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CELLULAR METABOLISM
Department of Biochemistry, Institute for Molecular Biology and Physiology, Copenhagen, Denmark
Submitted 4 July 2005 ; accepted in final form 11 July 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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osmotic stress; cell volume regulation; calcium-independent phospholipase A2; secretory phospholipase A2; nucleus
The PLA2 family, which hydrolyzes phospholipids to free fatty acids and membrane-bound lysophospholipids, includes 1) the low-molecular-mass secretory PLA2s (sPLA2s), which require millimolar concentrations of Ca2+ for their catalytic activity, 2) the high-molecular-mass (85–110 kDa) cytosolic Ca2+-dependent PLA2s (cPLA2s, also known as group IVA, -B, and -C), which do not require Ca2+ for catalysis but are dependent on a free intracellular Ca2+ concentration in the micromolar range to translocate to the membrane fraction, 3) the high-molecular-mass (85–90 kDa) Ca2+-independent PLA2s (iPLA2
and -
, also known as group VIA and VIB, respectively), and 4) the platelet-activating factor acetylhydrolases (14, 38). Whereas the role of cPLA2 in stimulus-induced arachidonic acid release is well established, the possible roles of sPLA2 and particularly iPLA2 in cellular signaling events are less well understood (see Ref. 14). In mammals, sPLA2 is involved in arachidonic acid metabolism, regulation of exocytosis, antimicrobial defense, phospholipid digestion, and has been implicated in the pathophysiology of inflammation and ischemia (see Ref. 14). A major function of iPLA2 is in basal phospholipid remodeling; however, recent studies also suggest the involvement of iPLA2 in a number of cellular signaling pathways associated with arachidonic acid release, as well as in the phospholipid reorganization occurring during apoptosis (14, 44). In accordance with these diverse roles, iPLA2 appears to localize to multiple cellular compartments, including mitochondria (4, 23), endoplasmic reticulum (13), plasma membrane lipid rafts (26), and, after activation, also to the nuclear membrane (37).
The available evidence indicates that the specific PLA2 enzymes and arachidonic acid metabolites involved in swelling-induced activation of osmolyte efflux vary between cell types (16, 31, 42). In NIH3T3 cells, swelling-induced taurine efflux was attenuated by the iPLA2 inhibitor bromoenol lactone (BEL) and potentiated by the calmodulin antagonist W7, suggesting a role for iPLA2, whereas cPLA2 did not appear to be involved (15). Preliminary data also indicated a contribution from sPLA2 activity (15). Consistent with this notion, direct activation of two different snake venom sPLA2s by osmotic swelling has been demonstrated in artificial lipid vesicles (21). However, neither the volume sensitivity of iPLA2 activation nor the potential role of sPLA2 in cell volume regulation has ever been directly addressed.
Melittin, a cationic amphiphilic bee venom peptide, potently stimulates sPLA2-mediated fatty acid release, apparently not by directly activating PLA2 but by increasing substrate availability (5). A role for PLA2 isoforms other than sPLA2 in melittin-induced arachidonic acid release also has been suggested (36, 45) but, to our knowledge, not comprehensively addressed. We have previously demonstrated that in NIH3T3 cells, nanomolar concentrations of melittin stimulate taurine efflux under isotonic conditions via a pathway that is pharmacologically similar to that activated by cell swelling (15). Melittin-induced taurine efflux was inhibited by both the iPLA2 inhibitor BEL and arachidonyl trifluoromethyl ketone (AACOCF3), which inhibits both cPLA2 and iPLA2, suggesting the involvement of several PLA2 isoforms (15). Further downstream to PLA2 activation, both swelling- and melittin-induced taurine efflux were dependent on arachidonic acid metabolites produced via the 5-lipoxygenase (5-LO) pathway (15).
Thus the present study was initiated to investigate the volume sensitivity and causal relationship between PLA2 activation and taurine efflux in NIH3T3 cells. The main questions asked were 1) does the volume sensitivity of taurine efflux reflect a similar volume sensitivity of PLA2 activity and, if so, by which PLA2 isoform(s)?, and 2) do cell swelling and melittin activate taurine efflux by similar pathways?
