©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Translocation of the 85-kDa Phospholipase A from Cytosol to the Nuclear Envelope in Rat Basophilic Leukemia Cells Stimulated with Calcium Ionophore or IgE/Antigen (*)

Sarah Glover (1) (2), Timothy Bayburt (2), Mechthild Jonas (3), Emil Chi (3), Michael H. Gelb (1) (2)(§)

From the (1)Departments of Chemistry, (2)Biochemistry, and (3)Pathology, University of Washington, Seattle, Washington 98195

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The rat mast cell line RBL-2H3.1 contains an 85-kDa cytosolic phospholipase A (cPLA) that is very likely involved in liberating arachidonate from membrane phospholipid for the synthesis of eicosanoids following stimulation with either calcium ionophore or IgE/antigen. In this study, the intracellular location of cPLA was determined using immunofluorescence microscopy and immuno-gold electron microscopy. In nonstimulated cells, cPLA is distributed throughout the cytosol and is excluded from the nucleoplasm. Following cell activation with calcium ionophore, most of the cPLA translocates to the nuclear envelope, and the enzyme remains there during the entire period that ionophore is present. With IgE/antigen stimulation for 5 min, approximately 20-30% of the cPLA translocates to the nuclear envelope, and after 30 min of stimulation, most of the enzyme returns to the cytosol. Measurement of intracellular calcium using the dye Fura-2/AM shows that the level of calcium rises immediately after antigen is added, remains high for about 30 s, and then declines back to resting levels. Activation with calcium ionophore produces a 10-fold larger release of arachidonate than does stimulation with IgE/antigen. Thus, the results suggest that the extent of membrane binding of cPLA correlates with the release of arachidonate and that the site of arachidonate liberation is the nuclear envelope where many of the enzymes that oxygenate this fatty acid are located.


INTRODUCTION

The 85-kDa cytosolic phospholipase A (cPLA)()has been identified and purified from a number of mammalian cells including rodent macrophage cell lines(1, 2, 3) , platelets(4, 5, 6, 7) , human monocytic cell lines(8, 9, 10, 11) , and rat kidney(12) . In contrast to the well-characterized 14-kDa secreted PLAs that require millimolar amounts of calcium as a catalytic cofactor(13) , cPLA is activated by submicromolar amounts of Ca. Furthermore, Ca is required for the binding of cPLA to cell membranes(9, 11, 14, 15, 16, 17) and to synthetic phospholipid vesicles(14, 18, 19) , but does not serve as a catalytic cofactor(14, 18, 20) . cPLA is found in the soluble fraction of cellular homogenates when the Ca concentration is low (typically <0.1 µM), and it is found in the particulate fraction in the presence of submicromolar to micromolar amounts of Ca. The enzyme in cells is also activated in part by phosphorylation(21, 22, 23, 24, 25, 26, 27) , although the molecular basis of this phenomenom is not known since the phosphorylated enzyme shows a very modest increase in catalytic turnover when examined in vitro.

The cDNA that encodes the cPLA from human U937 cells shows no homologous regions to 14-kDa secreted phospholipases A (14, 28). The amino acid sequence reveals a stretch of 45 residues in the amino-terminal region that shows homology to Ca-dependent forms of protein kinase C and other Ca-dependent membrane binding proteins. A 140-amino acid fragment of cPLA that contains the amino terminus was shown to bind to membranes in the presence of submicromolar amounts of Ca(14, 29) .

cPLA preferentially hydrolyzes phospholipids with an sn-2 arachidonyl chain, whereas 14-kDa phospholipases A display essentially no acyl-chain specificity(30, 31, 32, 33) . All of these results suggest the cPLA plays a role in signal-mediated release of arachidonic acid from membrane phospholipids for the genesis of eicosanoids, and thus cPLA may be a good target for antiinflammatory therapeutics.

Mast cells contain cPLA(34) and two additional phospholipases A. One is a 14-kDa enzyme of the type II variety that is secreted from activated mast cells(34, 35) , and the other is a 30-kDa enzyme that prefers phosphatidylserine(34) . The role of these enzymes in mast cell function is being clarified. Inhibitors of the 14-kDa enzyme cause suppression of histamine release, suggesting that this enzyme is a component of the mediator release pathway(34) . Furthermore, addition of purified 14-kDa phospholipase A to mast cells leads to histamine release(36) . The role of the 14-kDa enzyme in eicosanoid generation in mast cells is controversial. One report indicates that addition of exogeneous mast cell type II 14-kDa phospholipase A to mast cells does not lead to PGD production(36) , while another group provides evidence that addition of the 14-kDa phospholipase A from cobra venom leads to arachidonate liberation and production of cyclooxygenase products(35) . These differences are likely due to the different affinities of 14-kDa phospholipases A for mast cell membranes. The role of mast cell cPLA in arachidonate liberation is not clear, but recent studies show that this enzyme becomes phosphorylated in response to IgE cross-linking(37, 38) . In platelets, cPLA becomes phosphorylated in response to stimulation with thrombin(22) , and studies with potent cPLA inhibitors provide strong evidence for the role of this enzyme in arachidonic acid liberation(39, 40) . Antisense RNA technology has been used to show that cPLA is involved in eicosanoid generation in monocytes stimulated with bacterial lipopolysaccharide and platelet-activating factor(41) . No role for the phosphatidylserine-specific phospholipase A in mast cell function has been reported.

