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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
Department of Molecular Biology, University of Copenhagen, Copenhagen, Denmark
Submitted 15 March 2007 ; accepted in final form 29 April 2007
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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organic osmolytes; reactive oxygen species; vanadate; H2O2; tyrosine phosphatases; arachidonic acid mobilization
Osmotic perturbation in various cell types is accompanied by marked cytoskeletal rearrangement (shift in actin polymerization), translocation of specific enzymes [phospholipase A2 (PLA2) and 5-lipoxygenase (5-LO)], mobilization of second messengers (eicosanoids and nucleotides), and shift in the balance between protein tyrosine kinase (PTK) and protein tyrosine phosphatase (PTP) activity (see Refs. 5, 22, and 38). The initial osmo/volume sensor has not been identified yet, but in the case of NIH3T3 mouse fibroblasts, it has turned out that activation of PLA2 is an initial upstream event in the intracellular signaling cascade, which is elicited by osmotic cell swelling and leads to activation of volume-sensitive transporters for organic and inorganic osmolytes (22). Oxidation of arachidonic acid via the 5-LO system seems to be an additional permissive element for volume-sensitive taurine loss not only in NIH3T3 cells but also in Ehrlich ascites tumor cells, HeLa cells, C2C12 myotubes, and pig muscle (18–20, 24, 25, 29, 35). The PLA2 family constists of a diverse family of enzymes that, based on functional criteria, can be divided into 1) high-molecular-weight, intracellular, Ca2+-dependent PLA2 (cPLA2-
, cPLA2-
, and cPLA2-
); 2) low-molecular-weight, secretory, Ca2+-dependent PLA2 (sPLA2); and 3) high-molecular-weight, Ca2+-independent PLA2 (iPLA2- and iPLA2-) (for reviews, see Refs. 1, 7, and 47). The PLA2 isoform(s) involved in volume-sensitive taurine release has been investigated, and it appears that the particular isoform(s) activated depends on the cell type in question, i.e., cPLA2- distributes uniformly throughout Ehrlich ascites tumour cells under unpertubated conditions but aggregates at the nuclear area and releases arachidonic acid from the nuclear envelope within the first 2 min following hypotonic exposure (37, 42), whereas iPLA2- exhibits a similar role in NIH3T3 fibroblasts (39).
ROS, e.g., the superoxide anion and its derivatives plus the lipid-permeable, nonradical oxygen species H2O2, are recognized as intracellular signaling molecules in nonphagocytic cells (10, 14, 41, 44). Fibroblasts, in contrast to neutrophiles, produce superoxide radicals, mainly intracellularly (44), and an increase in ROS production has been demonstrated to occur within the first minute following hypotonic exposure in NIH3T3 fibroblasts (20), skeletal muscle (35), and HTC cells (45). In the case of NIH3T3 fibroblasts, it has been proposed that the swelling-induced ROS production involves NADPH oxidase (NOX) that is activated at a step downstream to iPLA2/sPLA2 activation (20, 39). Similarly, Colston and coworkers (6) demonstrated that NOX is activated downstream of cPLA2 in cardiac fibroblasts following an exposure to exogenous H2O2.
