Critical duration of intracellular Ca2+ response required for continuous translocation and activation of cytosolic phospholipase A2.

When cells are exposed to certain external stimuli, arachidonic acid (AA) is released from the membrane and serves as a precursor of various types of eicosanoids. A Ca2+-regulated cytosolic phospholipase A2 (cPLA2) plays a dominant role in the release of AA. To closely examine the relation between Ca2+ response and AA release by stimulation of G protein-coupled receptors, we established several lines of Chinese hamster ovary cells expressing platelet-activating factor receptor or leukotriene B4 receptor. Measurement of intracellular Ca2+ concentration ([Ca2+]i) demonstrated that cell lines capable of releasing AA elicited a sustained [Ca2+]i increase when stimulated by agonists. The prolonged [Ca2+]i elevation is the result of Ca2+ entry, because this elevation was blocked by EGTA treatment or in the presence of Ca2+ channel blockers (SKF 96365 and methoxyverapamil). cPLA2 fused with a green fluorescent protein (cPLA2-GFP) translocated from the cytosol to the perinuclear region in response to increases in [Ca2+]i. When EGTA was added shortly after [Ca2+]i increase, the cPLA2-GFP returned to the cytosol, without liberating AA. After a prolonged [Ca2+]i increase, even by EGTA treatment, the enzyme was not readily redistributed to the cytosol. Thus, we propose that a critical time length of [Ca2+]i elevation is required for continuous membrane localization and full activation of cPLA2.

Arachidonic acid (AA) 1 is a precursor for biosynthesis of eicosanoids, including prostaglandins, thromboxanes, leukotrienes, and lipoxins. In the resting state, the bulk of the AA in mammalian cells is esterified in glycerophospholipids at the sn-2 position (1). Liberation of AA occurs mainly by activation of phospholipase A 2 (PLA 2 ) in response to varieties of extracellular stimuli such as cytokines, hormones, neurotransmitters, mitogens, antigens, and endotoxins (2). Mammalian cells have structurally diverse forms of PLA 2 including secretory PLA 2 , Ca 2ϩ -independent PLA 2 , and cytosolic PLA 2 (cPLA 2 ) (3)(4)(5). Among these PLA 2 s, receptor-mediated AA release is primarily attributed to cPLA 2 because the enzyme preferentially hydrolyzes phospholipids containing AA and is regulated by physiological levels of intracellular calcium concentrations ([Ca 2ϩ ] i ) and phosphorylation of Ser-505 by mitogen-activated protein kinase (MAPK) (6 -9). Consistently, stimulated peritoneal macrophages derived from cPLA 2 -deficient mice fail to produce eicosanoids (10,11). Sensitivity of cPLA 2 to Ca 2ϩ is mediated through an amino-terminal CaLB domain which is homologous to the C2 domain of protein kinase C and several other Ca 2ϩdependent phospholipid-binding proteins (6,12). Immunofluorescent studies have shown that cPLA 2 translocates from the cytosol to the nuclear envelope and the endoplasmic reticulum in response to [Ca 2ϩ ] i increase (13,14). Other proteins involved in eicosanoid production, such as cyclooxygenase isoforms (15)(16)(17), 5-lipoxygenase, 5-lipoxygenase-activating protein, and leukotriene C 4 synthase (18 -20) are also localized to these sites constitutively or upon cell stimulation.
The mobilization of Ca 2ϩ can be elicited by ligand binding to cell-surface receptors that activate phospholipase Cs through heterotrimeric G protein-dependent or protein tyrosine kinasedependent pathway. Calcium signaling patterns occur as single transients, repetitive oscillations, or sustained plateaux. Although several investigators have noted the importance of Ca 2ϩ influx in receptor-mediated AA release (21)(22)(23), little is known about how these Ca 2ϩ signaling patterns regulate cPLA 2 translocation and AA release.
To address this question, we monitored Ca 2ϩ signaling pattern, cPLA 2 translocation, and AA release using Chinese hamster ovary (CHO) cell lines stably expressing platelet-activating factor (PAF) receptor (24) and/or leukotriene B 4 (LTB 4 ) receptor (25). In CHO cells, cPLA 2 is expressed endogenously and coupled to receptor-mediated release of AA (26,27). We found that a critical time length of [Ca 2ϩ ] i elevation is required for continuous translocation of cPLA 2 to the membrane and for the release of AA.

