflux pathway in NIH3T3 cells (38). Because the swelling-induced taurine release from NIH3T3 cells is significantly potentiated following exposure to Ca2+-mobilizing agonists [ATP (35)] and the PKC activator phorbol 12-myristate-13-acetate [PMA (36)], we initiated the present work to characterize the catalytic and putative NADPH oxidase components that mediate the swelling-induced increase in the ROS generation in NIH3T3 cells and their relationship with the swelling-induced taurine efflux.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chemicals. Antibiotics (penicillin and streptomycin), DMEM (GIBCO; high glucose, L-glutamine), fetal calf serum (GIBCO), pluronic F-127 (20% in DMSO), and trypsin (10x; GIBCO) were from Invitrogen. [3H]taurine and [3H]arachidonic acid were from Amersham; 5-(and-6)-carboxy-2', 7'-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA) and fura-2 AM were from Molecular Probes (Leiden, The Netherlands). All other compounds were from Sigma-Aldrich. The following stock solutions were prepared: DPI (10 mM, solvent ethanol), carboxy-H2DCFDA (50 mM, solvent DMSO), fura-2 AM (5 mM, solvent DMSO), N{omega}-nitro-L-arginine methyl ester (L-NAME, 100 mM, solvent DMSO), PMA (400 µM, solvent ethanol), tamoxifen (10 mM, solvent ethanol), clofilium (25 mM, solvent DMSO), 4-(2-aminoethyl)benzenesulfonyl fluoride [AEBSF, 100 mM, solvent deionized distilled H2O (ddH2O)], LPA (5 mM, solvent ethanol), wortmannin (50 µM, solvent DMSO), ATP (10 mM, solvent water), chelerythrine (1 mM, solvent water), and Gö-6976 (2.5 mM, solvent DMSO).

Inorganic media. The phosphate-buffered saline (PBS) contained (in mM) 137 NaCl, 2.6 KCl, 6.5 Na2HPO4, and 1.5 KH2PO4. Isosmotic NaCl medium (335 mosM) contained (in mM) 143 NaCl, 5 KCl, 1 Na2HPO4, 1 CaCl2, 0.1 MgSO4, and 10 N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid. Hyposmotic NaCl medium (200 mosM) was obtained by reduction of the NaCl in the isosmotic solutions to 95 mM, with the other components remaining unchanged. pH was in all solutions adjusted at 7.40.

Cell cultures. NIH3T3 mouse fibroblasts (wild type and RacV12A3) and Ehrlich Lettre cells were grown in 75-cm2 culture flasks as monolayer cultures in DMEM (NIH3T3 cells) or RPMI-1640 (Ehrlich Lettre) containing heat-inactivated fetal bovine serum (10%) and antibiotics. Cell cultures were kept at 37°C/5% CO2/100% humidity and split every 3–4 days using 0.5% trypsin in PBS to detach the cell lines. The RacV12A3 cell line was originally established by Kristine Beisner, Department of Molecular Biology, University of Copenhagen (56).

Estimation of ROS production. Cells were grown to 80% confluence on 10 x 50-mm coverslips that were pretreated with HCl and EtOH to reduce autofluorescence. All experiments were performed set wise i.e., two coverslips were grown in the same well in a four-well polyethylene dish, and during experiments, one coverslip served as a control for the other. Loading DMEM containing the ROS-sensitive probe (carboxy-H2DCFDA) and 20% pluronic F-127 was prepared by adding equal amounts of the probe and pluronic F-127 to 1 ml serum-free DMEM and sonicated for 20 s. Serum-free DMEM was added to a final concentration of 20 µM of the ROS probe. The coverslips were washed twice in PBS, and the cells were incubated in loading DMEM 2 h before initiation of the experiments. At the beginning of each experiment, the coverslip was washed in isotonic solution and placed in a cuvette containing experimental solution. ROS production was measured on a PTI Ratio Master spectrophotometer, and the experimental solution in the cuvette was continuously stirred by use of a Teflon-coated magnet, driven by a motor attached below the cuvette house. The excitation and emission wavelengths were 490 nm and 515 nm, respectively. Data were collected every 2 s for 200 s. The first 20 s of the curve was used to calculate a slope representing the initial rate of ROS production.

Estimation of intracellular Ca2+ mobilization. Cells were grown to a confluence of 80% on round coverslips (diameter = 15 mm), which were pretreated with HCl and EtOH to reduce autofluorescence. Loading medium was prepared by mixing 4 µl Ca2+-sensitive fura-2 AM probe with 5 µl pluronic F-127 and 1 ml isotonic solution. Loading medium was sonicated for 20 s and isotonic solution was added, giving a final concentration of the Ca2+ probe of 10 µM. Cells were washed once in isotonic solution and transferred to loading media 20 min before initiation of the experiments performed. At the beginning of each experiment, the coverslip was washed once in isotonic solution and mounted in a perfusion system, i.e., between two plastic rings with in- and outlet connections to the top ring to allow change in solution during experiments. Fura-2 fluorescence was measured on a PTI Ratiometer connected to a Nikon Diaphot TMD microscope with a Nikon Fluor 20 objective (0.75 numerical objective). At the beginning of each experiment, the cells were perfused with isotonic solution until a stabile trace was obtained. Experimental solutions were changed through the inlet in the top plastic ring. Emission was measured at 515 nm after excitation at 340 nm and 380 nm. Data are presented as the emission ratio between counts obtained at 340 nm and 380 nm.

Estimation of the rate constant for taurine efflux and the fractional arachidonic acid release. Taurine efflux and release of arachidonic acid were estimated at room temperature on NIH3T3 grown to 80% confluence in six-well polyethylene dishes (9.6 cm2/well) and loaded with growth media containing [3H]taurine (2 µCi/well, 2 h) or [3H]arachidonic acid (3 µCi/well, 24 h), respectively. Following the loading period, cells were washed four times with isosmotic media to remove excess extracellular 3H-labeled compound. The efflux and release experiments were initiated by aspiration of the medium followed by addition of 1 ml of experimental solution. In the case of arachidonic acid release experiments, 0.2% BSA was added to the experimental medium to trap released arachidonic acid. The cells were left for 2 min whereafter the entire medium was transferred to scintillation vial and rapidly substituted by 1 ml fresh medium. This procedure was repeated for 20 to 30 min with a shift from isotonicity to hypotonicity at time 6 or 8 min. At the end of the experiment, the cells were lysed by addition of 1 ml NaOH (0.5 mM, 1 h). The total pool of labeled compound was estimated as the sum of 3H activity (β-scintillation counting, Ultima Gold) in all the efflux samples, the NaOH lysate plus two final well wash outs with ddH2O. The release of taurine under the present experimental setup is assumed to follow a monoexponential equation, and the rate constant (k: min–1) for the taurine efflux at a given time point can accordingly be estimated as the negative slope between the time point and the proceeding time point on a graph were the natural logarithm to the fraction of 3H-labeled taurine remaining in the cells is plotted versus time (for details, see, e.g., Ref. 38). Arachidonic acid mobilization reflects the activity of several PLA2 isoforms (41), and the arachidonic acid release at a given time point was consequently estimated as the total fraction released (in %).