It has been estimated that 15% cell swelling is required for activation of the taurine efflux pathway in NIH3T3 cells and that exogenous addition of H2O2 potentiates the swelling-induced taurine release (20). H2O2 has no effect on taurine release from NIH3T3 cells when added under isotonic conditions, and H2O2 does not affect the volume set point for activation of the efflux pathway (20). Exposure of NIH3T3 cells to N-acetyl cysteine (NAC), which increases the intracellular level of reduced glutathione and thereby the elimination of ROS via the glutathione peroxidase system, reduces the maximal rate constant for swelling-induced taurine efflux marginally, although significantly, whereas the antioxidant butylene hydroxyl toluene (BHT) completely impairs volume-sensitive taurine release in NIH 3T3 cells (20). Furthermore, inhibition of NOX does not affect the initiation of the swelling-induced efflux pathway in NIH3T3 cells but accelerates dramatically its inactivation (20). These observations have been taken to indicate that ROS might have dual effects in NIH3T3 cells, i.e., ROS potentiate the volume-sensitive taurine efflux pathway and delay its inactivation. Exposure of NIH3T3 cells to genistein, a general PTK inhibitor, or to PD-153035, which inhibits PTK activity coupled to EGF, reduces swelling-induced taurine efflux, whereas the addition of EGF potentiates taurine efflux following hypotonic exposure (20). Addition of the PTP inhibitor vanadate mimics the potentiating effects of H2O2 on swelling-induced taurine release and delays the inactivation of the volume-sensitve efflux pathway in NIH3T3 cells (20). Furthermore, exogenous H2O2 shifts the tyrosine phosphorylation pattern of the PTK c-Src under hypotonic conditions (23). It has, accordingly, been suggested that the open probability of the volume-sensitive taurine release pathway in NIH3T3 cells is regulated by PTKs as well as by PTPs and that EGF receptor PTK and c-Src could be involved (23).
The present work was initiated to 1) elucidate how ROS and PTPs are involved in the potentiation and inactivation of the volume-sensitive taurine efflux pathway and 2) test whether the effect of ROS and PTPs on volume-sensitive taurine release is cell type dependent. Using NIH3T3 fibroblasts and Ehrlich Lettre cells, an adherent version of Ehrlich ascites tumor cells, we show that 1) multiple PLA2 isoforms, 5-LO, and NOX are involved in the activation of volume-sensitive taurine release in both cell types; 2) inhibition of PTP activity increases arachidonic acid release and ROS production and potentiates the swelling-induced taurine efflux in both cell types; and 3) inhibition of PTPs delays the inactivation of the volume-sensitive efflux pathway in NIH3T3 cells, whereas the inactivation in Ehrlich Lettre cells is unaffected. Inhibition of NOX leads to a rapid inactivation of the volume-sensitive taurine efflux pathway even in the presence of a PTP inhibitor. It is suggested that increased tyrosine phosphorylation of regulatory components of NOX leads to increased ROS production and a subsequent delay in the inactivation of the volume-sensitive taurine efflux pathway and that the expression and activity of NOX or the antioxidative capacity differ between NIH3T3 cells and Ehrlich Lettre cells.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Inorganic media. PBS contained (in mM) 137 NaCl, 2.6 KCl, 6.5 Na2HPO4, and 1.5 KH2PO4. Isosmotic NaCl medium (300 mosM) contained (in mM) 143 NaCl, 5 KCl, 1 Na2HPO4, 1 CaCl2, 0.1 MgSO4, and 10 HEPES. Isoosmotic, Na+-free, N-methyl-D-glucamine (NMDG)-Cl medium was prepared as the isotonic NaCl medium with NMDG-Cl being substituted for NaCl in equimolar amounts. Hypoosmotic NaCl/NMDG media (200 mosM) were obtained by reduction of NaCl/NMDG in isoosmotic solutions to 95 mM, with the other components remaining unchanged. pH was adjusted at 7.40 in all solutions.
Cell cultures. NIH3T3 mouse fibroblasts (clone 7) and Ehrlich Lettre ascites cells (American Type Culture Collection) were grown in 75-cm2 culture flasks as monolayer cultures in DMEM and RPMI-1640, respectively, containing heat-inactivated FBS (10%) and antibiotics. Both cells lines were kept at 37°C/5% CO2/100% humidity and split every 3–4 days using 0.5% trypsin in PBS to detach the cells.