In this report, studies are presented showing that cPLA in rat basophilic leukemia cells (RBL-2H3.1) is located mainly in the cytosol in unactivated cells, and this enzyme translocates to the nuclear envelope in response to stimulation with calcium ionophore or IgE/antigen. The results suggests that cPLA is involved in the release of at least some of the arachidonic acid from membrane phospholipid of activated mast cells.


EXPERIMENTAL PROCEDURES

Materials

Recombinant human cPLA was prepared from baculovirus-infected Sf9 cells as described(42) . The polyclonal antiserum R11683 to cPLA and preimmune serum were obtained as generous gifts from Prof. C. C. Leslie (National Jewish Center for Immunology and Respiratory Medicine, Denver, CO). R11683 was obtained by injection of rabbits with full-length cPLA that had been extracted from a gel following SDS-PAGE. Affinity purified FITC-conjugated, goat anti-rabbit IgG F(ab`) is from Organon Teknika Corp. (Durham, NC). Ionomycin is from Sigma. Anti--tubulin IgG and anti-BiP antisera were obtained as generous gifts from Prof. Frank Solomon (Massachusetts Institute of Technology) and Prof. Linda Hendershot (St. Jude's Childrens Hospital), respectively.

Cell Culture

RBL-2H3.1 cells were obtained as a generous gift from Professor B. A. Helm (University of Sheffield, United Kingdom). Cells were routinely cultured at 37 °C in a humidified atmosphere of 5% CO in dishes of minimal essential medium (Life Technologies, Inc., 330-1650-AJ) with heat-inactivated fecal calf serum (12% by volume), NaHCO (29.3 ml/liter of 7.5% solution), glutamine (2 mM, added every 2 weeks). Media also contained penicillin/streptomycin/Fungizone (JHR Biosciences). Adherent cells were routinely dislodged by treatment with trypsin/EDTA solution except as noted below.

Preparation of Cells for Fluorescence Microscopy

For all studies described in this report, cells were grown to approximately 50% confluence as described above. For fluorescence microscopy, cells were cultured in 10-cm dishes containing multi-well glass slides (10 wells/slide, Cell Line Associates, Inc., Newfield, NJ). Washing and fixation of cells in dishes were carried out by adding the appropriate solution to the cells (10 ml/10-cm dish), and after the desired time the solution was removed by aspiration followed by the immediate addition of the next solution. For stimulation with the Ca ionophore A23187 (Sigma), cells were first washed three times with Hank's BSS with CaCl (Life Technologies, Inc., 14025-019). Washed cells were covered with 10 ml of Hank's BSS with CaCl and stimulated by addition of A21387 (final concentration 10 µM, 5 mM stock solution in MeSO, control cells were treated with MeSO only). Cells were incubated at 37 °C for 30 min and then fixed as described below.

Cells were fixed by covering the multi-well slides in the dishes with 10 ml of freshly prepared 2% formaldehyde (Aldrich, 25,254-9) in PBS and incubating for 20 min at 37 °C. All subsequent steps were done at ambient temperature. The multi-well slides were removed from the dishes and washed twice with PBS (5 min/wash in Coplin jars), and then permeabilized for 5 min in a Coplin jar containing freshly prepared 0.1% Triton X-100 (Sigma) in PBS. Cells were washed five times with PBS (5 min/wash in Coplin jars). The slides were removed from the Coplin jars and placed on a stack of moist paper towels inside of a plastic basin fitted with an air-tight lid. The lid was kept closed except during the brief manipulation of the samples. The liquid between the wells on the slide was carefully removed by wiping with a paper towel. PBS was periodically applied to the wells with a pipette so that the wells did not become dry. The liquid in the wells was removed with a pipette, and then 3% BSA in PBS (10 µl) was applied to each well with a pipette. After 15 min, R11683 antiserum (10 µl of serum diluted 1000-fold with 3% BSA in PBS) was applied to each well. Controls were carried out in which either the same volume of preimmune serum was used or serum was omitted. After 2 h the cells were washed four times with PBS (5 min/wash in Coplin jars). Liquid was removed between the wells, and the cells were treated with 3% BSA in PBS as described above. FITC-conjugated anti-IgG F(ab`) antiserum (10 µl of serum diluted 300-fold with 3% BSA in PBS) was applied to each well. After 1 h, the cells were washed four times with PBS (5 min/wash in Coplin jars), in some cases the nuclei were stained (see below), and coverslips were mounted over the wells using Citifluor mounting solution (Ted Pella, Redding, CA).

As an additional control, a 20-µl portion of R11683 antisera (diluted 500-fold in PBS with 3% BSA, approximately 0.6 µg of IgG) was mixed with an equal volume of purified cPLA (0.5 µg/µl in PBS). After 30 min at room temperature the mixture was applied to the wells containing fixed cells as described above.