EXPERIMENTAL PROCEDURES
Materials-The sources of materials used in this work were as follows: PAF C- 16  Plasmid Construction-For a chimeric protein containing GFP at the carboxyl terminus of cPLA 2 , cDNA encoding human cPLA 2 was amplified by polymerase chain reaction with primers upstream (5Ј-GGAA-GATCTATGTCATTTATAGATCCTT-3Ј) and downstream (5Ј-TGCG-GTCGACTGCTTTGGGTTTACTTAGA-3Ј) using pSV2-cPLA 2 (provided by Drs. I. Kudo and M. Murakami, Showa University, Tokyo) as a template. The resulting polymerase chain reaction product was cloned into BglII-SalI sites of pEGFP-N3 vector (CLONTECH, Palo Alto, CA) to obtain a cPLA 2 -GFP fusion construct. The orientation of cPLA 2 and the integrity of the reading frame was verified by restriction analysis and DNA sequencing.
Cell Culture and Transfection-CHO-K1 cells were maintained under 5% CO 2 at 37°C in growth medium (Ham's F12 medium supplemented with 10% fetal calf serum, 50 units/ml penicillin, and 50 g/ml streptomycin). CHO cells stably expressing PAF receptor or LTB 4 receptor were generated as described previously (25,28). Briefly, cDNAs of guinea pig PAF receptor and human LTB 4 receptor were subcloned into mammalian expression vectors, pcDNAI/Neo and pcDNA3 (Invitrogen, Carlsbad, CA), respectively, and transfected into CHO-K1 cells. Clones resistant to Geneticin (1 mg/ml) were isolated by limiting dilution, tested for receptor expressions by binding assay, and maintained in the presence of 0.3 mg/ml Geneticin. PAF-LT cells were generated by co-transfection of PAF11 cells expressing PAF receptor (see below) with the expression vector for LTB 4 receptor and pPUR encoding the puro-mycin resistance gene (CLONTECH), and by selection with puromycin (10 g/ml, Sigma).
For cPLA 2 -GFP expression, cells were seeded at a density of 5 ϫ 10 5 cells/60-mm dishes and transiently transfected with 2 g of the expression vector encoding a cPLA 2 -GFP fusion protein (pcPLA 2 -GFP) or a control vector pEGFP-N3 with LipofectAMINE PLUS (Life Technologies, Inc.), according to the manufacturer's protocol, and used for experiments 48 -72 h after transfection. The amount of cPLA 2 -GFP was comparable to that of endogenous cPLA 2 , as determined by immunoblotting with anti-cPLA 2 antibody (data not shown).
Binding Assay-Binding assays for the PAF receptor or the LTB 4 receptor on CHO-K1 cells were performed as described previously (29 12 mM NaHCO 3 , 0.37 mM NaH 2 PO 4 , 10 mM Hepes (pH 7.4), and 0.1% (w/v) fatty acid-free BSA) and then lysed in 1% Triton X-100. The radioactivity of the cell lysate was determined by liquid scintillation counting. K d and B max values were calculated by Scatchard analysis.
Single Cell Calcium Imaging-4 ϫ 10 4 cells were seeded on coverslips (16-mm diameter) in the growth medium and incubated for 24 h. After another 24 h incubation in serum-free medium containing 0.1% (w/v) fatty acid-free BSA, the cells were washed twice with Hepes/ Tyrode's/BSA buffer and incubated at 37°C with 10 M fura-2 acetoxymethyl ester and 0.01% (w/v) cremophore EL for 1 h in the buffer. After loading, cells were washed with and maintained in the same buffer in the dark at room temperature. Fura-2 fluorescence intensity was measured by alternating excitation at 340 and 380 nm and detecting emission at 505 nm with a 40ϫ objective (Nikon UV-fluor), an SIT camera, and an ARGUS-50/CA image processor (Hamamatsu Photonics, Hamamatsu, Japan). To determine intracellular free Ca 2ϩ concentrations, the 340/380 ratio of fluorescence intensities were compared with the ratio for Ca 2ϩ standard in solution (Molecular Probes, Eugene, OR).
AA Release Assay-Cells were seeded onto 12-well culture plates at a density of 8 ϫ 10 4 cells/well in the growth medium. After 24 h incubation, the medium was removed, and the cells were labeled by incubation for 24 h in 0.75 ml of serum-free medium containing 3.7 kBq of [ 3 H] arachidonic acid (3.7 TBq/mmol) and 0.1% (w/v) fatty acid-free BSA. The cells were then washed three times with Hepes/Tyrode's/BSA buffer and stimulated with ligands or ionomycin in the same buffer at 37°C. Radioactivity of supernatants and cell lysates (in 1% Triton X-100) were measured by liquid scintillation counting. The amount of the radioactivity released into the supernatant was expressed as a percentage of the total incorporated radioactivity.