Estimation of ROS production. Cells were cultured for 24 h in serum containing growth media on HCl- and ethanol-washed glass coverslips (10–50 mm). Confluence at the time of ROS estimation was 80%. For the estimation of ROS, cells were washed two times with PBS and subsequently incubated in serum-free growth medium containing the ROS-sensitive fluorescent probe carboxy-H2DCFDA (20 µM, 2 h). Cells were subsequently washed with isotonic solution, and coverslips were placed vertically in a polystyrene cuvette (10-mm path length, 50° angle relative to the excitation light) containing experimental solution. ROS estimation was performed 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. Excitation and emission wavelengths were 490 and 515 nm, respectively, and data were collected every 2 s for 400 s. As the ROS-dependent fluorescence depends on cell density, dye concentration and incubation time were estimated as the effect of reduction in osmolarity and addition of diverse agents by comparing time traces obtained from the same cell preparation.
Estimation of cell volume.
Cells, grown to 
80% confluence in 175-cm2 culture flasks, were detached by trypsination (0.2% trypsin in PBS) at 37°C. Trypsination was terminated by the addition of 10 ml of growth medium, and the suspension was transferred to a Sorwal glass (50 ml), centrifuged gently (4 min, 700 g), and resuspended in 5 ml of isotonic Na+-free NMDG-Cl medium. Three milliliters of the resuspended cells were diluted 20 times in hypotonic NMDG-Cl medium, and the cell volume was estimated by electronic cell sizing in a Coulter counter. All media were filtered (0.45-µm filters). Absolute cell volumes (fluid 
10–15 liters) were obtained from the median of the distribution curves after calibration with latex beads (14.25 µm diameter, Coulter Electronics).
Estimation of the rate constant for taurine efflux and fractional arachidonic acid release.
Efflux measurements were performed as described previously (15, 20, 39). Briefly, taurine efflux was estimated at room temperature on NIH3T3/Ehrlich Lettre cells grown to 80% confluence in six-well polyethylene dishes (9.6 cm2/well) and loaded with 1 ml of growth media containing [3H]taurine (2 µCi/well, 2 h). Cells were washed four times with isosmotic media to remove excess extracellular [3H]taurine and cellular debris. Efflux was initiated by aspiration of the medium followed by the addition of 1 ml of experimental solution. Cells were left for 2 min, whereafter the entire medium was transferred to a scintillation vial and rapidly substituted by 1 ml of fresh medium. This procedure was repeated for 20–30 min. At the end of the experiment, cells were lysed by the addition of 1 ml NaOH (0.5 mM, 1 h). The total pool of labeled taurine in the cell system was estimated as the sum of 3H activity (-scintillation counting, Ultima Gold) in all the efflux samples (the NaOH lysate plus two final well washouts with double-distilled H2O). Release of taurine under the present experimental conditions follows a monoexponential equation, and the natural logarithm to the fraction of [3H]taurine remaining in the cells at a given time was plotted versus time. The rate constant (k; in min–1) for the taurine efflux at each time point was subsequently estimated as the negative slope of the graph between the time point and the proceeding time point. Inactivation of swelling-induced taurine efflux at time t following hypotonic exposure (%) was estimated as follows: 100 x (kt – kinit)/(kmax – kinit), where kt, kinit, and kmax are the rate constants estimated under hypotonic conditions at time t, under isotonic conditions prior to hypotonic exposure, and under hypotonic conditions at maximal activation, respectively. Arachidonic acid release was estimated using a similar protocol as the one used for estimation of taurine efflux with the exception that cells were loaded with [3H]arachidonic acid (3 µCi/well, 24 h), 1% BSA was added to the efflux medium to trap released arachidonic acid, and an additional washout was performed with methanol. The release of [3H]arachidonic at a given time point is shown as the total fraction released (%). It has been previously verified by reverse-phase HPLC analysis that the released label represents [3H]arachidonic acid (42).
Statistical analysis. Data are presented either as individual experiments that are representative of at least three independent sets of experiments or as mean values ± SE. Statistical significance was estimated by a paired Student's t-test. For all statistical evaluations, P values of <0.05 were taken to indicate significant differences.