To stain nuclei, cells were processed as described above. Just prior to mounting coverslips, the wells were treated with 4`,6-diamidino-2-phenylindole (1 µg/ml in PBS containing 3% BSA, Molecular Probes) for 1 min. The cells were washed four times in PBS (5 min/wash in Coplin jars) and coverslips were mounted as above.

Slides were immediately viewed by microscopy or wrapped with aluminum foil and stored at 4 °C for up to 24 h prior to microscopy. Fluorescence microscopy was carried out with a Nikon Microphot-FXA epifluorescence microscope (300 magnification, oil immersion). Cells were observed with either a fluorescein filter (excitation 480 nm, barrier 535 nm) or a rhodamine filter (Nikon DM580 filter; excitation 546 nm, barrier 590 nm) depending on the dye probe used. Confocal fluorescence microscopy was carried out with a Bio-Rad MRC-600 confocal laser scanning microscope using the 488 line of a krypton/argon laser (for viewing fluorescein) and a 60 objective lens.

Preparation of Cells for Electron Microscopy

For electron microscopy, cells were grown in 12-well plastic dishes as described above. Stimulation with A23187 was carried out as described above. For stimulation with DNP-HSA, cells were first primed by adding anti-DNP IgE (final concentration 0.5 µg/ml, obtained as a generous gift from Professor B. A. Helm, or purchased from Sigma) directly to the culture medium, and the cells were incubated at 37 °C for 12-24 h. Cells were washed three times with pre-warmed RBL-BSS (1 liter contains 8.67 g of NaCl, 0.365 g of KCl, 1.64 g of D-sorbitol, 0.6 g of KHPO, 0.14 g of KHPO, 4.83 g of HEPES, pH 7.40) containing 0.1% BSA and 1 mM CaCl. Cells were covered with the same solution, and DNP-HSA was added (final concentration 100 ng/ml, 3 mg/ml stock solution, obtained as a generous gift from Professor B. A. Helm). Cells were incubated at 37 °C for various times, and then fixed.

Cells were processed as described for fluorescence microspopy (using 2 ml of solutions per well) up to and including the washing steps following the treatment with anti-cPLA antisera (R11683 antiserum was used at a dilution of 150-fold rather than the 1000-fold dilution used for fluorescence microscopy). At this stage, the prepared cells were covered with PBS, the dish covers were sealed to the dishes by wrapping with Parafilm, and the dishes kept at 4 °C in an air-tight container for up to 1 week.

The cells were then processed for electron microscopy. PBS was removed from the wells, and the cells were covered with anti-rabbit IgG-labeled 15-nm gold particles (Janssen Life Science Products, Olen, Belgium, diluted 10-fold in PBS). After 16 h at 4 °C, the fluid was removed, and the cells were covered with 2% glutaraldehyde in PBS for 20 min at 20 °C. The cells were washed several times with PBS, and then post-fixed in 1% osmium tetroxide in water (Ted Pella, Inc.), dehydrated with ethanol, and embedded in Medcast (Ted Pella, Inc.) The samples were cut by a diamond knife at an oblique angle of 30°. Sections were stained in uranyl acetate and lead citrate, and then viewed with a JEOL 100B electron microscope at 60 kV as described previously(43) .

Visualization of Organelles

To visualize mitochondria, cells were grown on multi-well slides. Mitotracker (Molecular Probes) was added to the culture medium to give a final concentration of 25 nM. After incubation at 37 °C for 15-45 min, the cells were washed twice with PBS and then fixed as described above. Coverslips were mounted with Citifluor as described above, and the cells were viewed by fluorescence microscopy using the rhodamine filter.

To visualize the Golgi apparatus, cells were grown on multi-well slides. The medium was removed, and the cells were washed twice with PBS. Cells were covered with PBS containing 40 µM C-DMB-ceramide (Molecular Probes) and incubated for 20 min at 37 °C. Cells were washed with PBS, and coverslips were mounted with Citifluor as described above. Cells were viewed immediately by fluorescence microscopy using the fluorescein filter. To visualize tubulin, cells were prepared as described above for cPLA visualization except that the anti-tubulin antiserum was used (1:250 dilution).

Arachidonate Release-Cells were grown in 12-well plates as described above. To each well was added 0.25 µCi of [H]arachidonic acid (60-100 Ci/mmol, DuPont NEN), and to some of the wells was added anti-DNP IgE (final concentration 0.5 µg/ml). Cells were incubated for 24 h at 37 °C. Cells that were to be treated with Ca ionophore were washed three times with prewarmed Hank's BSS containing 0.1% BSA, and those that were to be treated with DNP-HSA were washed with prewarmed RBL-BSS containing 0.1% BSA and 1 mM CaCl. Cells were stimulated with A23187 or DNP-HSA as described above. Cells were incubated at 37 °C for variable times, and then the culture media above the cells was transferred to microcentrifuge tubes. After centrifugation of the samples to remove small amounts of dislodged cells (20 s at 16,000 g), 90% of the supernatants were transferred to scintillation vials. To the wells containing the remaining adherent cells was added 1 ml of CHCl:MeOH (2:1 by volume). This extract was transferred to the microcentrifuge tubes containing small amounts of pelleted cells, and after brief mixing with a vortexer, the samples were transferred to scintillation vials. Scintillation fluid was added to all samples, and the samples were analyzed by scintillation counting in a Beckman LS 1801 counter. Release of radiolabeled arachidonic acid is expressed as the percentage of the total cellular radiolabeled arachidonate released into the culture medium for each culture well.