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Unless otherwise indicated, reagents were of the highest analytical grade and obtained from Sigma Aldrich (St. Louis, MO). Rhodamine-conjugated phalloidin was obtained from Molecular Probes (Leyden, The Netherlands). Paraformaldehyde was prepared fresh regularly as a 20% stock solution in double-distilled H2O (ddH2O), filtered, and kept at 4°C. The standard isotonic medium contained (in mM) 143 NaCl, 5 KCl, 1 Na2HPO4, 1 CaCl2, 0.1 MgSO4, 10 HEPES, and 5 glucose, pH 7.4, 300 mosmol/l. Hypo- (200 mosmol/l) and hypertonic media (600 mosmol/l) were prepared by adjusting the NaCl concentration. Na+-free and KCl media were prepared by substituting N-methyl-D-glucamine (NMDG)-Cl or KCl, respectively, for NaCl in equimolar amounts. Tris-buffered saline (TBS) contained (in mM) 150 NaCl, 10 Tris·HCl, 1 MgCl2, and 1 EGTA, pH 7.4. The iPLA2-VIA antibody used for immunocytochemistry was obtained from Cayman Chemical (Ann Arbor, MI).
Cells and Cell Culture
NIH3T3 mouse fibroblasts (clone 7), a kind gift from Prof. B. Willumsen (Dept. of Molecular Cell Biology, University of Copenhagen) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum and 100 U/ml penicillin-streptomycin (both from Invitrogen Life Technologies, Taastrup, Denmark), in a 37°C, 5% CO2, 100% humidity incubator. Cells were passaged every 3–4 days by gentle trypsination, and only passages 10–30 were used for experiments.
Estimation of Taurine Efflux and Arachidonic Acid Release
Taurine efflux was estimated at room temperature on cells grown to 80% confluence and loaded in DMEM containing [3H]taurine (80 nCi/ml, 2 h). The cells were washed five times with isosmotic solution, and the efflux was initiated by aspiration of the medium followed by addition of experimental solution. The cells were left for 2 min, after which the entire medium was transferred to a scintillation vial and rapidly substituted by 1 ml fresh medium. This procedure was repeated for 20–30 min. The cells were lysed at the end of the experiment (NaOH, 0.5 M). The total 3H activity in the cell system was estimated as the sum of 3H activity (beta scintillation counting; Ultima Gold) in all the efflux samples, the NaOH lysate, plus two final washouts with ddH2O. The natural logarithm to the fraction of 3H activity remaining in the fibroblast was plotted against time, and the rate constant for the taurine efflux (min–1) at each time point was estimated as the negative slope of the curve between the time point and the proceeding time point (see Refs. 15, 30). Arachidonic acid release was estimated using a similar protocol with the exception that cells were loaded with [3H]arachidonic acid (150 nCi/ml, 24 h) and 0.5% BSA was added to the efflux medium to trap arachidonic acid. The release of [3H]arachidonic at a given time point is shown as the total fraction released (%).
Estimation of Release of Arachidonic Acid From Nuclei
[3H]arachidonic acid release from nuclei was estimated using a Sigma NUC-201 nuclei isolation kit. NIH3T3 fibroblasts were grown in 75-cm2 culture flasks to 80–90% confluence and preincubated with [3H]arachidonic acid 24 h before initiation of the experiments. After preincubation, the cell monolayer was washed three times in 10 ml of isotonic Ringer containing 1% BSA. BEL (10 µM) was added for the inhibition experiments, and all flasks were left to rest for 30 min. Ringer was removed, and cells were incubated in either isotonic or hypotonic Ringer for 5 min. After stimulation, the Ringer was removed and cells were lysed in 8 ml of lysis buffer containing Nuclei PURE lysis buffer, 1 mM DTT, and 10% Triton X-100. Cells were harvested using a cell scraper and collected in 50-ml tubes, briefly vortexed, and incubated on ice for 5 min. Samples (100 µl) were taken for protein content estimations. Nuclei were purified by centrifuging through a sucrose cushion solution: 14 ml of 1.8 M cushion solution were added to the lysate and gently mixed. Next, 8 ml of 1.8 M cushion solution were added to the bottom of an ultracentrifuge tube, and the 22 ml of lysate-cushion solution mix were gently layered on top of the 8 ml of cushion solution. The tubes were centrifuged at 30,000 g at 4°C for 45 min and placed on ice. Nuclei were visible as a small pellet at the bottom of the tubes. The supernatant was transferred to scintillation vials for [3H]arachidonic acid activity estimations, and the remaining nuclei were washed three times in 2 ml of PBS and lysed in 2 ml of 1 M NaOH. [3H]arachidonic acid in the nuclei was calculated as the fraction of the total [3H]arachidonic acid activity present in the cell system per milligram of protein. Total [3H]arachidonic acid activity was estimated as the sum of 3H activity in the supernatants, the washes, and the NaOH lysate.