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RESULTS |
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50% to 10% in NIH3T3 cells, whereas inactivation was even slightly increased by vanadate from 60% and 70% in Ehrlich Lettre cells (Fig. 1D). Thus, ROS (H2O2) delayed inactivation of the volume-sensitive taurine efflux pathway in NIH3T3 cells as well as in Ehrlich Lettre cells, whereas inhibition of PTPs delayed inactivation of the efflux pathway in NIH3T3 fibroblasts but apparently had no effect on the inactivation of the efflux pathway in Ehrlich Lettre cells. It has previously been demonstrated that the release of arachidonic acid from the nuclear envelope is increased in NIH3T3 cells following hypotonic exposure, although no concomitant release to the extracellular compartment was detected (39). Figure 2 shows that in contrast to NIH3T3 cells (Fig. 2, A and C), a small but significant increase in the release of arachidonic acid to the extracellular space was demonstrated in Ehrlich Lettre cells following hypotonic cell swelling (Fig. 2, B and D). Exposure to 1 mM H2O2 stimulated arachidonic acid release under isotonic as well as hypotonic conditions in both cell types (Fig. 2, C and D). Furthermore, arachidonic acid release was significantly increased by vanadate under hypotonic conditions, although the release seemed to be quantitatively larger in NIH3T3 cells compared with Ehrlich Lettre cells (Fig. 2, C and D). The swelling-induced release of arachidonic acid and taurine in NIH3T3 cells involves manoalide-sensitive sPLA2 activity and BEL-sensitive iPLA2 activity (27, 39), and the experiments shown in Fig. 3 were initiated to characterize the PLA2 isoforms involved in swelling-induced taurine efflux in Ehrlich Lettre cells and to test whether the vanadate-induced potentiation of swelling-induced taurine efflux shown in Fig. 1 reflected the vanadate-induced increase in arachidonic acid release, i.e., increased availability of lipid substrate for downstream signaling. Figure 3A shows that manoalide as well as BEL significantly reduced the maximal rate constant for volume-sensitive taurine efflux in Ehrlich Lettre cells. Furthermore, addition of the 5-LO inhibitor ETH-615139 also reduced the maximal rate constant for swelling-induced taurine release in Ehrlich Lettre cells (Fig. 3A). Thus, sPLA2 and iPLA2 as well as 5-LO activities are required for activation of the volume-sensitive taurine efflux in NIH3T3 cells and Ehrlich Lettre cells. Figure 3 also shows that BEL reduced the maximal rate constant for volume-sensitive taurine efflux in vanadate-treated Ehrlich Lettre cells (Fig. 3A) and vanadate-treated NIH3T3 cells (Fig. 3B). Hence, the potentiation of volume-sensitive taurine efflux following inhibition of PTPs requires BEL-sensitive PLA2 activity, i.e., the potentiation could be secondary to the concomitant increase in arachidonic acid release.