Ca Mobilization

Increase in intracellular Ca was measured essentially as described(44) . Cells were grown in a 75-cm flask as described above, and primed with anti-DNP IgE as described above. Nonenzymatic cell dissociation solution (5-10 ml/flask, Sigma catalog number C-5789) was added, and after 10-15 min the cells were dislodged by banging the plate several times against the lab bench. Cells were collected by centrifugation and resuspended at a density of 2 10/ml in BSS. Fura-2/AM (Molecular Probes) was added to a final concentration of 2 µM, and the cells were incubated at 37 °C for 20 min with periodic swirling. Cells were washed three times with BSS containing 0.1% BSA (Sigma A-7906) and resuspended to a density of 1 10/ml in BSS containing 0.1% BSA and 1 mM CaCl. Cells were kept on ice and in the dark for up to 2 h.

Cell suspension (0.33 ml) was transferred to a 1-ml fluorescence cuvette containing 0.66 ml of BSS, 0.1% BSA, 1 mM CaCl. The sample was equilibrated in a thermostated holder to 21 °C and stirred with a small Teflon stir bar. Fluorescence was monitored with excitation at 340 nm and emission at 490 nm. Other additives are described in the legend to Fig. 3.


Figure 3: Ca mobilization in RBL-2H3.1 cells following stimulation with Ca ionophore or IgE/antigen. The fluorescence emission from Fura-2/AM is plotted as a function of time. Bottom, cells that were not primed with anti-DNP IgE were treated with DNP-HSA (100 ng/ml) and then with ionomycin (7 µM, final concentration) at the second arrow and finally with a second portion of ionomycin (14 µM total final concentration). Top, anti-DNP IgE-primed cells were treated with DNP-HSA and then with ionomycin. Other conditions are described under ``Experimental Procedures.''



-Hexosaminidase Release-Essentially the procedure of Tanaka et al.(45) was followed. Cells were grown in 12-well plates. Cells were treated with anti-DNP IgE, washed, and stimulated with DNP-HSA as described above. After 5 or 15 min at 37 °C, aliquots of culture media (200 µl) were removed and added to an assay mixture (900 µl) consisting of 4 mMp-nitrophenyl-2-acetamido-2-deoxy--D-glucopyranoside (Sigma) in 40 mM sodium citrate, pH 4.5. The mixtures were incubated at 37 °C for 30 min, and the reactions were stopped by adding 1 ml of 0.2 M glycine, pH 10.5. The amount of product was determined by measuring the absorbance at 410 nm using a calibration curve prepared by adding known amounts of p-nitrophenol to the assay mixture.

Cell Fractionation

Cells were grown in 15-cm culture dishes, the media was aspirated, and cells were scraped into 4 ml of RBL-BSS. Cells from three plates were pooled, and the suspension was centrifuged at 300 g for 10 min at room temperature in a Sorvall RC-5 centrifuge using an SS-34 rotor. The cell pellet was resuspended in 4 ml of RBL-BSS containing 1 mM CaCl, and divided among 4 tubes. Five µl of a 2 mM MeSO solution of A23187 was added to two of the tubes, and 5 µl of MeSO was added to the remaining tubes. After 5 min at room temperature, the cells were spun down and resuspended in 250 µl of 20 mM Tris, pH 7.4, 10 mM KCl, 2 mM MgCl, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, 20 µg/ml aprotinin, and either 10 mM EGTA or 0.2 mM EGTA plus 0.3 mM CaCl. After 20 min on ice, 3 volumes of 50 mM Tris, pH 7.4, 25 mM KCl, 5 mM MgCl, 0.25 M sucrose, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, 20 µg/ml aprotinin, and either 10 mM EGTA or 0.2 mM EGTA and 0.3 mM CaCl was added, and the cells were immediately homogenized using a 2-ml glass Dounce homogenizer with a loose fitting pestle (Kontes, B pestle). Greater than 95% of the cells were lysed after 15-20 vigorous strokes, based on trypan blue exclusion. The lysates were centrifuged at 1000 g for 10 min, and the pellets were resuspended in 300 µl of buffer (low speed pellet fraction). The supernatant was centrifuged at 100,000 g for 1 h, and the membrane pellet was resuspended in 300 µl of buffer (high speed pellet fraction). Under microscopic examination using either trypan blue or Diff-Quik Stain (Baxter), the low speed pellet contained crude nuclei, while the high speed pellet and high speed supernatant (cytosolic fraction) contained no visible nuclei. Cellular fractions were flash frozen in liquid nitrogen and stored at -80 °C. Equal volumes of cell fractions were thawed and applied to a 10% SDS-PAGE minigel. cPLA was detected by immunoblot analysis as described below.