Visualization of iPLA2 Localization
Cells were seeded 24–48 h before experiments on HCl- and ethanol-washed no. 1 glass coverslips. After stimulation, cells were fixed in 2% paraformaldehyde for 15 min at room temperature, followed by 30 min on ice, three washes in TBS, permeabilization for 10 min in TBS containing 0.1% Triton X-100, and three washes in TBS. For simultaneous visualization of iPLA2, F-actin, and nuclei, cells were fixed and permeabilized as described above, and unspecific binding sites were blocked for 30 min in TBS containing 2.5% BSA. Subsequently, cells were incubated with the primary antibody against iPLA2-VIA (1:100 in TBS + 1% BSA) overnight at 4°C, washed three times in TBS + 1% BSA, incubated with secondary antibody (1:400 in TBS + 1% BSA) plus rhodamine-phalloidin (2 U/ml) for 1 h at room temperature, washed three times as before, and incubated with 4,6-diamidino-2-phenylindole (1:600 in TBS + 1% BSA) for 5 min at room temperature, followed by three final washes. Coverslips were mounted (N-propyl-galleate 2% wt/vol in PBS/glycerin) and visualized using a x40/1.25 NA plan apochromat objective and the UV and 488- and 568-nm argon-krypton laser lines of a Leica DM IRB/E microscope equipped with a Leica TSC NT confocal laser scanning unit (Leica Lasertechnik, Heidelberg, Germany). Optical slice thickness was 1 µm, and pinhole size was 1 Airy disc. Images shown are frame-averaged and presented in RGB pseudocolor.
Cell Volume and Intracellular pH Measurements
Large-angle light scattering and estimation of intracellular pH. Relative cell volume was estimated using large-angle light scattering, as previously described (32), except that intracellular pH (pHi) was simultaneously detected using BCECF-tetraacetoxy methyl ester (BCECF-AM). Briefly, cells were seeded on rectangular coverslips, with 70–80% confluency at the time of experiments. Cells were incubated with 1.6 µM BCECF-AM for 30 min, washed twice, and incubated for a further 15 min to allow AM-ester cleavage, all in isotonic saline. Cells were placed at a 50° angle relative to the excitation light in a cuvette in a PTI RatioMaster spectrophotometer with a 75-W xenon lamp. Light was collected in 7-s cycles of excitation at 577 nm with emission measured at 580 nm for detection of light scattering, and excitation at 445 and 495 nm with emission measured at 525 nm for detection of BCECF fluorescence. The cells were continuously perfused with the experimental media at 1.6 ml/min (solutions were changed by briefly increasing flow to 3.5 ml/min). BCECF fluorescence was converted to pHi values essentially as previously described (34) and had no effect on the light scattering signal. Light scattering data are given as the reciprocal of the emission intensity (I) normalized to that obtained under steady-state isotonic conditions (I0), i.e., I0/I (because emitted light intensity correlates inversely with cell volume).
Coulter counter measurements. Cells grown to 80–90% confluency in 150-cm2 culture flasks were detached by treatment with a Ca2+- and Mg2+-free trypsin-EGTA solution for 2 min at 37°C. The cell suspension was transferred to a Sorwal glass, centrifuged gently (30 s, 700 g), and resuspended in isotonic saline. Resuspended cells (2 ml) were diluted 25 times in filtered (0.45-µm filters; Millipore) experimental saline in the cell chamber of a Coulter Multisizer II with a 100-µm tube orifice. Absolute cell volumes were calculated from the median of the cell volume distribution curves after calibration with latex beads (14.1-µm diameter; Coulter Electronics).
Estimation of K+ Content
Cells grown to 80% confluency in 75-cm2 flasks were washed three times in isotonic NaCl and incubated for 10 min in iso- or hypotonic NaCl medium in the absence or presence of 1 µg/ml melittin. At the end of the incubation period, the cells were washed rapidly with ice-cold PBS, the PBS was aspirated completely, and the cells were lysed in 2 ml of ddH2O before deproteinization with 100 µl of perchloric acid (70%). The cell homogenate was centrifuged to pellet debris (5 min, 20,000 g), and K+ content was estimated using emission flame photometry.