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10% to 90% in the presence of H2O2 (Fig. 7C, light gray bars), and from 40% to 90% in the presence of vanadate (Fig. 7C, dark gray bars). As shown Fig. 7B, swelling-induced taurine release from NIH3T3 cells was also reduced in the presence of vanadate under hypotonic, Na+ conditions following the addition of gramicidin, i.e., inactivation of the taurine pathway in NIH3T3 cells was in the presence of vanadate estimated to increase from 35% to 100% and from 35% to 70% within 8 min following the substitution of isotonic solution for the hypotonic solution or substitution of the hypotonic gramicidin-containing solution for the hypotonic solution, respectively (Fig. 7D). In the case of Ehrlich Lettre cells, restoration of the original cell volume following exposure to the hypotonic, Na+-free, vanadate-containing medium had no greater effect on the inactivation of the volume-sensitive taurine efflux pathway (Fig. 7D), most probably because the inactivation of the efflux pathway in the presence of vanadate was already significant and close to the control values in nontreated cells (Fig. 7D). Taken together, the lack of an effect of H2O2 and vanadate on taurine release under isotonic conditions and following restoration of the cell volume indicates that inhibition of PTP activity in NIH3T3 cells modulates the activity of the volume-sensitive efflux pathway for taurine once it is activated and not the activation per se.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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) at the nuclear envelope within the first minutes following hypotonic exposure and a concomitant mobilization of arachidonic acid and oxidation of arachidonic acid to leukotrienes via the 5-LO system have similarly been demonstrated in Ehrlich ascites tumor cells (26, 37). Activation of iPLA2 isoforms has been previously reported to involve ROS (17, 32), and it seems plausible that ROS via peroxidation of lipids, shift in membrane fluidity, or oxidative modification of proteins could affect iPLA2 activity and thus account for the swelling-induced release of arachidonic acid from the nuclear envelope and the concomitant release of taurine in NIH3T3 cells. However, the swelling-induced ROS production in NIH3T3 cells is impaired in the presence of BEL (20), indicating that stimulation of ROS-producing enzymes is downstream from PLA2 activation. It is noted that the addition of vanadate under isotonic conditions increases ROS production (Fig. 4), which, according to the results shown in Fig. 2, should elicit an increase in arachidonic acid mobilization. However, no detectable effect on arachidonic acid mobilization (Fig. 2) or taurine release (Fig. 1) was seen in vanadate-treated cells under isotonic conditions, presumably because the ROS production following vanadate exposure was quantitatively insufficient to activate PLA2. NOX and swelling-induced ROS production. Phagocyte NOX is acknowledged for its role in host defense, whereas NOX in nonphagocytes has been assigned a role in intracellular cell signaling (30) and cell volume restoration (20, 45). The NOX system comprises catalytic, membrane-integrated gp91phox homologs (NOX1, NOX3, NOX4, NOX5, DUOX1, and DUOX2) plus the regulatory units Rac1/2, p22phox, p47phox (NOXO1), p40phox, and p67phox (NOXA1) (30, 40). Osmotic cell swelling is accompanied by increased ROS production in, e.g., NIH3T3 cells (20) (Fig. 4), pig skeletal muscle cells (35), HTC cells (45), and Ehrlich Lettre cells (Fig. 4). PLA2 products, e.g., arachidonic acid and lysophosphatidyl choline, in themselves increase ROS production (22), and, as the swelling-induced ROS production in NIH3T3 cells is impaired in the presence of the NOX inhibitor DPI (M. B. Friis, K. G. Vorum, and I. H. Lambert, unpublished data), it is conceivable that NOX contributes to the swelling-induced ROS production in NIH3T3 cells. It is noted that DPI interferes with at least two components of the NOX complex that differ by their sensitivity to the inhibitor (8), i.e., the negligible versus pronounced effect of DPI on the maximal rate constant for the swelling-induced taurine efflux in control NIH3T3 cells (Fig. 5B) and control Ehrlich Lettre cells (Fig. 5A) could reflect variation in the expression of regulatory and catalytic components of NOX in the two cell types.
Several PTKs are involved in the regulation of the cellular pool of organic osmolytes in mammalian cells (see Ref. 36), and it has been demonstrated that the swelling-induced taurine efflux as well as the potentiating effect of H2O2 and vanadate on the swelling-induced taurine efflux in NIH3T3 are strongly impaired in the presence of the general protein kinase inhibitor genistein (20). Furthermore, nonreceptor PTK c-Src has been shown to stimulate NOX-driven generation of ROS in a process that involves tyrosine phosphorylation and subsequent translocation of the regulatory unit p47phox (4, 43). It is thus conceivable that increased net tyrosine phosphorylation and activation of p47phox could explain the increased ROS production in vanadate-exposed cells (Fig. 4).