Estimation of the cellular amino acid content. The amino acid content was estimated by o-phthaldialdehyde derivatization followed by reversed phase high-pressure liquid chromatography (HPLC, Gilson: 322-Pump, 234-Autoinjector, 155-UV/VIS) (see Ref. 73). Briefly, cells were lysed and deproteinized by addition of 1.2 ml 4% sulfosalisylic acid, and the cell homogenate was subsequently sonicated on ice. Aliquots were denatured with NaOH and used for estimation of the protein content [Lowry procedure (47), BSA as protein standard]. The residual homogenate was centrifuged (20,000 g, 10 min), and the supernatant was filtered (Milex-GV, 0.45 µm) before separation of amino acids on a Nucleosil column (Macherey-Nagel, C18, 250/4, 5 µM) using gradient elution with acetonitrile/phosphate buffer (12.5 mM, pH 7.2) and ultraviolet absorption (330 nm). Amino acid standards (0.1 mM) were used for quantitative estimation of the content of amino acids. The cellular amino acid content (µmol/mg protein) was estimated from the amino acid and protein content.

Detection of NADPH oxidase components and LPA receptors in NIH3T3 fibroblasts. mRNA was isolated from subconfluent NIH3T3 fibroblast cultures using RNeasy Protect mini kit (catalog no. 74124, Qiagen) according to the manufacturer's description. RT-PCR was performed using a JumpStart RED HT RT-PCR kit from Sigma (catalog no. J 3520). Primers for NOXO1, p47phox, NOXA1, p67phox, NOX1, gp91phox, NOX3, NOX4, Edg2, Edg4, Edg7, and LPA4 were designed in Primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) on the basis of sequences obtained from GenBank. The following mouse-specific primers were generated: NOXO1: forward, 5'-ATA GTC ATG GCA AGC CCA AG-3'; reverse, 5'-GGGT TAC AAA GAA GCC GTG AA-3'; forward, 5'-TCC CTG TGT ACA GCC TTT CC-3'; reverse, 5'-TCC TGT TTT CTT GGT GAG GT-3'; p47phox: forward, 5'-CAC CGA GAT CTA CGA GTT CCA-3'; reverse, 5'-TGT CAA GGG GCT CCA GAT AG-3'; forward, 5'-TCC TGG TTA AGT GGC AGG AC-3'; reverse, 5'-ATG ACC TCA ATG GCT TCA CC-3'; NOXA1: forward, 5'-CCC AGG CGA TAC CTA AAA CA -3'; reverse, 5'-CAC AGA ACA TCC ACC GTG TC-3'; reverse, 5'-AAG CAT GGC TTC CAC ATA GG-3'; p67phox: forward, 5'-GCC ACA GTC ATG TTC AAT GG-3'; reverse, 5'-ACA AAA GCC TTC GGG AAA AT-3'; NOX1: forward, 5'-GGG ATG ACC ATA AGG GGA GT-3'; reverse, 5'-CCA GCC AGT GAG GAA GAG AC-3'; forward, 5'-AGC CAT TGG ATC ACA ACC TC-3'; reverse, 5'-ACA GAG GAG AGC TTG GGT GA-3'; gp91phox: forward, 5'-CTT TCT CAG GGG TTC CAG TG-3'; reverse, 5'-TCT TCC AAA CTC TCC GCA GT-3'; NOX3: forward, 5'-TGC CTT ATG CCC TGT ACC TC-3'; reverse, 5'-TTC ACT CAT CCG TGT TTC CA-3'; NOX4: forward, 5'-GCA TCT GCA TCT GTC CTG AA-3'; reverse, 5'-TGG AAC TTG GGT TCT TCC AG-3'; forward, 5'-CCA GAA TGA GGA TCC CAG AA-3'; reverse, 5'-AAA ACC CTC GAG GCA AAG AT-3'; p22phox: forward, 5'-TGG ACG TTT CAC ACA GTG GT-3'; reverse, 5'-TAG GCT CAA TGG GAG TCC AC-3'; forward, 5'-AAA GAG GAA AAA GGG GTC CA-3'; reverse, 5'-TAG GCT CAA TGG GAG TCC AC-3'; reverse 5'-ACC GAC AAC AGG AAG TGG AG-3'; Edg7: forward, 5'-AGG GCT CCC ATG AAG CTA AA-3'; reverse, 5'-AGC CGT TTT TAT TGC ACA CC-3'; Edg4: forward, 5'-CAC TGC CTC TGT GAC TTG GA-3'; reverse, 5'-ACC ACT GCA TTG ACC AGT GA-3'; Edg2: forward, 5'-ACA CCA GCC TGA CAG CTT CT-3'; reverse, 5'-CTG TAG AGG GGT GCC ATG TT-3'; LPA4: forward, 5'-ACC CTG GCC CTC TCT GAT TT-3'; reverse, 5'-CGA TCG GAA GGG ATA GAC AA-3'. The RT-PCR reactions were performed with the following settings: reverse transcription at 50°C for 30 min, hot start and denaturing at 94°C for 3 min, denaturing at 94° for 15 s, annealing at 50–65°C for 15 s, and extension at 72°C for 1 min. Steps were repeated 40 times starting with denaturing at 94°C for 15 s. Final extension was at 72°C for 10 min followed by cooling to 4°C. To avoid amplification of contaminating genomic DNA, PCR was performed on RNA samples treated with DNase. Furthermore, a control PCR amplification of some of the expressed isoforms was performed after treating the sample with RNase, to make sure the observed bands were amplified from RNA. PCR fragments were verified by sequencing (MWG Biotech).

Construction of p47phox full-length clone. Full-length p47phox was PCR amplified from human embryonic kidney (HEK) cell cDNA, kindly provided by Dr. Stine Falsig Pedersen, Department of Molecular biology, University of Copenhagen, using the following primers: p47phox: forward, 5'-GAA TCC TGG GGG ACA CCT TCA TTC GCC-3'; reverse, 5'-GGT ACC TCA CAC GGA CGT CAG CTT CC-3'. This incorporates an EcoRI site to the 5'-prime end and a KpnI site to the 3'-prime end. The PCR fragment was isolated from a 1% agarose gel using E.Z.N.A gel extraction kit (catalog no. D2500-01, Omega) according to the manufacturer's instructions. The fragment was cloned into a pCMV-Myc and a pCMV-HA vector (catalog no. 631604, Clontech) at the EcoRI and KpnI sites. The constructs were transformed into DH5{alpha} competent cells and subsequently isolated using E.Z.N.A Fastfilter Midi Kit (catalog no. D690503, Omega). NIH3T3 cells were grown to 50% confluence in six-well dishes before transfection. Transient transfection was performed using Lipofectamine2000 (catalog no. 11668-027, Invitrogen) according to the manufacturer's instructions. Briefly, 1 µg of plasmid was mixed with 5 µl Lipofectamine2000 and 200 µl serum-free DMEM without penicillin/streptomycin and left at room temperature for 30 min. The cells in one well were incubated in 2 ml serum-free DMEM without penicillin/streptomycin, and 200 µl transfection solution was added. The transfection medium was substituted with 2 ml of DMEM containing serum 2.5 h later. Mock transfection was performed using a plasmid containing myc-tagged CAP350 kindly provided by Dr. Lotte B. Pedersen, Department of Molecular Biology, University of Copenhagen. Cells were used 24 h after transfection. Expression of myc-p47phox was verified by Western blot analysis.

Small interfering RNA silencing of p47phox. MWG online small interfering (si)RNA design tool (http://www.mwg-biotech.com) was used to design siRNA oligos from the p47phox coding sequence. Cells were transfected using Lipofectamine2000 as described above, 150 nM of p47phox siRNA (5'-UAA CGU AGC UGA CAU CAC A-3'), or control siRNA, containing approximately the same GC content. The latter was kindly provided by Dr. Linda Schneider, Department of Molecular Biology, University of Copenhagen. Cells were used for experiments 24 h after transfection. Silencing was confirmed by Western blot analysis.

siRNA silencing of NOX4. NOX4 siRNA was purchased at Dharmacon as ON-TARGETplus SMARTpool (L-058509-00-0005, Dharmacon). According to the manufacturer's instructions, a final concentration of 100 nM siRNA was used for transfection of cells grown in four-well dishes (described above). Briefly, 3 µl FuGENE (catalog no. 1814443, Roche) was mixed gently with 92 µl serum-free DMEM. NOX4 SMARTpool siRNA (15 µl, 20 µM) was added and mixed gently. The transfection solution was left at room temperature for 20 min. Meanwhile, the cells were washed and 3 ml serum-free DMEM was added. The transfection solution was added drop wise to the cells. After 3 h incubation, 300 µl serum was added to the cells. The cells were used for experiments 24 h after transfection. As mentioned above a control siRNA provided by Dr. Linda Schneider was used to rule out any nonspecific effects of siRNA transfection.