Isolation of RNA, Reverse Transcription, and PCR
Total RNA was isolated from NIH3T3 fibroblasts using an RNeasy mini kit from Qiagen according to the manufacturer’s protocol. Before cDNA synthesis, the quality of the RNA was evaluated using agarose gel electrophoresis. cDNA was synthesized in a total volume of 40 µl by hybridization of 500 ng of random primers to 4 µg of RNA at 65°C for 5 min, followed by extension at 42°C for 50 min in the presence of 200 units of Superscript II RT, 500 µM dNTPs, 10 µM DTT, 50 mM Tris·HCl, pH 8.3, 75 mM KCl, and 3 mM MgCl2. Finally, the RT was inactivated at 70°C for 15 min. PCR was performed in a total volume of 20 µl containing 1 µl of the RT reaction cDNA, 0.5 mM dNTPs, 0.5 µM of each primer, 2 mM MgCl2, and 2 units of Taq polymerase in PCR buffer (50 mM KCl and 10 mM Tris·HCl, pH 9.0). The conditions for PCR were an initial denaturation step at 95°C for 2 min, followed by 35 cycles of 95°C for 30 s, 58°C for 30 s, 72°C for 2 min, and a final extension step at 72°C for 10 min. Primers used for PCR were iPLA2 (forward, 5'-GGAGTTCCTGAAGCGGGAGT-3'; reverse, 5'-TGAGTCGGTGGCTTCAGGTT-3'), iPLA2 (forward, 5'-GAATAACCCTTCGGCCTTGG-3'; reverse, 5'-AGAAGGCAACAGGCCATCAA-3'), cPLA2
(forward, 5'-GTGGGCGAAAATGAACAAGC-3'; reverse, 5'-CGATTCGGGGTCATCAAAAA-3'), cPLA2 (forward, 5'-GGCACTGGCCAACCTCTATG-3', reverse, 5'-ATTGCTCCAGATGCCTTCCA-3'), cPLA2 (forward, 5'-ACCCTGCACTTGGGGCTTAT-3; reverse, 5'-TCCTTGATGCTGGGGTCATT-3'), sPLA2-GIIA (forward, 5'-CAGTTTGGGGAAATGATT-3'; reverse, 5'-CTGGTTTGCAGAACAGGTGA-3'), and sPLA2-G5 (forward, 5'-GATGCACGACCGTTGTTATG-3'; reverse, 5'-CAGGCAGTAGACCAGCTTCC-3'). The origin of cPLA2 and iPLA2 PCR products was confirmed by PCR cloning and sequencing.
In Vitro PLA2 Activity Assay
NIH3T3 cells were lysed in 50 mM HEPES, pH 7.4, 1 mM EDTA, 1 mM sodium orthovanadate, and a protease inhibitor cocktail (Sigma) diluted 1:100 and sonicated on ice twice for 10 s. Cell homogenates were then cleared by centrifugation (10 min, 14,000 g) at 4°C, and supernatants were spun through 30-kDa cutoff filters (Microcon YM-30; Millipore) (12 min, 14,000 g). Activity was measured using the PLA2 substrate arachidonoyl thiophosphatidylcholine and following the protocol recommended by the manufacturer (Cayman Chemical). Aliquots (10 µl) of supernatants containing 25–30 µg of protein were added to 96-well plates together with 5 µl of assay buffer (80 mM HEPES, 150 mM NaCl, 10 mM CaCl2, 4 mM Triton X-100, 30% glycerol, and 1 mg/ml BSA). Arachidonoyl thiophosphatidylcholine was dissolved in 160 mM HEPES, 300 mM NaCl, 20 mM CaCl2, 8 mM Triton X-100, 60% glycerol, and 2 mg/ml BSA to a final concentration of 1.5 mM. The reaction was initiated by addition of 200 µl of substrate to each well and was run for 60 min. Where applicable, melittin and/or PLA2 inhibitors were added to both the assay buffer and the substrate 10 min before start of the experiments. The reaction was stopped by addition of 10 µl of a solution containing 25 mM 5,5'-dinitrobis(2-nitrobenzoic acid) (DTNB) plus 475 mM EGTA in 0.5 M Tris·HCl (pH 8.0). The absorbance was measured at 405 nm in a Fluostar Optima plate reader (BMG LabTechnologies). PLA2 activity was expressed as micromoles per minute per milligram of protein, determined from the extinction coefficient of DTNB using Lambert-Beer’s law and the protein content in the supernatants.
Statistics and Data Presentation
Data are presented either as means ± SE of at least three independent experiments or as individual experiments representative of at least three independent experiments. Statistical significance was estimated using Student’s t-test, with 0.05 as the level of significance.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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We previously found swelling-induced taurine efflux in NIH3T3 cells to be BEL sensitive, suggesting a role for iPLA2 in the activation process (15). In the present study, we further evaluated the causal relationship among cell volume, arachidonic acid release, and taurine efflux in NIH3T3 cells. Figure 1A shows the rate constant for taurine efflux over time under isotonic (300 mosmol/l), hypotonic (200 mosmol/l), and hypertonic (600 mosmol/l) conditions. As shown, efflux rate constants were robustly increased in hypotonically swollen cells and reduced in hypertonically shrunken cells. In contrast, arachidonic acid release to the extracellular compartment was unaltered under hypotonic conditions compared with that measured in isotonic control cells but was significantly reduced under hypertonic conditions (Fig. 1B).