Immunoblotting

Analysis of cell extracts by SDS-PAGE/immunoblotting was carried out essentially as described(24) . Cells were grown in 10-cm dishes and washed twice with PBS. Cells were scrapped into 200 µl of ice-cold lysis buffer (50 mM HEPES, 150 mM NaCl, 10% glycerol, v/v, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 20 µM sodium orthovanadate, 10 mM tetradsodium pyrophosphate, 100 mM sodium fluoride, 3 µMp-nitrophenyl phosphate, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride, pH 7.4; protease and phosphatase inhibitors were added fresh from frozen stocks. Lysates were incubated on ice for 30 min and then centrifuged (10 min at 13,000 g) for 10 min. The protein concentration in the supernatant was estimated using a BCA protein assay kit (Pierce), Laemmli sample buffer containing dithiothreitol was added, and 8 µg of protein was loaded on each lane of a 7.5% SDS-polyacrylamide gel (Laemmli, Bio-Rad Mini-Protean-II). Prestained molecular weight markers (Bio-Rad) and known amounts of purified cPLA (typically 3 ng) were also loaded in separate lanes. After electrophoresis, protein was transferred to a nitrocellulose membrane using an electroblotter (Bio-Rad). Following transfer, the gel was stained with Coomassie Blue to verify the efficiency of transfer. The membrane was rinsed once for 15 min with TTBS (20 mM Tris, 137 mM NaCl, 500 µl/liter Tween 20, pH 7.6) and then incubated in TTBS containing 5% nonfat dry milk overnight at 4 °C on a rotary shaker. After blocking, the membrane was incubated in fresh TTBS, 5% nonfat dry milk containing R11683 antisera (diluted 12,000-fold) for 2 h at room temperature on a rotary shaker. The membrane was rinsed four times (15 min/rinse) with TTBS on a rotary shaker at room temperature. The immunoblot was visualized using the ECL kit from Amersham using horseradish peroxidase-conjugated anti-rabbit IgG diluted 5,000-fold. The blot was exposed to Kodak X-AR x-ray film for up to 10 min.

In some cases high background staining was seen on the immunoblots. This problem was alleviated by submerging the membrane in stripping buffer (100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris, pH 6.7) at 50 °C for 30 min with occasional agitation. The membrane was rinsed twice (10 min each) with TTBS on a rotary shaker at ambient temperature. Blocking was carried out with TTBS, 5% nonfat dry milk for 1 h at ambient temperature on a rotary shaker. The membrane was treated with primary antibody and processed for ECL detection as described above.


RESULTS

RBL-2H3.1 Cells Contain cPLA

The RBL-2H3.1 cells studied in this report is a subclone (46) of the well established RBL-2H3 cell line of mast cell lineage. RBL-2H3.1 cells support high levels of mediator secretion in response to stimulation with IgE and antigen compared to the subclone RBL-2H3.2 which supports 10-fold lower mediator release(44) . Biochemical characterization of these subclones has shown that the low secretor displays a reduced extent of phosphorylation of the cell surface, high affinity IgE receptor complex (44). This low level phosphorylation leads to subnormal production of inositol phosphate and thus a reduced increase in intracellular Ca. For the purposes of the present study, the high secreting variant was used in order to guard against the possibility that signal transduction between the IgE receptor complex and cPLA is blocked in low secreting variants.

Immunoblot analysis of total protein (soluble + membrane-bound) extracted from RBL-2H3.1 cells clearly reveals the presence of cPLA as a major band of apparent molecular mass of approximately 100-kDa (Fig. 1); the actual molecular mass of cPLA is nominally 87-kDa(14, 28) , and its anomalously slow migration on SDS-PAGE gels is well documented(2, 8, 10) . The R11863 anti-cPLA antisera used in this analysis has relatively high affinity for cPLA since it can be routinely used for immunoblotting at a dilution of 12,000-fold (Fig. 1). Even 50,000-fold dilution of this antibody gives a strong immunblot response (not shown). The additional minor bands of higher mobility seen in the immunoblots are also seen in highly purified cPLA expressed as a recombinant protein in insect cells (Fig. 1), suggesting that they are proteolytic degradation products of cPLA. The presence of cPLA in RBL-2H3 cells has also been demonstrated by Inoue and co-workers (47) who partially purified this enzyme from these cells.


Figure 1: Immunoblot analysis of cPLA in RBL-2H3.1 extracts probed with the R11683 anti-cPLA antiserum. Lanes 1-3, protein (8 µg) extracted from RBL-2H3.1 cells. Lane 4, purified cPLA (3 ng) expressed as a recombinant protein in insect cells. The positions of the molecular weight markers are indicated. Other conditions are as described under ``Experimental Procedures.''



Response of RBL-2H3.1 Cells to Stimulation with CaIonophore or IgE/Antigen

Several studies have shown that RBL-2H3 cells liberate arachidonic acid in response to stimulation with Ca ionophore or IgE/antigen (for example, see Refs. 48-51). In order to demonstrate that the subclone RBL-2H3.1 is capable of stimulus-mediated arachidonate mobilization, these cells were labeled with [H]arachidonate for 24 h and unincorporated fatty acid was removed by extensive washing with buffer containing BSA. Cells that were stimulated with antigen (DNP-HSA) were primed overnight with anti-DNP IgE. The results are shown in Fig. 2. Stimulation with Ca ionophore results in arachidonate release over 30 min approaching 30% of the total radiolabeled fatty incorporated into the cells, whereas very little release was seen in the absence of ionophore. Compared to ionophore, stimulation with IgE/antigen resulted in a lower level of arachidonate release over 30 min but well above that measured in cells that were stimulated with antigen but were not primed with IgE. Similar trends have been reported previously for RBL-2H3 cells(49, 52, 53, 54) .