Verification of protein expression and silencing by Western blot analysis. Proteins from whole cell lysates from cells overexpressing myc-p47phox or -p47phox knockdown were separated on a 10% acrylamide gel by SDS-PAGE and transferred to nitrocellulose membranes. Mouse monoclonal anti-myc antibody (Clontech) was used to identify myc-p47phox and goat polyclonal anti-p47phox antibody (catalog no. sc-7660, Santa Cruz) was used to identify cellular p47phox. Alkaline phosphatase-conjugated goat anti-mouse and donkey anti-goat secondary antibodies were from Jackson ImmunoResearch. Membranes were developed using BCIP/NBT phosphatase substrate (Kem-En-Tech).

Statistical analysis. Data are presented either as individual experiments that are representative of at least three independent sets of experiments or as means ± SE. Statistical significance was estimated by paired and unpaired Student's t-test. For all statistical evaluations, P < 0.05 was taken to indicate a significant difference.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Catalytic and regulatory NADPH oxidase subunits expressed in NIH3T3 fibroblasts. It has previously been shown that fibroblasts originating from different tissues and species express subunit homologs to the neutrophilic NADPH oxidase (5, 9, 10, 52). To determine the oxidase subunits expressed in NIH3T3 fibroblasts, we performed RT-PCR, using primers directed against known subunits of the NADPH oxidase identified in the mouse genome. As shown in Fig. 1, NIH3T3 fibroblasts express the regulatory units p67phox and p47phox as well as p22phox. Moreover, we amplified a band of the catalytic subunit, NOX4 (~800 bp), and the regulatory unit, NOXO1 (~1,050 bp) (Fig. 1). The predicted size of the two fragments was 650 bp, and it is plausible that the difference in size between the calculated value and the amplified size reflects the expression of NOX4 and NOXO1 splice variants. The expression of subunits is in agreement with data obtained from the mouse embryonic fibroblasts (MEF) (5). As with the MEF cells (5), we found no expression of the catalytic subunit gp91phox, normally found in neutrophils and some nonphagocytic cells. Furthermore, NIH3T3 cells did not express NOXA1 or the catalytic subunits NOX1, NOX3, DUOX1, and DUOX 2 (Fig. 1).


Figure 1
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  		Fig. 1. Expression of NADPH oxidase components in NIH3T3 cells. mRNA was isolated from NIH3T3 fibroblasts, and RT-PCR was performed using mouse-specific primers as described in MATERIALS AND METHODS. Lane M represents the band sizes of the DNA ladder used for the electrophoresis.

 
NADPH oxidase activity is increased as a function of the magnitude of osmotic stress. It was previously demonstrated that the ROS production in NIH3T3 cells is increased following a 33% reduction in the extracellular osmolarity (35). This is confirmed in Fig. 2A, where it is shown that the initial increase in the fluorescence of the ROS-sensitive probe carboxy-H2DCFDA is larger in cells exposed to hypotonic medium compared with cells exposed to isotonic medium. Using the increase in fluorescence within the initial 20 s as an estimate of the ROS production, it was estimated that reducing the extracellular osmolarity by 35 mosM and 95 mosM led to a 44% (P < 0.05) and 84% (P < 0.01) increase in the ROS production, respectively (Fig. 2B). Reducing the extracellular osmolarity below 220 mosM did not seem to amplify the ROS production further, indicating that a maximal rate of ROS production is achieved at 220 mosM. Assuming that NIH3T3 cells respond as perfect osmometers and that 10% of the cell volume is osmotically inactive, it is estimated that the exposure to 220 mosM corresponds to a 1.5-fold increase in cell volume.


Figure 2
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  		Fig. 2. Swelling-induced reactive oxygen species (ROS) production in NIH3T3 cells involves the NADPH oxidase. Cells grown to 80% confluence on coverslips were loaded for 1–2 h with the ROS-sensitive probe, 5-(and-6)-carboxy-2', 7'-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA; 20 µM). The coverslips were washed in isotonic solution (335 mosM) and transferred to isotonic NaCl (335 mosM) or hypotonic NaCl (320-200 mosM), and fluorescence emission followed with time. A: typical ROS trace for cells exposed to an isotonic or a hypotonic solution (220 mosM). B: relative increase in ROS production after exposure to hypotonic solutions (320-200 mosM). The ROS production was estimated as the initial slope of the ROS trace (0–20 s) for each hypotonic condition and given relative to the isotonic values. C: ROS production was estimated in the presence and absence of diphenylene iodonium chloride (DPI; preincubated with 25 µM DPI, 1 h) in hypotonic media (320-200 mosM). The ROS production was in each case estimated as the initial slope (0–20 s) of the ROS trace. At each tonicity, the ROS production in the presence of DPI is presented relative to the production in the absence of DPI (control). D: ROS production was estimated in the presence and absence of tamoxifen (Tamox; 10 µM), clofilium (100 µM), 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF; 0.5 mM, preincubated for 5 min), and N{omega}-nitro-L-arginine methyl ester (L-NAME; 1 mM, preincubated for 2 h) in isotonic and hypotonic media (200 mosM). The ROS production was in each case estimated as the initial slope (0–20 s) of the ROS trace. The relative increase in ROS production was calculated for control and experiments with inhibitors and is presented relative to the increase in control cells. All data represent at least 3 separate sets of experiments, and data in B, C, and D are presented as mean relative values ± SE. *P < 0.05; **P < 0.01.

 
To determine the source of ROS production during increasing hypotonic stress, we estimated the ROS production under hypotonic conditions in the presence of DPI, which is a specific flavoprotein inhibitor (18), and L-NAME, which blocks the nitric oxide synthase (61). As seen from Fig. 2C, DPI (25 µM) reduced the ROS production by 20% (P < 0.05) when cells were exposed to 280 mosM. Maximal DPI-induced inhibition in the ROS production was measured at 200 mosM, where 53% reduction was obtained (P < 0.01). It is noticed that DPI reduced ROS production by 20%, although not significantly, when cells were exposed to a solution with an osmolarity of 320 and 300 mosM (Fig. 2C). Because the fractional DPI-induced decrease in ROS production approximately equals the fractional increase at 260 mosM or lower tonicities, it is likely that the NADPH oxidase is the main source of ROS during hypotonic stress. In the following we have used 200 mosM as the working hypotonic solution because of the significant increase in ROS production and the significant contribution from the NADPH oxidase. Exposing NIH3T3 cells to L-NAME (1 mM) had no significant effect on the increase in the ROS production following hypotonic exposure (Fig. 2D), eliminating contribution from the nitric oxide synthase to the ROS production. Furthermore, exposing NIH3T3 cells to the NADPH oxidase inhibitor AEBSF, which is structurally distinct from DPI and interferes with the assembly/activation of the NADPH oxidase complex (15), also had no effect on the increase in the ROS production under hypotonic conditions (Fig. 2D). Hence, assembly of NADPH oxidase components is not a prerequisite for the swelling-induced increase in oxidase activity in NIH3T3 cells. In this context it is noted that ROS is produced by NIH3T3 cells even under isotonic conditions (Fig. 2A) and even in the presence of DPI (data not shown), indicating that other DPI-insensitive ROS-producing systems are constitutively active in NIH3T3 cells.