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iPLA2 has been reported to translocate to the nuclear membrane upon activation (37), Hence, to address the second possibility, we exposed the cells to hypotonic swelling in the absence and presence of BEL, isolated the nuclei, and measured the arachidonic acid content of the nuclear fraction under isotonic conditions and after swelling with and without preincubation with the iPLA2 inhibitor BEL (Fig. 2). As shown, hypotonic swelling was associated with a loss of arachidonic acid from the nuclear fraction, which was completely inhibited by BEL. Consistent with the notion that this reflects an increase in iPLA2-mediated arachidonic acid release from the nuclear fraction, iPLA2 appeared to translocate to the nuclear membrane of swollen NIH3T3 cells (Fig. 3). As shown, under isotonic conditions (Fig. 3B), iPLA2-VIA immunostaining in NIH3T3 cells was found predominantly as a punctuate labeling in lamellipodia-like regions at the plasma membrane and in endoplasmic reticulum-like vesicular structures. After a 5 min exposure to hypotonic conditions (Fig. 3D), nuclear localization was detectable in a significant fraction of the cells, and after 60 min of hypotonic exposure (Fig. 3E), marked nuclear localization was evident in essentially all cells. In contrast, after 5 min of exposure to hypertonic conditions (Fig. 3C), nuclear labeling was completely absent and the localization to lamellipodia-like regions at the plasma membrane more pronounced. Moreover, the size of the iPLA2-iPLA2-VIA-labeled vesicular structures appeared to increase upon cell swelling and decrease upon cell shrinkage, respectively. Thus, taken together, the data in Figs. 2 and 3 indicate that iPLA2-mediated arachidonic acid occurs at least in part from the nuclear membrane in swollen NIH3T3 cells. With regard to the specificity of the vesicular labeling pattern, it may be noted that 1) staining of C2C12 myoblasts with this antibody and 2) staining of NIH3T3 cells using another iPLA2-VIA antibody (Santa Cruz Biotechnology; not shown) yielded a similar pattern, 3) there was no detectable staining in the absence of primary antibody, and 4) staining of NIH3T3 cells with numerous other antibodies by identical procedures never elicited this staining pattern.
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350 nM), arachidonic acid release was robustly increased by cell swelling and decreased by shrinkage, respectively, compared with isotonic conditions (Fig. 4A). Comparison with Fig. 1 reveals that melittin potentiates arachidonic acid release to the cell exterior modestly under isosmotic conditions and substantially in swollen cells, yet not at all or only marginally in shrunken cells. Similarly, melittin potentiated taurine efflux in a volume-sensitive manner, resulting in a robust difference in efflux between isotonic and shrunken cells (Fig. 4B).
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The above-described findings indicate that increased substrate availability causes an increase in a volume-sensitive PLA2 activity and a concomitant increase in taurine efflux. A number of effects of melittin unrelated to substrate availability, however, have been reported (7, 28, 43); hence, it was important to further analyze the effects of melittin on arachidonic acid release and osmolyte efflux in NIH3T3 cells. If exposure to melittin stimulates release of taurine and/or other osmolytes, this should be associated with a cell shrinkage, which, similar to the melittin-induced arachidonic acid release and taurine efflux, should be potentiated in swollen cells and inhibited in shrunken cells. Under isotonic conditions (Fig. 5, A and B), exposure to 1 µg/ml melittin elicited a rapid and transient swelling of NIH3T3 cells, followed by cell shrinkage. Hypotonic challenge (Fig. 5C) elicited cell swelling and RVD under control conditions. In melittin-treated hypotonic cells, the shrinkage following the initial swelling was strongly potentiated compared with that in hypotonic control cells (Fig. 5, C and D), resulting in a final volume considerably smaller than the original steady-state volume. Hypertonic challenge (Fig. 5E) elicited rapid osmotic cell shrinkage with no recovery but rather a slight continued shrinkage. Melittin had no detectable effect on cell volume in the osmotically shrunken cells (Fig. 5F). To confirm and quantify these effects, we also analyzed cell volume changes by electronic cell sizing, from which it is seen that 8 min after melittin exposure under isotonic conditions, cell volume was reduced to 
64% of the initial value (Table 1). At an estimated intracellular concentration of 10 mM (27), the taurine efflux is unlikely to be of a sufficient magnitude to alone explain the substantial cell shrinkage following the initial swelling in cells exposed to melittin under iso- and hypertonic conditions. In two sets of experiments, each performed in duplicate, it was estimated that over a period of 10 min, 1 µg/ml melittin reduced the cellular K+ content under isotonic conditions by 4% and increased the K+ loss following hypotonic exposure (200 mosmol/l) from 6 to 20%. This indicates that the massive cell shrinkage induced by melittin under hypotonic exposure reflects net loss of taurine, K+, and, for reasons of electroneutrality, also Cl–. Cl– lost from swollen cells via volume-sensitive pathways is recycled back into the cell via the Cl–/HCO3– exchanger, resulting in cellular acidification (24). In congruence with this, melittin treatment was associated with a reduction of pHi, which was modest under isotonic, substantial under hypotonic, and absent under hypertonic conditions, consistent with the expected loss of Cl– under these conditions (n = 3–4 for each condition; not shown). The melittin-induced cell shrinkage was inhibited by ETH 615-139 but not by the cyclooxygenase pathway inhibitor indomethacin (Table 1). Thus, similar to swelling-induced taurine efflux (15), melittin-induced osmolyte loss is dependent on 5-LO metabolites.