Figure 2: Release of radiolabeled arachidonic acid from RBL-2H3.1 cells stimulated with Ca ionophore or IgE/antigen. Release is expressed as the percent of total cellular radiolabeled arachidonic acid that was released into the culture medium. Filled triangles, with A23187; open triangle, without A23187; filled circles, anti-DNP IgE-primed cells stimulated with DNP-HSA; open circle, stimulated with DNP-HSA but without IgE priming. Other conditions are described under ``Experimental Procedures.''



The results in Fig. 3show that RBL-2H3.1 cells mobilize Ca in response to stimulation with IgE/antigen as reported previously(44) . The fluorescence emission of Fura-2/AM-loaded cells was monitored in real time before and after the addition of stimulus. Stimulation of the cells that were not primed with IgE with the Ca ionophore ionomycin led to an immediate rise in intracellular Ca, and this increased Ca level was persistent and was not altered by the addition of a second portion of ionomycin (Fig. 3, bottom). A23187 was not used for these experiments because of its high autofluorescence. In the absence of ionophore, antigen promoted an increase in intracellular Ca but only if the cells were primed with IgE. In this case, the Ca level started to rise 5-10 s after the addition of antigen, and the increase lasted 10-15 s. Ca remained high for about 25 s and then started to fall over 15-20 s to nearly the unstimulated level at which point addition of ionomycin resulted in an immediate increase in intracellular Ca (Fig. 3, top).

RBL-2H3.1 cells when activated release a number of hyrolytic enzymes including -hexosaminidase. Release of this enzyme from RBL-2H3.1 cells was monitored with a colorimetric substrate as described under ``Experimental Procedures.'' Released enzyme levels from cells primed with anti-DNP IgE and stimulated with DNP-HSA for 15 min were 5-6-fold higher than those measured in the absence of DNP-HSA and 5-6-fold higher than those measured in the presence of DNP-HSA but in the absence of priming with IgE (not shown).

Cell Fractionation Studies

Locating cPLA in cells by subcellular fraction studies is likely to be problematic because the distribution of enzyme between membranes and cytosol depends on the concentration of Ca in the homogenation buffer. For example, when RAW 264.7 macrophages are homogenized in the presence of EGTA most of the cPLA appears in the cytosol, but when the homogenation buffer contains >200-300 µM Ca, 60-70% of the enzyme is recovered in the membrane fraction(15) . Similar results have been reported for U937 cells(8, 9, 10) , neutrophils(55) , rat liver macrophages(16) , and platelets(56) . Furthermore, membrane-bound enzyme can be released in variable yields by treatment with EGTA(15, 16) . Thus, translocation studies involving cell homogenation may be more a reflection of the conditions of the homogenation rather than of the events that occur in an intact and stimulated cell. In addition, since the membrane binding is reversible, there could be scrambling of cPLA between different cellular membranes during the fraction procedure.

Despite these concerns, subcellular fraction studies with RBL-2H3.1 cells were carried out. Cells bathed in RBL-BSS containing 1 mM CaCl were treated either with A23187 or vehicle as described under ``Experimental Procedures.'' In both cases, cells were homogenized either in the absence of Ca (10 mM EGTA) or in the presence 100 µM free Ca, and cytosol, nuclear pellet, and crude membrane pellet were isolated as described under ``Experimental Procedures.'' Surprisingly, under all conditions essentially all of the cPLA detected by immunoblotting was found in the cytosolic fraction even when the cells were homogenized in the presence of 100 µM free Ca. This result differs significantly from results reported with other cell types as referenced above. Given this result and the problems inherent in using subcellular fractionation to determine the cellular site of binding of cPLA to membranes, attempts were made to localize cPLA in fixed and permeabilized cells using immunocytochemistry combined with microscopy. Such an approach has been used to localized other Ca-dependent membrane binding proteins such as protein kinase C(57, 58) .

Visualization of cPLA2 in RBL-2H3.1 Cells by Immunofluorescence Microscopy

Fixation and permeabilization of RBL-2H3.1 cells for microscopy was carried out essentially as described previously for the RBL cell line(59) . Fluorescent anti--tubulin IgG was used to evaluate the integrity of the prepared cells. Among the fixatives formaldehyde, paraformaldehyde, and glutaraldehyde, formaldehyde was found to give the best results, and permeabilization with Triton X-100 was superior to that obtained with methanol.