Osmotic cell swelling is accompanied by activation of volume-sensitive, separate Cl– and K+ channels, and because the increase in the Cl– conductance is often larger than the increase in the K+ conductance, this leads to a depolarization of the plasma membrane in, e.g., Ehrlich ascites tumor cells (39). Ahluwalia (2) has indicated that Cl– current activated by cell swelling in human neutrophiles counteracts a depolarization induced by the NADPH oxidase. Furthermore, DPI has been reported to inhibit K+ channels in, e.g., isolated pulmonary smooth muscle cells (74) and type I cells from neonatal rat carotid body (76). To test whether inhibition of the volume-sensitive K+ and Cl– channels would have an impact on the ROS production following hypotonic exposure, we estimated the change in fluorescence with time in NIH3T3 cells after hypotonic exposure in the absence and presence of tamoxifen and clofilium, which are recognized as potent blockers of the volume-sensitive Cl– and K+ channels, respectively (33, 58). From Fig. 2D it is seen that the inhibitors do not affect the increase in ROS following osmotic exposure, indicating that inhibition of the volume-sensitive efflux pathways for K+ and Cl–, i.e., interference with ion channel activity and hence the membrane potential, does not affect ROS production in hypotonically exposed NIH3T3 cells.

Role of the NADPH oxidase in Ca2+-, PKC-, and LPA-mediated potentiation of volume-sensitive taurine efflux. The swelling-induced taurine efflux is potentiated in the presence of H2O2 and attenuated in the presence of antioxidants as well as DPI in NIH3T3 fibroblasts, and it has been suggested that the swelling-induced taurine release is modulated by ROS, produced by a NADPH oxidase (35, 37). The swelling-induced release of taurine is in various cell types potentiated also by the PKC activator PMA, the lipid messenger LPA, and ATP (24, 29, 42, 50). Because LPA and ATP mobilize intracellular calcium (54, 55) and some isoforms of the NADPH oxidase are known to be regulated by Ca2+-sensitive PKC isoforms (25), we tested whether the LPA- and ATP-mediated potentiation of the volume-sensitive taurine efflux could reflect an increased NADPH oxidase activity. The taurine efflux was estimated under hypotonic conditions in the absence or presence of PMA, ATP, or LPA. Figure 3, A and B, show that addition of LPA (10 nM), PMA (50 nM), and ATP (10 µM) to the experimental solution potentiated the release of taurine from NIH3T3 cells during hypotonic stress. Using the maximal rate constant for the swelling-induced taurine efflux, obtained within 4 min following hypotonic exposure (Fig. 3A), as an estimation of the swelling-induced taurine release, it was estimated that PMA, ATP, and LPA increased the taurine efflux by 95% (P < 0.01), 27% (P < 0.05), and 45% (P < 0.001), respectively (Fig. 3B). Incubation of the NIH3T3 cells with DPI before the hypotonic exposure led to a significant reduction in the taurine release by 44% (P < 0.01), 51% (P < 0.01), 35% (P < 0.05), and 72% (P < 0.01) in control cells, PMA-, ATP-, and LPA-treated cells, respectively (Fig. 3B). It is noticed that the PMA- and LPA-induced potentiation of the volume-sensitive taurine efflux was completely impaired by DPI, i.e., there was no significant difference in the maximal rate constant for taurine efflux in the absence or presence of PMA or LPA when cells were preincubated with DPI (Fig. 3B). The DPI-induced reduction in the swelling-induced taurine efflux seen in cells exposed to ATP equaled the reduction seen in cells exposed to hypotonic solution alone, which is taken to indicate that the ATP-induced potentiation is unaffected by NADPH oxidase inhibition. Repeating the experiments in the presence of L-NAME (1 mM) instead of DPI indicated that L-NAME had no effect on the rate constant of the swelling induced taurine efflux (data not shown). Hence, ROS generated by the NADPH oxidase seem to modulate the swelling-induced taurine efflux in NIH3T3 cells. Furthermore, PMA and LPA seem to interfere at a step upstream to the NADPH oxidase since the potentiation of the taurine efflux is abolished in the presence of DPI (Fig. 3B). Alternatively, the LPA- and PMA-induced signaling pathway, which leads to potentiation of the swelling-induced taurine efflux, could be tightly regulated by ROS. ATP, on the other hand, still potentiates the taurine efflux in the presence of DPI, which indicates that the ATP-mediated potentiation of the taurine efflux does not involve the NADPH oxidase.


Figure 3
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  		Fig. 3. Effect of phorbol 12-myristate 13-acetate (PMA), ATP, and lysophosphatidic acid (LPA) on the swelling-induced taurine release and ROS production in NIH3T3 cells. A: NIH3T3 cells grown to 80% confluence in six-well dishes were loaded with [3H]taurine for 2 h. The cells were subsequently washed, and the efflux experiments were performed in isotonic NaCl medium (335 mosM) with a shift to hypotonic NaCl medium (200 mosM) at time 8 min as indicated by the arrow. Release was followed in control cells ({circ}), cells exposed to 10 nM LPA ({triangleup}), 25 µM DPI (bullet), or DPI plus 10 nM LPA ({blacktriangleup}). Rate constants were estimated as indicated in MATERIALS AND METHODS and plotted as a function of the time. B and C: maximal rate constants for the taurine efflux, obtained 4 min following hypotonic exposure, were estimated in control cells and cells exposed to PMA (50 nM), ATP (10 µM), or LPA (10 nM). The efflux experiments were performed in the absence or presence of 25 µM DPI (B), 0.5 µM Gö-6976 (C), or 10 µM chelerythrine (C). Cells were preincubated with inhibitors for 30 min, and all inhibitors were present throughout the whole efflux experiment. Data are presented as mean absolute values ± SE of at least 3 experiments. D: ROS experiments were performed and ROS production was estimated as outlined in Fig. 2. ROS production was estimated in the absence or presence of ATP (10 µM), LPA (10/100 nM), and PMA (100 nM) under hypotonic conditions (200 mosM). E: ROS was estimated on LPA (10 nM)- or PMA (100 nM)-stimulated cells under hypotonic conditions in the absence or presence of 10 µM chelerythrine. Values are given relative to cells not treated with the kinase inhibitor. Data are given relative to the value in hypotonic control cells and represent means ± SE of at least 3 experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