Confocal Microscopy-Cells transfected with pcPLA 2 -GFP were seeded on coverslips (14-mm diameter) of glass-bottomed culture dishes (Matsunami, Osaka, Japan) at a density of 2.6 ϫ 10 4 and serumstarved as described above. The culture medium was replaced with Hepes/Tyrode's/BSA buffer, and fluorescence images were taken in a FLUOVIEW confocal laser scanning microscope system (Olympus, Tokyo) with a 100ϫ oil immersion objective (NA 1.35), 488-nm laser line for excitation and a 520 Ϯ 20-nm band pass filter for emission. 4 Receptor-Guinea pig PAF receptor and human LTB 4 receptor were expressed in CHO-K1 cells that have no detectable endogenous receptors. Six cell lines PAF11, PAF12, PAF14, LT13, LTF2, and PAF-LT were examined for binding assay, agonist-induced Ca 2ϩ response (Fig. 1), and AA release. Results obtained using these cell lines are summarized in Table I. In cells with low levels of receptor expression (PAF12 and LT13 cells), stimulation with agonists caused a rapid and transient [Ca 2ϩ ] i increase, which returned to the base line within 2-5 min (Fig. 1, B and F). These cell lines did not release AA in response to PAF or LTB 4 . On the other hand, in cells with high levels of receptor expression (PAF11, PAF14 and LTF2 cells), stimulation with agonists induced a sustained [Ca 2ϩ ] i increase which remained elevated at least for 5 min (Fig. 1, A, C, and E) and AA release. In PAF-LT cell line with a high level of PAF receptor and a low level of LTB 4 receptor expression, PAF induced a sustained [Ca 2ϩ ] i increase (Fig. 1D) and AA release, whereas LTB 4 induced a transient [Ca 2ϩ ] i increase (Fig. 1G) and little AA re-lease. These results suggest that the sustained [Ca 2ϩ ] i increase is necessary for receptor-mediated AA release from CHO cells.

Agonist-induced Calcium Response and AA Release in CHO Cells Expressing PAF or LTB
Requirement of Calcium Influx for AA Release-PAF11 cells with a relatively higher receptor expression were used in the following studies. 100 nM PAF induced AA release in the time   Fig. 2A. It had a 2-min lag phase, followed by a linear increase up to 10 min. PAF induced AA release in a dose-dependent manner, and ED 50 was around 4 ϫ 10 -10 M (Fig. 2B). We next examined contributions of intracellular and extracellular Ca 2ϩ sources in the agonist-induced Ca 2ϩ response and AA release. PAF stimulation in the presence of either Ca 2ϩ chelator EGTA (10 mM) or Ca 2ϩ channel blockers (50 M SKF 96365 or 100 M methoxyverapamil) (30, 31) evoked a transient [Ca 2ϩ ] i increase which returned to the base line within 2 min (Fig. 3, A-D). Thus, the initial rise in [Ca 2ϩ ] i is attributed to release from the intracellular Ca 2ϩ pool, whereas the sustained elevation requires Ca 2ϩ influx. Fig. 4A shows that lowering extracellular Ca 2ϩ concentration or depriving Ca 2ϩ by EGTA reduced PAF-stimulated AA release, whereas increasing its concentration enhanced the release. Pretreatment of the cells with SKF 96365 or methoxyverapamil inhibited PAF-induced AA release (Fig. 4B), indicating that the Ca 2ϩ influx is required for AA release.

Critical Length of [Ca 2ϩ ] i Elevation for AA Release-EGTA
was applied at various time points before and after PAF stimulation in order to terminate [Ca 2ϩ ] i elevation. Single cell Ca 2ϩ imaging confirmed that addition of EGTA after PAF stimulation created a transient [Ca 2ϩ ] i increase, which returned to the base line within 1 min following the addition (Fig. 5A). Fig. 5C shows that EGTA added 1 min after PAF stimulation substantially inhibited the PAF-induced AA release but did not alter the AA release when added 5 min after the stimulation.