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Identity of the PLA2 Isoform(s) Activated by Increases in Cell Volume or Exposure to Melittin
We have previously shown that the taurine efflux pathway(s) activated by cell swelling and melittin are pharmacologically highly similar (15); however, the relative roles of different PLA2 isoforms under these conditions have not been analyzed in detail. PCR analysis indicated that NIH3T3 cells express at least three cPLA2 isoforms (cPLA2-IVA, cPLA2-IVB, and cPLA2-IVC), two iPLA2 isoforms (iPLA2-VIA and iPLA2-VIB), and one sPLA2 isoform (sPLA2-V), whereas sPLA2-IIA could not be detected (Fig. 6). We therefore next evaluated the relative roles of these PLA2 isoforms in swelling and arachidonic acid release. Well-characterized pharmacological inhibitors were used to inhibit iPLA2 [BEL (1)] and sPLA2 [manoalide (12)]. AACOCF3, which is most commonly reported to inhibit both cPLA2 and iPLA2 (1), but which has no effect on at least some forms of iPLA2 (4), was also applied in some experiments (regarding PLA2 inhibitor specificity, see also Ref. 2). Under control conditions, arachidonic acid release in intact NIH3T3 cells under iso- and hypotonic conditions was partially inhibited by both BEL (30 µM) and manoalide (5 µM) (Fig. 7A). Similarly, both BEL and manoalide significantly inhibited melittin-induced arachidonic acid release in intact NIH3T3 cells (Fig. 7A). AACOCF3 had no effect on arachidonic acid release from intact cells under any of the conditions tested. Thus, in the presence of 40 µM AACOCF3, the relative release at time 14 min was estimated at 0.89 ± 0.17 (isotonic conditions, n = 3), 1.09 ± 0.15 (hypotonic conditions, n = 4), 0.97 ± 0.03 (isotonic conditions plus melittin, n = 5), and 1.12 ± 0.19 (hypotonic conditions plus melittin, n = 5).