 
It is well known that some isoforms of the NADPH oxidase are regulated by PKC and that mutation of putative PKC motifs on the regulatory subunit p47phox reduces the swelling-induced release of ions in, e.g., HeLa cells (71). To test the impact of PKC activity, we estimated the taurine efflux and the ROS production under hypotonic conditions in the presence of chelerythrine, a pan-PKC inhibitor, or Gö-6976, an inhibitor of conventional PKC isoforms. Figure 3C shows that preincubation with chelerythrine significantly reduced the taurine efflux in the absence (P < 0.001) and presence of PMA (P < 0.05) or LPA (P < 0.01). However, chelerythrine had no effect on the ATP-induced potentiation of the taurine efflux (Fig. 3C). Moreover, Gö-6976 had no significant effect on the taurine efflux in the absence or presence of PMA, LPA, or ATP (Fig. 3C). Thus, PKC is involved in the PMA- and LPA-induced potentiation of the swelling-induced taurine efflux in NIH3T3 cells, although the PKC in question is not a conventional PKC isoform. To test whether the signaling events, activated by LPA, PMA, and ATP and leading to potentiation of the volume-sensitive taurine release from NIH3T3 cells, involved the NADPH oxidase, we estimated the ROS production during hypotonic stress (200 mosM) in the absence or presence of LPA, PMA, and ATP. Figure 3D shows that both LPA (10 nM or 100 nM) and PMA (50 nM) potentiated the ROS production under the hypotonic conditions significantly, i.e., PMA and LPA increased the ROS production 2.5- and 1.75-fold, respectively, compared with the cells exposed to hypotonic solution alone. ATP (10 µM), on the other hand, had no effect on the ROS production under hypotonic conditions (Fig. 3D). The data in Fig. 3 indicate that the PMA-/PKC- and the LPA-mediated, but not the ATP-mediated, potentiation of the swelling-induced taurine release from NIH3T3 cells could reflect a concomitant potentiation of the ROS production. To see whether the PKC-mediated potentiation of the taurine efflux reflects a PKC-mediated effect through the NADPH oxidase complex, as appears to be the case when cells are stimulated with either PMA or LPA, we estimated the effect of chelerythrine and DPI on ROS production following hypotonic stress. Chelerythrine had no effect on the ROS production when cells were exposed to hypotonic media alone (data not shown). However, repeating the experiments under hypotonic conditions (200 mosM) in the presence of PMA or LPA showed that chelerythrine reduced the PMA- and LPA-mediated potentiation of the ROS production by 40–50% (Fig. 3E). Thus, although chelerythrine is not able to affect the basal ROS production under hypotonic conditions, it impairs the PMA- and LPA-mediated potentiation of the ROS production, i.e., the NADPH oxidase activity under standard hypotonic conditions is not PKC dependent, but PKC amplifies its activity.

Exogenous ATP triggers Ca2+ and arachidonic acid mobilization under hypotonic conditions. Mobilization of arachidonic acid by various PLA2 isoforms plays a role in the initiation of the swelling-induced taurine release in, e.g., Ehrlich ascites tumor cells, NIH3T3 fibroblasts, and Ehrlich Lettre cells (38, 41, 57, 69), and because ATP is known to release Ca2+ from intracellular stores, stimulation with ATP could lead to an activation of Ca2+-dependent PLA2 isoforms, arachidonic acid mobilization, and thus account for a NADPH oxidase-independent potentiation of the volume-sensitive taurine release. From Fig. 4A it is seen that ATP (20 µM) induces an immediate, transient increase in the cellular Ca2+ concentration in NIH3T3 cells under hypotonic conditions. The Ca2+ transient is more pronounced in the absence than in the presence of the Ca2+ chelator EGTA (data not shown), indicating that ATP mobilizes Ca2+ from intracellular stores in NIH3T3 cells as well as the extracellular compartment. ATP (20 µM) increases the release of [3H]arachidonic acid under hypotonic conditions from preloaded NIH3T3 (Fig. 4B), and using the fraction of the total [3H]arachidonic acid released 22 min after hypotonic exposure, it was estimated that the release of arachidonic acid under hypotonic conditions was increased by 25 ± 6% in the presence of ATP (n = 3, P = 0.015). Arachidonic acid had no effect on the swelling-induced ROS production (data not shown). Thus, the ATP-induced potentiation of the taurine release under hypotonic conditions could reflect an increased PLA2 activity, i.e., an increased mobilization of arachidonic acid for downstream signaling.


Figure 4
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  		Fig. 4. ATP-induced Ca2+ and arachidonic acid mobilization in NIH3T3 cells. A: NIH3T3 cells grown to 80% confluence on coverslips were loaded for at least 20 min with fura-2 AM before the experiment. The coverslips were washed in isotonic NaCl (335 mosM), placed on the experimental perfusion setup, and perfused with isotonic NaCl medium. Cellular Ca2+ concentration was followed with time under isotonic and hypotonic (200 mosM) conditions as indicated by the bars. Cells were exposed to ATP (10 µM) under hypotonic conditions as indicated by the arrow. Traces are representative of at least 3 sets of independent experiments. B: NIH3T3 cells grown to 80% confluence were loaded with [3H]arachidonic acid for 24 h in growth media. The cells were washed, and the efflux experiments were subsequently performed in hypotonic NaCl Ringer solution (200 mosM) containing 1% BSA. The arachidonic release was estimated as the fraction of the total pool in the absence ({circ}) or presence (bullet) of ATP (10 µM) and is presented as a function of time. Data are given as means ± SE and represent 3 sets of experiments.

 
LPA-induced ROS production and taurine release under hypotonic conditions are Ca2+ independent. LPA is known to be a potent Ca2+ mobilizing agonist in various cell types (31, 68), and four known LPA receptors have been cloned: Edg2 (LPA1), Edg4 (LPA2), Edg7 (LPA3), and LPA4 (60). Using RT-PCR and specific primers for the different LPA receptors revealed that NIH3T3 cells express Edg2, Edg4, and LPA4 and that Ehrlich Lettre cells express Edg2, Edg4, LPA4 plus Egd7 (Fig. 5). The Ehrlich Lettre cells were included in the present investigation as a control for Edg7 expression. Edg4, Edg7, and LPA4 use Ca2+ and small GTP-binding proteins for intracellular, downstream signaling, whereas Edg2 relays on Rac1/2, phosphatidylinositol 3-kinase (PI3K) (31, 60, 68). Figure 6A shows that 10 nM LPA, i.e., a concentration that potentiates taurine release (Fig. 3B) and ROS production (Fig. 3D), did not mobilize Ca2+ in NIH3T3 cells under isotonic or hypotonic conditions. Increasing the LPA concentration to 100 nM, on the other hand, elicited an immediate, transient increase in the cellular Ca2+ concentration under isotonic as well as hypotonic conditions (Fig. 6B). Buffering extracellular Ca2+ with EGTA reduced the Ca2+ mobilization, induced by 100 nM LPA (data not shown), indicating that the LPA-induced increase in cytosolic Ca2+ in the NIH3T3 cells is a result of release from intracellular stores and influx from the extracellular compartment. In Fig. 3D it was shown that the ROS production in the presence of 100 nM LPA equaled the production elicited by 10 nM LPA, and it is therefore suggested that the potentiation of ROS production, i.e., the NADPH oxidase activity and the volume-sensitive taurine release obtained with 10 nM LPA, does not involve Ca2+ mobilization. Preincubation of NIH3T3 cells with the PI3K inhibitor wortmannin (0.5 µM, 30 min), which in itself does not affect the swelling-induced taurine release under hypotonic conditions (56), reduced the LPA-induced potentiation of the swelling-induced taurine release; i.e., the maximal rate constant was increased by 50 nM LPA from 0051 ± 0.003 min–1 to 0.75 ± 0.003 min–1 and from 0.046 ± 0.003 min–1 to 0.065 ± 0.004 min–1 in the absence or presence of wortmannin, respectively (3 sets of experiments). On the other hand, wortmannin treatment did not affect the LPA-induced potentiation of the ROS production under hypotonic conditions (two sets of experiments, data not shown). Thus, LPA seems to potentiate the volume-sensitive taurine release via Edg2 and PI3K and the ROS production in a PI3K-independent way.


Figure 5
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  		Fig. 5. Expression of LPA receptors in NIH3T3 cells. mRNA was isolated from NIH3T3 fibroblasts and Ehrlich Lettre cells (ELA). RT-PCR was performed using mouse-specific primers as described in MATERIALS AND METHODS. Lane M represents the band sizes of the DNA ladder used for the electrophoresis.

 

Figure 6
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  		Fig. 6. Effect of LPA on Ca2+ mobilization in NIH3T3 cells. Calcium was measured as outlined in Fig. 4A. LPA (10 nM, A; 100 nM, B) or ATP (10 µM; A) were added as indicated by the arrows. Traces represent at least 3 sets of experiments.