The volume-sensitive taurine efflux is potentiated following exposure to Ca2+-mobilizing agonists (20, 29) and the PKC-activating agent PMA (21). It has been shown that the Ca2+-induced potentiation in the case of HeLa cells involves calmodulin and PKC (novel PKC) activity (9), whereas PMA-induced potentiation in the case of NIH3T3 cells involves NOX (21). NOX activity is regulated by Ca2+ (2) and by phosphorylation of p47phox by classic/novel, Ca2+-sensitive PKCs (11). Furthermore, exposure to vanadate under hypotonic conditions elicits an instantaneous increase in the cellular Ca2+ concentration in NIH3T3 and Ehrlich Lettre cells (fura2 estimation; M. B. Friis, K. G. Vorum, and I. H. Lambert, unpublished data) as well as an increase in ROS production (Fig. 4). It is currently under investigation whether the effect of vanadate on the swelling-induced taurine release reflects increased NOX activity provoked by Ca2+ mobilization or a shift in the net tyrosine phosphorylation of the p47phox (NOXO1) unit.
ROS potentiate volume-sensitive taurine efflux. Several lines of evidence have indicated that ROS are not involved in the activation of the volume-sensitive taurine efflux pathway per se but modulate volume-sensitive taurine efflux once it has been activated: 1) H2O2 does not induce taurine efflux under isotonic conditions but potentiates taurine release under conditions where PLA2 activity is ensured by either osmotic cell swelling or exposure to melittin (20, 27, 39); 2) H2O2 does not affect the volume set point for the efflux pathway, i.e., the degree of cell swelling required for the activation of volume-sensitive taurine efflux (20); 3) the volume-sensitive taurine efflux pathway is inactivated following restoration of cell volume even in the presence of H2O2 and vanadate (Fig. 7); 4) H2O2 has no effect on swelling-induced taurine release in the presence of a 5-LO inhibitor (20); 5) exposure to the antioxidant BHT or NAC reduces swelling-induced taurine efflux (20); and 6) exposure to the NOX inhibitor DPI accelerates the inactivation of volume-sensitive taurine efflux (20) (Fig. 8). H2O2 and the phosphate analog vanadate are assumed to reduce cellular PTP (subtype PTP1B) activity via oxidation of a cystein hydroxyl group in the catalytic site of the phosphate or by competitive inhibition, respectively (13, 33). As shown in Figs. 3 and 5, the iPLA2 inhibitor BEL and the NOX inhibitor DPI significantly reduced the swelling-induced taurine efflux in vanadate-treated NIH3T3 and Ehrlich Lettre cells. Thus, it is conceivable that the potentiation of the rate constant for the volume-sensitive taurine efflux following inhibition of PTP activity reflects a concomitant increase in the activity of BEL-sensitive iPLA2 (arachidonic acid mobilization), NOX (ROS production), and/or the 5-LO system (production of second messengers for downstream signaling). In this context, it is noted that ROS are reported to stimulate iPLA2 isoforms (17, 32) and NOX (45), and they are required for the activation of the 5-LO system (34). Thus, swelling-induced ROS production could stimulate 5-LO activity and, at the same time, constitute a positive feedback loop.