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If taurine efflux is a simple downstream function of arachidonic acid release to the cell exterior, then the pharmacological profiles of the measured arachidonic acid release and taurine efflux should be similar. Swelling-activated taurine efflux was potently inhibited by BEL and partially by manoalide (Fig. 8), in agreement with Lambert (15). Melittin-induced taurine efflux was inhibited potently by manoalide and partially by BEL under both isotonic and hypotonic conditions. The effect of AACOCF3 was not investigated, because this compound has previously been shown to be without effect on swelling-activated taurine efflux and to partially inhibit melittin-activated taurine efflux in NIH3T3 cells (15).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Swelling-induced taurine efflux has been proposed to be downstream from PLA2-mediated arachidonic acid release in several cell types, including NIH3T3 cells, based on pharmacological evidence (3, 15, 19, 22, 41; see Ref. 9). In the present study, we further studied the causal relationship between PLA2 activity and taurine efflux after cell volume perturbation. Under control conditions, taurine efflux was inhibited by osmotic shrinkage in addition to its previously demonstrated activation by swelling, whereas arachidonic acid release was similarly inhibited by shrinkage but not significantly increased by swelling. However, three lines of evidence indicated that PLA2 activity in NIH3T3 cells was increased by osmotic swelling, despite the fact that arachidonic acid release to the cell exterior was not detectably increased. Thus 1) swelling-induced arachidonic acid release was potentiated by a 5-LO inhibitor, indicating that arachidonic acid release was masked by rapid oxidation, similar to findings in Ehrlich cells (18, 31); 2) swelling was associated with loss of arachidonic acid from the nuclear fraction, to where iPLA2-VIA appeared to translocate in swollen cells. If the majority of the arachidonic acid is released from the nucleus, where it is also metabolized [as 5-LO also localizes to the nuclear membrane (25)], this will further reduce the likelihood that the release will be detectable in the cell exterior. Finally, 3) in the presence of melittin, which increases substrate availability for PLA2 (5), arachidonic acid release was potentiated and exhibited marked volume dependence, and taurine efflux and osmolyte loss (estimated as cell shrinkage) were augmented in a similar, volume-dependent manner. Inhibition of 5-LO also blocked melittin-induced osmolyte efflux from NIH3T3 cells, consistent with the effect of this compound on swelling- and melittin-induced taurine efflux (15). In aggregate, these findings are most consistent with the interpretation that cell swelling increases PLA2 activity, and thus arachidonic acid release, at least in part from the nuclear membrane and that, conversely, cell shrinkage reduces PLA2 activity and arachidonic acid release. The arachidonic acid is metabolized to 5-LO products, resulting in parallel changes in osmolyte efflux. An important implication of these findings is that the volume-sensitive PLA2 activity, and consequently the taurine efflux pathway it stimulates, has a set point (i.e., the volume at which it is fully inactive) below the steady-state volume. Thus this PLA2 activity is well suited for fine-tuning cell volume to the set point value: slight shrinkage will further inhibit taurine efflux and favor cell volume increase, whereas slight swelling will activate taurine efflux and favor cell volume decrease.
Mechanisms of Melittin-Induced Arachidonic Acid Release and Taurine Efflux
The potentiating effects of melittin on arachidonic acid release, taurine efflux, and osmolyte loss were absent or marginal in shrunken cells, modest under isotonic conditions, and dramatic in swollen cells. This indicates that melittin-induced potentiation of arachidonic acid release requires that PLA2 is already activated in a volume-dependent manner. The effect of melittin was not secondary to melittin-induced cell swelling but appeared to be dependent on Vm, in accordance with the higher affinity of melittin for anionic than for zwitterionic lipids (5). However, the effect of Vm may also be at the PLA2 level, e.g., several mammalian sPLA2s are cationic and exhibit preference for anionic phospholipids (29). Notably, a similar dependence on Vm has previously been demonstrated for the swelling-induced taurine efflux in Ehrlich cells (17) and in HeLa cells (40).
Identity of the PLA2 Isoform(s) Activated by Osmotic Swelling and Melittin Exposure
Six PLA2 isoforms, cPLA2-IVA, cPLA2-IVB, cPLA2-IVC, iPLA2-VIA, iPLA2-VIB, and sPLA2-V, were detected in the NIH3T3 cells at the mRNA level. The swelling-induced increase in arachidonic acid release from the nuclear fraction was fully blocked by BEL, indicating that an iPLA2, possibly iPLA2-VIA, is volume sensitive and mediates swelling-induced release of arachidonic acid from the nuclear membrane. Swelling-induced arachidonic acid release to the cell exterior was only inhibited partially by BEL and partially by the sPLA2 inhibitor manoalide. Since melittin-induced potentiation of arachidonic acid release to the cell exterior was volume sensitive, and since swelling-induced arachidonic acid release and taurine efflux were partially inhibited by manoalide, it seems likely that an sPLA2 activity is also volume sensitive; however, this cannot be unequivocally determined from the present findings.
Melittin-induced arachidonic acid release from NIH3T3 cells was also slightly but significantly inhibited by BEL. Moreover, BEL-sensitive, melittin-induced PLA2 activity was detected in NIH3T3 cell lysates cleared of sPLA2, indicating that iPLA2 activity can be directly potentiated by melittin. This indicates that in addition to the expected potentiation of sPLA2-mediated arachidonic acid release by this compound (e.g., Ref. 39), at least some of the melittin-induced arachidonic acid release in vivo may be iPLA2-mediated. AACOCF3 had no effect on either swelling- or melittin-induced arachidonic acid release in intact cells, yet similarly to BEL, AACOCF3 blocked melittin-induced PLA2 activity in NIH3T3 lysates. The reason for the differential effect of AACOCF3 in intact cells and lysates is not clear but may relate to a restricted accessibility to the relevant PLA2 in vivo.