Images obtained by conventional fluorescence microscopy are shown in Fig. 4, and those obtained by confocal fluorescence microscopy are shown in Fig. 5. Visualization of microtubules with anti--tubulin IgG (Fig. 4, upper left) shows the expected pattern of strands radiating outward from the microtubule organizing center located near the center of the cell. Fig. 4(upper right) shows the mitochondria (visualized with the dye Mitotracker) scattered throughout the cytosol. Most of the Golgi complex (visualized with the dye C-DMB-ceramide) appears close to and on one side of the nucleus (Fig. 4, middle left). The confocal image of the endoplasmic reticulum (Fig. 5, upper right) shows the expected fluorescence emission from the nuclear envelope and regions radiating away from the nucleus and throughout the cytosol. This organelle was visualized with antisera to the immunoglobulin heavy chain binding protein (BiP) previously shown to reside in the endoplasmic reticulum(60) .


Figure 4: Conventional fluorescence microscopy of RBL-2H3.1 cells. Top left, microtubules visualized with the anti--tubulin IgG antibody; top right, mitochondria visualized with Mitotracker; middle left, the Golgi complex visualized with C-DMB-ceramide; middle right, cPLA in A23187-activated cells visualized with anti-cPLA antiserum and FITC-conjugated, goat anti-rabbit IgG F(ab`); lower left, nuclei visualized with 4`,6-diamidino-2-phenylindole.




Figure 5: Confocal fluorescence microscopy of RBL-2H3.1 cells. Top left, cPLA in resting cells visualized with anti-cPLA antiserum and FITC-conjugated, goat anti-rabbit IgG F(ab`). The white bar has a length of 20 µm; top right, the endoplasmic reticulum visualized with anti-BiP antiserum and FITC-conjugated, goat anti-rabbit IgG F(ab`); lower left, cPLA in A23871-activated cells visualized with anti-cPLA antiserum and FITC-conjugated, goat anti-rabbit IgG F(ab`); lower right, same as lower left except a different cell was viewed (this image is zoomed a factor of 1.5 relative to the other images).



Fig. 4(middle right) shows the image obtained with the anti-cPLA antiserum in A23187-treated cells. It can be seen that cPLA tends to collect on the nuclear envelope; the nucleus was viewed with the stain 4`,6-diamidino-2-phenylindole (Fig. 4, lower left). This cell has two nuclei, but similar nuclear envelope staining was seen in fluorescence images of cells with a single nucleus. The confocal images of cPLA location are very striking. In the resting cell, cPLA exists throughout the cytosol but excluded from the nucleoplasm (Fig. 5, upper left). Following stimulation with A23187, cPLA translocates to the nuclear envelope (Fig. 5, bottom left and right). The pattern of fluorescence is significantly different than that seen with the endoplasmic reticulum marker antibody in that most of the membrane-bound cPLA appears on the perimeter of the nucleus rather than fanning out into the cytosol. However, it is difficult to rule out that a sizeable portion of cPLA is bound to the endoplasmic reticulum.

The following controls were carried out. Cells were treated with secondary antibody (FITC-conjugated, goat anti-rabbit IgG F(ab`)) in the absence of anti-cPLA antiserum; cells were treated with preimmune serum followed by secondary antibody; cells were treated with anti-cPLA antiserum that had been previously incubated with an excess of purified cPLA, and the cells were then treated with secondary antibody. In all cases, conventional and confocal fluorescence images obtained with the same film exposure as those shown in Fig. 4and Fig. 5were featureless (not shown).

Visualization of cPLA2 in RBL-2H3.1 Cells by Immuno-gold Electron Miscroscopy

RBL-2H3.1 cells were treated with either calcium ionophore or IgE/antigen, fixed, and permeabilized as described for the fluorescence microscopy studies, and then further processed for electron microscopy using anti-rabbit IgG-labeled gold particles. The images are shown in Fig. 6. In the absence of stimulation, nearly all of the cPLA (>95% of the gold particles, seen in three independent cell preparations) appears to be in the cytosol (Fig. 6A). Stimulation with IgE/antigen for 5 min results in translocation of some of the cPLA (20-30%, seen in five independent cell preparations) to the nuclear envelope (Fig. 6B). After prolonged treatment with IgE/antigen (30 min) most of the cPLA (85-95%, seen in three independent cell preparations) is found in the cytosol (not shown). Stimulation with calcium ionophore for 5 min results in the translocation of most of the cPLA (>80% the gold particles, seen in three independent cell preparations) to regions on or near the nuclear envelope (Fig. 6C). Occasionally, clusters of cPLA are seen in the images of stimulated cells for unknown reasons (Fig. 6, A and B).


Figure 6: Electron microscopy images of RBL-2H3.1 cells. A, unstimulated cells; B, cells stimulated for 5 min with IgE/antigen; C, cells stimulated for 5 min with calcium ionophore. Fifteen-nm immuno-gold particles appear as black dots (circled). Particles clearly attached to the nuclear envelope are marked with an arrow, those that form small aggregates are marked with an arrowhead.




DISCUSSION

Translocation of cPLA to membranes was not seen in RBL-2H3.1 cell homogenates prepared in the presence or absence of calcium. This is in contrast to other reports with macrophages(15, 16) , brain(17) , neutrophils(61) , and mouse mammary gland-derived cells (62) showing that cPLA is found mainly in the particulate fraction when the cells are homogenized in the presence of micromolar amounts of calcium. The reason for this difference is not known. Although it may be possible to identify homogenation conditions that render cPLA membrane bound, the physiological relevance of such studies would be highly questionable. Thus, microscopic studies using immunolocalization of cPLA was warranted.