 
Overexpression of the small GTP-binding protein Rac does not increase taurine release. It was previously demonstrated that overexpression of the monomeric GTP binding protein Rac in NIH3T3 cells accelerated volume restoration following exposure to hypotonic medium (56). Because Rac1/2 constitutes a subunit of the NADPH oxidase complex (43, 66), we investigated the effect of overexpression of Rac1 on swelling-induced increase ROS production as well as taurine and arachidonic acid release. From Table 1 it is seen that release of arachidonic acid under isotonic and hypotonic conditions is unaffected by overexpression of the GTP-binding protein Rac1. The increase in the ROS production following a mild (35 mosM) and a more pronounced (135 mosM) hypotonic challenge is likewise unaffected by the overexpression of Rac1 (Table 1). Furthermore, overexpression of Rac1 does not affect the rate constants for taurine release under isotonic conditions or the maximal rate constant for taurine release under hypotonic conditions in the absence or presence of LPA (10 nM) or PMA (50 nM) (Table 1). It is noticed that the taurine efflux can be estimated as the product of the fractional rate constant and the cellular taurine pool and that an unaffected rate constant does not necessarily reflect an unaffected efflux. However, using HPLC technique, we estimated the cellular taurine, alanine, and glycine pool (µmol/mg protein) at 0.003 ± 0.001 (n = 6), 0.004 ± 0.0004 (n = 5), and 0.015 ± 0.001 (n = 6) in wild-type NIH3T3 cells, respectively, and at 0.003 ± 0.001 (n = 4), 0.002 ± 00002 (n = 5), and 0.009 ± 0.002 (n = 6) in Rac1 cells, respectively. Hence, release of organic osmolytes is not affected by Rac1 overexpression, indicating that Rac1 expression under normal, unpertubated conditions is sufficient to maintain NADPH oxidase activity. It is emphasized that a quantitative correlation of the ROS production between the two cell lines is not possible because of the fact that it is impossible to obtain the exact same cell density, degree of probe loading when the experiments are performed on two different cell lines.


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  		Table 1. Effect of Rac1 overexpression on arachidonic acid mobilization, ROS production, and taurine release in NIH3T3 cells following hypotonic exposure

 
Overexpression or silencing of the regulatory NADPH oxidase component p47phox does not affect swelling-induced ROS production or taurine release. The NADPH oxidase in neutrophiles is regulated by the subcomponents p47phox and p67phox, whereas p47phox is regulated by PKC (43, 66). To evaluate the role of p47phox in swelling-induced ROS production, we overexpressed p47phox, cloned from human kidney epithelial cells, in the NIH3T3 cells. The fragment was fused with a myc-tag, and Fig. 7A verifies that a band around 49 kDa that corresponds to the size of Myc-p47phox is expressed. Using the volume-sensitive taurine efflux as a functional parameter for the NADPH oxidase activity, we found that the myc-p47phox did not affect the magnitude of the swelling-induced taurine release or the volume set-point for its unset. This is seen from Fig. 7B, where the maximal rate constant for the volume-sensitive taurine release in nontransfected cells, myc-p47phox transfected cells, and mock transfected cells (CAP350) is plotted against the hypotonic challenge (320 mosM to 220 mosM). Thus, overexpression of p47phox did not potentiate the swelling-induced taurine release nor the extracellular tonicity/degree of cell swelling required for activation of the efflux pathway. This is in agreement with data obtained by Martyn and coworkers (49), who showed that HEK-293 cells expressing NOX4 did not generate more ROS when cells were transfected with p47phox. To eliminate the possibility that the lack of response from p47phox overexpression simply reflected that p47phox was already abundant in the cell, we knocked down p47phox using siRNA technique. Figure 7C shows that p47phox was reduced by the procedure, and using the histone band as reference, it was estimated that p47phox protein expression in NIH3T3 cells was reduced by 50% using 150 ng siRNA. The swelling-induced potentiation of the ROS production in NIH3T3 cells was not affected by p47phox knockdown (Fig. 7D); neither was the swelling-induced taurine release, i.e., the maximal rate constant obtained after exposure to hypotonic solution (200 mosM) in nontransfected control cells, significantly different from the rate constant in siRNA-transfected cells (Fig. 7E). Furthermore, LPA- and PMA-mediated potentiation of the volume-sensitive taurine efflux in hypotonic medium (200 mosM) was also unaffected by the siRNA knockdown of p47phox (Fig. 7E). These data indicate that although p47phox is expressed in the NIH3T3 cells, it seems not to be required for the swelling-induced ROS production nor its potentiation by LPA and PMA/PKC.


Figure 7
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  		Fig. 7. Effect of p47phox overexpression and knockdown on the swelling-induced taurine release in NIH3T3 cells. A: NIH3T3 cells grown to 50% confluence were transfected with 0.5 µg plasmid per millileter medium (24 h). Verification of myc-p47phox expression was performed by Western blot analysis using antibodies against the myc-tag. Lane 1 represents cells transfected with plasmid containing myc-tagged p47phox. Lane 2 represents nontransfected cells, i.e., cells transfected with Lipofectamine2000 alone. Lane 3 represents cells transfected with an empty vector, i.e., a plasmid containing the myc-tag alone. Lane 4 is empty, cell-free, and serves as a control for nonspecific antibody binding. B: taurine efflux was performed under isotonic (335 mosM) and hypotonic (318–224 mosM) conditions on nontransfected control cells and cells transfected with either myc-p47phox or a control-plasmid expressing the protein CAP350, which has no known effect on the NADPH oxidase. Taurine release at each given tonicity is given as the maximal rate constant obtained following hypotonic exposure. C: NIH3T3 cells grown to 50% confluence were transfected with 0–200 ng p47phox small interfering RNA per 2 ml medium. Knockdown of p47phox was verified by Western blot analysis using antibodies against p47phox. Histone H3 was used as loading control. D: ROS production under isotonic (335 mosM) and hypotonic (300–200 mosM) conditions was estimated as outlined in Fig. 2 in control cells and p47phox knockdown cells. ROS production in knockdown cells was estimated and is given relative to the respective hypotonic control. E: taurine release under hypotonic (200 mosM) conditions was estimated as outlined in Fig. 3 in control cells and p47phox knockdown cells in the absence or presence of LPA (10 nM) or PMA (50 nM). Efflux is presented as the absolute maximal rate constant obtained under the hypotonic conditions. A and C represent typical blots. Data in B, D, and E are given as means ± SE of at least 3 sets of experiments.