Hence, taken together, these data are consistent with the interpretation that both swelling- and melittin-induced arachidonic acid release involves iPLA2 and sPLA2 isoforms, yet their relative roles differ between the two conditions. Of course, this conclusion is subject to the usual limitations of the use of pharmacological inhibitors. For example, BEL also inhibits the phosphatidate phosphohydrolase-1 (see Ref. 14), and effects of melittin on ion homeostasis and signaling that are not secondary to PLA2 activation cannot be excluded (6, 8, 28, 35). The incomplete inhibition by the PLA2 inhibitors of arachidonic acid release to the cell exterior compared with the complete inhibition of nuclear release and in in vitro assays thus reflects at least in part that some fraction of the arachidonic acid release occurs in intracellular compartments. In this regard, it is interesting that the presence of a functional sPLA2 in intracellular compartments, including the perinuclear membrane, has been reported (29).
Taurine efflux was inhibited more potently by BEL and manoalide than was the arachidonic acid release to the cell exterior. This is unlikely to reflect a direct block of the taurine efflux pathway by BEL, which has no effect on the otherwise very similar taurine efflux in HeLa cells (16). In contrast, arachidonic acid release from the nucleus was potently inhibited by BEL, similar to the taurine efflux, suggesting that swelling-induced taurine efflux is preferentially dependent on BEL-sensitive iPLA2 activity in the nuclear region. The finding that swelling-induced taurine efflux is more strongly inhibited by BEL than by manoalide, whereas the reverse is true for melittin-induced taurine efflux, supports this interpretation. The nuclear localization of iPLA2-VIA in swollen cells is consistent with the previously reported iPLA2 translocation to the nuclear membrane upon activation in Chinese hamster ovary cells (37). In addition, we detected the enzyme in other, as yet unidentified, vesicular compartments and in distinct plasma membrane regions. Although the functional consequences remain to be determined in NIH3T3 cells, this pattern also appears consistent with previous studies reporting the detection of functional iPLA2 in mitochondria (4, 23), endoplasmic reticulum (13), and lipid rafts (26).
Possible Mechanism of Swelling-Induced Activation of Arachidonic Acid Release
How might PLA2 activity be modulated by cell volume perturbations? Mechanism(s) of osmosensing have yet to be fully understood in mammalian cells (see Ref. 10), making identification of upstream activating mechanisms difficult. By analogy with the finding that sPLA2 activity in liposomes is increased in response to osmotic swelling (21), it is tempting to suggest a role for reduction in lateral lipid packing in the membrane(s). However, although such a simple mechanism is enticing, osmotic swelling of real cells may not be associated with membrane stretch but rather with loss of membrane invaginations (see Ref. 33). Thus additional elements are likely to regulate this response in vivo, one example being RhoA activity, which we previously showed to strongly stimulate efflux of taurine and other osmolytes from NIH3T3 cells (32). The actin-based cytoskeleton is reorganized after osmotic stress and regulates other volume-sensitive transport pathways (33). However, arguing against a role for the actin cytoskeleton, neither cytochalasin D nor latrunculin B significantly affected arachidonic acid release and taurine efflux from NIH3T3 cells either under basal conditions or after osmotic swelling or shrinkage (Pedersen SF and Lambert IH, unpublished observations). Another possibility is that the signal would be one of ionic strength, for example, rather than of mechanical stress; however, at least some iPLA2 isoforms appear to be stimulated by increased ionic strength at constant osmolarity (4), which would be counter to activation by cell swelling. Hence, further studies are required to elucidate the mechanisms by which cell volume modulates PLA2 activity.
In conclusion, taurine efflux from NIH3T3 cells is increased by osmotic swelling and decreased by osmotic shrinkage, respectively. The swelling-induced stimulation of taurine efflux appears to predominantly reflect the swelling-induced stimulation of a BEL-sensitive iPLA2 activity, possibly iPLA2-VIA, at the nuclear membrane, although sPLA2 activity may also contribute. Arachidonic acid release to the cell exterior was potentiated in a volume-sensitive manner in the presence of melittin, apparently via effects on both sPLA2 and, to a lesser extent, iPLA2. Accordingly, melittin-induced taurine efflux from NIH3T3 cells was dependent on both sPLA2 and, to a lesser extent, iPLA2 activity. It is suggested that cell swelling and melittin both stimulate taurine efflux at least in part via potentiation of iPLA2- and sPLA2-mediated arachidonic acid release, yet the relative contributions of the two isoforms differ in the two conditions.
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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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