ROS delay inactivation of the volume-sensitive taurine efflux pathway. Exposure to ROS delays the inactivation of the volume-sensitive taurine efflux pathway in NIH3T3 cells and, to a minor extent, in Ehrlich Lettre cells (Fig. 1D). On the other hand, vanadate-induced inhibition of PTPs delays the inactivation of volume-sensitive taurine efflux in NIH3T3 cells significantly, whereas it has no or even a stimulating effect on the inactivation of the efflux pathway in Ehrlich Lettre cells (Fig. 1). Volume-sensitive efflux pathways for K+ and Cl– and thus the general volume-restoring process following hypotonic exposure are also modulated by PTKs and PTPs (see Ref. 36), and, as taurine release increases with cell volume (20), the vanadate-induced delay in the inactivation of the volume-sensitive taurine efflux pathway in NIH3T3 cells could reflect impaired volume restoration. As shown in Fig. 7, the volume-sensitive taurine efflux pathway is inactivated when the original cell volume is restored either by reexposure to isotonic medium or by exposure to gramicidin under hypotonic, Na+-free conditions. As vanadate has a minor accelerating effect on volume restoration following hypotonic cell swelling in both cell lines (Fig. 6), it seems reasonable to assume that the vanadate-induced delay of the inactivation of the volume-sensitive taurine efflux pathway in NIH3T3 cells reflects an increased tyrosine phosphorylation of components that modulate the open probability of the volume-sensitive taurine efflux pathway and not an indirect effect due to inhibition of K+ and Cl– conductance and thus the overall ability to restore the cell volume. However, the vanadate-induced delay of the inactivation of the swelling-induced taurine efflux pathway in NIH3T3 cells was completely abolished in the presence of DPI (Fig. 8). The PTP inhibitor pervanadate has previously been shown to activate NOX via a tyrosine kinase-dependent pathway (48). Hence, an increase in the net tyrosine phosphorylation of a regulatory component of NOX and a concomitant increase in the oxidase activity (Fig. 4) could explain the vanadate-induced impairment of the inactivation of the volume-sensitive taurine efflux pathway in NIH3T3 cells. Whether the open probability of the yet-unidentified taurine efflux pathway is directly increased by ROS-mediated oxidation or an increase in the net tyrosine phosphorylation is unknown. We are currently investigating whether the difference in the inactivation of the volume-sensitive taurine pathway in vanadate-treated NIH3T3 and Ehrlich Lettre cells can be explained by differences in either the expression of regulatory components and thus the activity of multicomponent NOX or an antioxidative capacity in the two cell lines.
In conclusion, inhibition of PTP activity in NIH3T3 and Ehrlich Lettre cells, i.e., a putative increase in the net tyrosine phosphorylation of various proteins/enzymes, increases arachidonic acid mobilization, stimulates ROS production, and potentiates taurine efflux under hypotonic conditions. PTP inhibition impairs the inactivation of the volume-sensitive taurine efflux pathway in NIH3T3 cells but has no effect on the inactivation of the efflux pathway in Ehrlich Lettre cells. However, the potentiation of swelling-induced taurine release is impaired and inactivation of the volume-sensitive taurine efflux pathway is accelerated dramatically following inhibition of NOX. It is suggested that 1) increased tyrosine phosphorylation of regulatory components of NOX leads to increased ROS production and subsequent potentiation of the swelling-induced taurine efflux and a delay in the inactivation of the volume-sensitive taurine efflux pathway, and 2) the opposing effect of PTP inhibition on inactivation of the volume-sensitive taurine efflux pathway in NIH3T3 and Ehrlich Lettre cells could reflect differences in the expression/activity of NOX or the antioxidative capacity.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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) or hypotonic conditions in the absence (
) or presence (
) of vanadate, is shown as a function of time for NIH3T3 (A) and Ehrlich Lettre cells (B). C and D: arachidonic acid release from NIH3T3 (C) or Ehrlich Lettre cells (D) obtained after a 24-min incubation in isotonic NaCl medium (open bars) or hypotonic NaCl medium (closed bars) in the absence or presence of H2O2 or vanadate. All data are given as mean values ± SE of 3 sets of experiments. #Significantly larger compared with isotonic control; *significantly larger than hypotonic control.
isotonic). Inactivation was estimated at time = 24 min. D: taurine release from NIH3T3 and Ehrlich Lettre cells was estimated in Na+-free NMDG-Cl media in the absence (control) or presence of 50 µM vanadate according to the protocol in B, i.e., isotonic NMDG-Cl medium was at time = 6 min shifted to hypotonic NMDG-Cl medium and the hypotonic medium was at time = 16 min either kept hypotonic or shifted to hypotonic NMDG-Cl medium containing 2 µM gramicidin (Hypo