 
Silencing of NOX4 reduces the swelling-induced ROS production and taurine release. To pinpoint the role of NOX4 in the swelling-induced increase in ROS production in NIH3T3 cells, we measured the ROS production following NOX4 knockdown. From Table 2 it is seen that 100 nM siRNA had no significant effect on the ROS production under isotonic conditions (335 mosM). However, NOX4 silencing effectively reduced the swelling-induced increase in ROS production following exposure to 300 mosM and 200 mosM hypotonic solutions to 50% and 40% of the production seen in nontransfected cells, respectively (Table 2). Furthermore, we observed that in several ROS time traces from NOX4 knockdown cells, the stabile face, normally following the initial increase in ROS production (see, e.g., Fig. 2A), was followed by a decrease in the cellular ROS level (data not shown), which could be taken to indicate that elimination of ROS in NIH3T3 cells exceeds the ROS production under hypotonic conditions following NOX4 silencing. The increase in ROS production following hypotonic exposure (200 mosM) was unaffected in cells transfected with control siRNA (mock), i.e., ROS was increased 1.2 ± 0.1-fold (n = 3) in mock transfected cells compared with nontransfected cells. From Fig. 2, it is seen that swelling in hypotonic medium (200 mosM) increases the ROS production 1.5-fold compared with isotonic cells (Fig. 2B) and that DPI reduces the ROS production under the hypotonic conditions (200 mosM) to 50% of the ROS production seen in nontreated control cells (Fig. 2C). Hence, DPI inhibition and NOX4 silencing impair the swelling-induced increase in ROS in NIH3T3 cells, i.e., NOX4 seems to account for the major fraction of the swelling-induced increase in the ROS production in NIH3T3 cells. From Table 2 it is also seen that NOX4 silencing reduces the volume-sensitive taurine release significantly following a reduction in the extracellular tonicity from 335 mosM to 200 mosM, i.e., to the same extent as seen in cells exposed to DPI (Fig. 3B). On the other hand, exposing the cells to a mild hypotonic shock (300 mosM) increases the rate constant for the taurine release by a factor less than 2 and now NOX4 silencing seems to stimulate taurine release, but only to the same extent as seen for NIH3T3 cells under isotonic conditions. Hence, NOX4-generated ROS only seem to affect the volume-sensitive taurine release under conditions where the taurine efflux pathway has been activated.


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  		Table 2. Effect of NOX4 silencing on the swelling-induced increase in ROS production and taurine release in NIH3T3 cells

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Potentiation of the volume-sensitive NADPH oxidase activity in NIH3T3 fibroblasts. The multicomponent NADPH oxidase was first identified as the ROS-producing protein complex in the phagosomes of neutrophils—one of the participants in the cell defense against pathogens in the process designated as the respiratory burst. With the cloning of homologs to the neutrophilic catalytic subunit, gp91phox/NOX2 in nonphagocytic cells, it was revealed that the NADPH oxidase was also implicated in ligand/receptor signaling, stress responses, apoptosis, proliferation, and cell volume regulation (9, 16, 37, 59, 63).

NIH3T3 fibroblasts were previously shown to increase the ROS production following hypotonic exposure and osmotic cell swelling (35). The present data confirm this observation and also indicate that the potentiation in the ROS production increases with the magnitude of the osmotic challenge. Similar observations have been made with HTC cells (71). The swelling-induced increase in the ROS production in NIH3T3 cells involves the NADPH oxidase because it is inhibited by the flavoprotein inhibitor DPI and unaffected by the nitric oxide synthase inhibitor L-NAME. Moreover, our experiments indicate that NOX4 silencing impairs the swelling-induced increase in ROS production, indicating that NOX4 is a major contributor to the swelling-induced increase in ROS production in NIH3T3 cells. However, NOX4 silencing only reduces taurine release from NIH3T3 cells following a hypotonic challenge, which is sufficient to trigger the volume-sensitive taurine efflux pathway. Thus, NOX4 in NIH3T3 cells seems to be volume sensitive and to play a role in the potentiation of the volume-sensitive taurine efflux pathway once this pathway has been activated.

The swelling-induced ROS production is unaffected by AEBSF, which is taken to indicate that assembly of catalytic and regulatory units in a NADPH oxidase complex is not a prerequisite for the increase in oxidase activity following hypotonic exposure. Inhibition of the volume-sensitive K+ and Cl– channels does not affect the swelling induced increase in the ROS production, indicating 1) that the effect of DPI is not secondary to inhibition of the K+ current as previously indicated (74, 76) and 2) that the inhibition of the Cl– current/anion channel activity does not affect the output of the NADPH oxidase activity. Release of the organic osmolyte taurine from NIH3T3 cells following hypotonic exposure is potentiated by exogenous addition of H2O2 and impaired by antioxidants (35). On the other hand, ROS have no effect on taurine release from NIH3T3 cells when added under isotonic conditions, and the ROS-evoked potentiation of taurine release vanishes when the cell volume has returned to its normal value (38). Thus, increasing the osmotic challenge improves the swelling-induced increase in the ROS production via the NADPH oxidase system in NIH3T3 cells and consequently potentiates the swelling-induced taurine release.

The ROS production in NIH3T3 cells under hypotonic conditions is potentiated following exposure to the PKC activator PMA, the lysophospholipid LPA but not by addition of the Ca2+-mobilizing agonist ATP. The swelling-induced taurine release is, on the other hand, potentiated by addition of PMA, LPA as well as by ATP. Inhibition of the NADPH oxidase with DPI impairs the swelling-induced (35, 37, 38) as well as the PMA- and LPA-induced potentiation of the volume-sensitive taurine efflux. The effect of DPI on the PMA-/PKC-induced potentiation of the swelling-induced taurine release from NIH3T3 cells is in agreement with previously published data (36). On the other hand, NADPH oxidase inhibition seems not to affect the ATP-induced potentiation of the volume-sensitive taurine release, i.e., the DPI-induced reduction in the maximal rate constant for the volume-sensitive taurine release in the presence of ATP corresponds to the reduction seen when cells were exposed to hypotonic solution alone. Thus, PMA-/PKC- and the LPA-mediated potentiation of the volume-sensitive taurine efflux from NIH3T3 cells seems to reflect an effect of an increased/potentiated NADPH oxidase activity, whereas ATP is still able to potentiate the taurine release following inhibition of the NADPH oxidase. Activation of PLA2 isoforms (iPLA2 and sPLA2) and release of arachidonic acid from the nuclear envelope is part of the intracellular signaling cascade in NIH3T3 cells that is activated by osmotic cell swelling and that leads to activation of the volume-sensitive taurine release pathway (40, 41, 57). Because ATP mobilizes Ca2+ in NIH3T3 cells and increases the release of arachidonic acid under hypotonic conditions, it is speculated that the ATP-induced potentiation of the volume-sensitive taurine efflux reflects an increased availability of the precursor arachidonic acid for downstream signaling rather than a Ca2+-induced potentiation of the ROS producing system. Ca2+ is previously shown to potentiate the volume-sensitive activation of the taurine efflux in HeLa cells through activation of calmodulin and PKC isoforms belonging to the novel subclass (24). However, the lack of an effect of Gö-6976 on the ATP-induced potentiation of the swelling-induced taurine release excludes the involvement of Ca2+-sensitive PKC isoforms in the Ca2+-mediated potentiation in NIH3T3 cells.