The results of this study provide strong evidence for the translocation of cPLA from the cytosol to the nuclear envelope in calcium ionophore- or IgE/antigen-stimulated cells of mast cell lineage (RBL-2H3.1). This is supported by both fluorescence and electron microscopies of fixed and permeabilized cells. In a recent study using immunoblot analysis, cPLA has been preferentially detected in nuclei isolated from cavitated rat peritoneal macrophages stimulated with calcium ionophore(63) . It is interesting to note that treatment of RBL-2H3.1 cells with calcium ionophore produced a 10-fold larger release of arachidonate than did stimulation with IgE/antigen (Fig. 2), and that immuno-gold electron microscopy reproducibly showed a larger fraction of cPLA bound to membranes in calcium ionophore- versus IgE/antigen-stimulated cells. This may be due, at least in part, to the fact that following IgE/antigen treatment, the rise in intracellular calcium is transient (Fig. 3); after an initial rise, intracellular calcium returns to resting levels in about 1-2 min. In contrast, with ionophore treatment, the calcium level remains elevated. Indeed after a 30-min treatment with IgE/antigen the amount of membrane-bound cPLA, as seen by electron microscopy, is reduced to 10-20% of the total immunologically detected enzyme (not shown). Furthermore, as seen by fluorescence microscopy, cPLA remains on the nuclear envelope even after 30 min of stimulation with calcium ionophore (not shown). It is also possible that the phosphorylation state of cPLA is different in IgE/antigen- versus calcium ionophore-stimulated cells and that this results in a change in the fraction of membrane-bound enzyme. The addition of okadaic acid or phorbol myristate, which are known to enhance the release of arachidonate in mouse peritoneal macrophages probably by increasing the level of cPLA phosphorylation(24) , resulted in no change or only a 1.7-fold increase, respectively, in released arachidonic following a 5-min stimulation with IgE/antigen (data not shown). Further work is needed to fully understand all of the elements that control the translocation of cPLA to the nuclear membrane.

Pulse-chase experiments have shown that radiolabeled arachidonate when added to the mouse fibrosarcoma cell line (HSDMC) most rapidly is found in nuclear membranes and that this pool is selectively hydrolyzed in response to bradykinin-induced production of prostaglandin E(64) . Although this work is interesting in light of the present studies, the enzyme responsible for arachidonate liberation in HSDMC cells has not been identified. It is interesting to note that some of the enzymes that oxygenate liberated arachidonate are located, at least in part, in nuclear membranes. The enzyme cyclooxygenase has been detected in nuclear and endoplasmic reticulum membranes(65, 66, 67) . The enzyme 5-lipoxygenase in unactivated RBL cells is located in the nucleus(68) . In a variety of stimulated cells the enzyme is found in the nuclear membrane(63, 66, 69, 70) . The protein 5-lipoxygenase activating protein, which is required for the function of 5-lipoxygenase in cells, is also located in the nuclear membrane(69) . The enzymes 12- and 15-lipoxygenase are found in nuclear membranes(66) , endoplasmic reticulum(66) , and bound to chromatin(66, 71) . If the eicosanoids are biosynthesized in the nuclear membrane and endoplasmic reticulum, as appears to be the case, these compounds can very likely spontaneously exchange between intracellular membranes as well as be released into the extracellular fluid. This stems from their finite solubility in both membranes and aqueous phases; the same is true of arachidonic acid.

Further work will be needed to determine if the selective binding of cPLA to the nuclear envelope is the result of an endogenous protein receptor or of other factors such as the specific phospholipid composition of this membrane. Although the binding of cPLA to phosphatidylcholine vesicles is enhanced by the presence of negatively charged lipids(18, 32) , the fraction of phospholipids that are acidic in the nuclear membrane is not significantly different that in microsomes or plasma membrane (see, for example, Refs. 72-74). The possibility of an asymmetric distribution of acidic phospholipids between inner and outer leaflets of the nuclear membrane should be considered, but there is little, if any, data to support such a proposal.


FOOTNOTES

*
This work was supported by Grant HL50040 and Research Career Development Award GM562 (to M. H. G.) and Grants AI17758 and AI34578 (to E. C.) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Depts. of Chemistry and Biochemistry, University of Washington, Mail Stop BG-10, Seattle, WA 98195. Tel.: 206-543-7142; Fax 206-685-8665.

The abbreviations used are: cPLA, 85-kDa cytosolic phospholipase A found in RBL-2H3.1 and other mammalian cell types; BSA, bovine serum albumin; BSS, balanced salt solution; DNP-HSA, dinitrophenol conjugated to human serum albumin; PBS, phosphate-buffered saline (pH 7.2); PAGE, polyacrylamide gel electrophoresis; FITC, fluorescein isothiocyanate.


ACKNOWLEDGEMENTS

We are grateful to C. C. Leslie for helpful discussions and to R. L. Wright and P. M. Brunner for help with microscopy.


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