Regulation of the NADPH oxidase in NIH3T3 fibroblasts. Data presented in this article show that NIH3T3 fibroblasts express the necessary components to form a functional NADPH oxidase complex. We found that the regulatory subunits, p47phox and p67phox, as well as the membrane-bound subcomponent p22phox are expressed in NIH3T3 cells. Moreover, a band was amplified of NOXO1 and the catalytic subunit NOX4, which was larger in size than the calculated value and most probably reflects NOX4 and NOXO1 splice variants. DNA contamination seems unlikely, since we were still able to amplify a band of the same larger size from samples that were DNase treated. The NADPH oxidase expression pattern resembles the pattern seen from unstimulated MEF cells, although no NOXO1 is expressed in the MEF cells (5). NOX4 shares the least homology (39%) with the neutrophilic gp91phox (26, 79) and the other known isoforms, creating a discrete phylogenetic group (44). Little is known about the regulation of NOX4 (32, 45), although an increase in ROS production is seen in NOX4 and p22phox cotransfected HEK-293 cells compared with NOX4 transfected HEK-293 cells (3, 49). Furthermore, it has been shown by Martyn and coworkers (49) that, of the known regulatory subunits, p47phox, p67phox, NOXO1 or NOXA1 plus the GTP-binding protein Rac1 are required for activation of NOX4 and that ROS production is not stimulated by PMA (49). Thus, the presence of the NOX4 and p22phox seems to be sufficient to form a functional ROS-producing complex. It is emphasized that some NOX isozymes are regulated by a phosphatidylinositol 4,5-bisphosphate-dependent activation of PKC, arachidonic acid mobilization, and the subsequent activation/recruitment of p47phox or Rac to the membrane (11, 78). The lack of effect of AEBSF, which prevents assembly of NADPH components, on the swelling-induced increase in ROS production could be taken to indicate that NOX4 plus p22phox alone could account for the volume-sensitive oxidase activity in NIH3T3 cells. This notion is supported by the fact that overexpression of p47phox did not affect the ROS production upon hypotonic challenge and did not change the volume set point for the unset of the volume-sensitive taurine release from NIH3T3 cells. Moreover, p47phox knockdown by siRNA had no effect on the ROS production or the taurine efflux when the NIH3T3 cells were exposed to hypotonic solution alone or following potentiation with either LPA or PMA. It should be noted that we cannot exclude that the remaining p47phox expression following p47phox silencing could be sufficient to maintain the regulation of the oxidase activity. Rac1 has been shown to facilitate the stabilization of the p67phox at the membrane bound catalytic subunits of the NADPH oxidase complex (7, 12, 16). However, we found that overexpression of Rac1 had no effect on swelling-induced ROS production nor the concomitant taurine release. These data are in agreement with previous reports, showing that NOX4 is constitutively active and not regulated by conventional NADPH oxidase subcomponents, i.e., by the p47phox or Rac (26, 49). However, Mahadev and coworkers (48) showed an increase in ROS production upon insulin stimulation of 3T3-L1 adipocytes, which express endogenous NOX4, and that this ROS production was elevated in NOX4 overexpression. (48). Furthermore, the insulin-induced ROS production was blocked in NOX4 mutants mutated in their FAD- or NADPH-binding regions, indicating that the NOX4 mutants function as a dominant negative, which is unlikely for regulated proteins (48). Although this could be explained by competition for p22phox, this was not the case, because overexpression of NOX4 increased ROS production, which excludes p22phox as the limiting factor (48). Likewise, we find that p22phox is not the limiting factor in the ROS production in NIH3T3 cells under hypotonic conditions because NOX4 overexpression leads to an increase in the swelling-induced increase in ROS production (Friis MB and Lambert IH, unpublished observations). Moreover, Gorin and coworkers (27) showed that, in mesangial cells, the ROS production could be stimulated by angiotensin II and arachidonic acid, whereas NOX4 siRNA and expression of a dominant negative Rac1 attenuated the effect of angiotensin II and arachidonic acid (27). Finally, it has been shown by Park and coworkers (53) that NOX4 directly interacts with lysophosphatidyl serine receptors, creating a direct link between receptor stimulation and ROS production in human aortic endothelial cells (53). Taken together, these observations indicate that NOX4 can be stimulated by means different from the conventional regulatory subunits.

LPA-induced potentiation of the volume-sensitive taurine release. LPA has within recent years turned out to be an important second messenger that exerts its effect via high-affinity receptors. Low concentration of LPA is produced continuously during membrane synthesis, and some cells, e.g., activated platelets, produce significant amounts of extracellular LPA during ischemia-reperfusion injury (20, 77), i.e., under conditions where significant cellular swelling occurs (46). NIH3T3 fibroblasts express the high-affinity LPA receptors LPA1, LPA2, and LPA4, which have all been shown to stimulate Ca2+ release with EC50 values around 20 nM, as well as arachidonic acid release at 1 µM LPA in receptor-transfected cells (4, 6, 34). LPA has also been shown to stimulate the activation of Rho and the MAP-kinase pathway (6, 8, 14, 23, 34) as well as the PI3K (75). Overexpression of Rho is previously demonstrated to potentiate dramatically the swelling-induced taurine release from NIH3T3 cells (56). Furthermore, a Ras/PI3K/Rac1/NADPH-oxidase-dependent signaling pathway has been demonstrated to be involved in the enhancement of DNA repair enhancement by oncogenic H-ras expression in NIH3T3 cells (12). Exposing the NIH3T3 cells to 10 nM LPA potentiates the swelling-induced taurine release and the concomitant increase in the ROS production, whereas 10 nM LPA has no detectable effect on the intracellular Ca2+ concentration. Increasing the LPA concentration from 10 nM to 100 nM, on the other hand, induces a transient increase in the cellular Ca2+ concentration, which reflects influx from the extracellular compartment and release from intracellular stores. However, the effect of LPA on the ROS production or the taurine release is not improved when the LPA dose is increased from 10 nM to 100 nM, indicating that the LPA-induced amplification of the ROS production and taurine release in swollen NIH3T3 cells does not involve Ca2+ mobilization. Because the PI3K inhibitor wortmannin reduces the LPA-induced potentiation of the swelling-induced taurine efflux but leaves the ROS production unaffected, it seems reasonable to assume that the LPA signaling in NIH3T3 cells activates more than one signaling pathway. The Edg2 receptor, Rac1, and PI3K seem to be coupled to the taurine release, whereas the mechanism behind the LPA-induced potentiation of the ROS production in NIH3T3 cells is unclear. Ca2+-insensitive PKC isoforms could be involved because the LPA- and PMA-mediated potentiation of the ROS production is inhibited by the pan-PKC inhibitor chelerythrine to the same extent as by DPI but not by Gö-6976. Yamamori and coworkers (78) demonstrated that stimulation of neutrophils with the tripeptide N-formylmethionyl-leucyl-phenylalanine leads to the sequential activation of PI3K, an increase in phosphatidylinositol 4,5-bisphosphate, activation of PLC, increase in diacylglycerol, and, subsequently, activation of the Ca2+-insensitive PKC{delta}. LPA induces a receptor-mediated activation of PI3K (75), and various PKC isoforms, including PKC{delta}, lead to the phosphorylation and activation of p47phox (78). Thus, it seems reasonable to assume that LPA mediates its effect via a Edg2/PI3K/PKC-mediated stimulation of a yet unidentified regulatory unit of the NADPH oxidase. It is emphasized that our studies showed that p47phox is not required for NADPH oxidase activity, but this does not exclude that the catalytic components of the NOX isozyme expressed in NIH3T3 fibroblasts are subject to indirect volume-dependent regulation by PKC, since neither NOX4 nor p22phox contain putative PKC motifs.

From the present data, it is suggested that a NOX4 isotype plus the p22phox constitute the catalytic components of the volume-sensitive NADPH oxidase in NIH3T3 fibroblasts and that the oxidase activity is potentiated under hypotonic conditions by PKC and LPA but not by Ca2+. LPA in the low-nanomolar range stimulates the volume-sensitive ROS production and the concomitant taurine release in NIH3T3 cells, and because activation of various PLA2 isoforms is reported to be involved in the swelling-induced release of taurine in, e.g., Ehrlich ascites tumor cells (69), HeLa cells (42), NIH3T3 cells (40, 41, 57), and Ehrlich Lettre cells (38), it seems plausible that the PLA2 product LPA could stimulate the NOX4-p22phox system and hence potentiate release of organic osmolytes following osmotic cell swelling.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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The present work was supported by The Danish Natural Sciences Research Council (Grants 21-01-0507; 21-02-0358; 21-04-0535).


   ACKNOWLEDGMENTS
 
The technical assistance of Dorthe Nielsen and Jonathan Rahlff Rogersen is gratefully acknowledged.


   FOOTNOTES
 

Address for reprint requests and other correspondence: I. H. Lambert, Department of Biology, The August Krogh Building, University of Copenhagen, Universitetsparken 13, DK-2100, Copenhagen Ø, Denmark (e-mail: ihlambert{at}bio.ku.dk)

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.


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