Since stimulation of the PAF receptor results in various signals besides [Ca 2ϩ ] i elevation (32), similar experiments were carried out using a Ca 2ϩ ionophore ionomycin to study the role of Ca 2ϩ signaling in a more direct manner. The prolonged [Ca 2ϩ ] i increase was generated by 2 M ionomycin and had about the same magnitude as the PAF-stimulated plateau (Fig.  5B). The transient [Ca 2ϩ ] i increase was created by ionomycin with the following addition of EGTA, and this increase returned to the base line within 1 min after the addition (Fig. 5B). The ionomycin-induced sustained [Ca 2ϩ ] i rise caused AA release, whereas the brief [Ca 2ϩ ] i rise did not (Fig. 5D), confirming that a prolonged [Ca 2ϩ ] i increase (over 2 min) is essential for AA release.
Translocation and Reversal of cPLA 2 by the Change in [Ca 2ϩ ] i -Due to its inherent fluorescence and unique compact structure, green fluorescent protein (GFP) has been reported in many studies to serve as a valuable reporter in the localization of various proteins without interfering with their biological activity (33). We constructed a chimeric protein cPLA 2 -GFP by fusing EGFP to the carboxyl terminus of cPLA 2 . To ensure that the fusion of GFP did not affect structure and enzyme activity of cPLA 2 , the cPLA 2 -GFP construct was analyzed for AA release. The expression of cPLA 2 -GFP enhanced PAF-induced AA release in PAF11 cells by about 1.5-fold at 10 min. The correlation of AA release with the duration of [Ca 2ϩ ] i increase was essentially identical to that in untransfected or GFP-transfected cells (Fig. 5, C and E). In addition, gel shift of cPLA 2 -GFP in response to agonist stimulation was comparable with that of endogenous cPLA 2 (data not shown). The gel shift has been established to result from phosphorylation at Ser-505 (7). These results suggest that the fusion of GFP to the carboxyl terminus of cPLA 2 does not perturb cPLA 2 function.
We then monitored the localization of the cPLA 2 -GFP in living cells by confocal laser fluorescence microscopy. In the resting state, cPLA 2 -GFP was almost homogeneously present in the cytosol (Fig. 6, A and B, 0 min). Stimulation of the cells with PAF or ionomycin triggered translocation of cPLA 2 -GFP to the perinuclear region within 1 min, and the cPLA 2 -GFP fluorescence was retained for over 10 min (Fig. 6, A and B). When PAF12 cells transiently expressing cPLA 2 -GFP were stimulated with PAF, the translocation of cPLA 2 -GFP to the perinuclear region was transient and reversed within 5 min (Fig.  6C). In contrast to cPLA 2 -GFP, unconjugated GFP was distributed homogeneously in the cytosol and nucleus, and its distribution was not affected by PAF or ionomycin (data not shown).
Next, the effect of termination of [Ca 2ϩ ] i increase by EGTA on PAF-induced translocation of cPLA 2 -GFP was examined. cPLA 2 -GFP translocation was reversed rapidly when EGTA was added 1 min after PAF stimulation (Fig. 7A, upper panels).
In contrast, the reversal was much slower when EGTA was added 5 min after stimulation; a large portion of cPLA 2 -GFP was retained at the perinuclear region for another 5 min after the addition of EGTA (Fig. 7A, lower panels). Similar redistributions of cPLA 2 -GFP were obtained by application of ionomycin instead of PAF (Fig. 7B). These results agree with the finding that AA release was inhibited when EGTA was added 1 min after PAF application, while it was not inhibited after 5 min (Fig. 5C), which means that a prolonged [Ca 2ϩ ] i increase for about 5 min induces stable localization of cPLA 2 at the perinuclear region and continuous hydrolysis of AA from membrane phospholipids. DISCUSSION cPLA 2 plays a major role in releasing AA upon cell stimuli and in production of eicosanoids (10,11,26). This enzyme is activated by [Ca 2ϩ ] i increases in physiological ranges. Although previous reports have shown by immunohistochemical and biochemical methods that cPLA 2 is translocated to membranes in response to [Ca 2ϩ ] i increases, the dynamic change of the enzyme localization in the living cell has not been reported. The question we addressed here was how different Ca 2ϩ signaling patterns control cPLA 2 translocation and AA release. We found that a critical time length of [Ca 2ϩ ] i elevation is required for continuous membrane localization and full activation of cPLA 2 .
Optimal concentrations of PAF and LTB 4 triggered comparable maximal increases in [Ca 2ϩ ] i in CHO cell lines expressing PAF and/or LTB 4 receptor. However, duration of the [Ca 2ϩ ] i increase varied with the cell line (Fig. 1). Correlations between Ca 2ϩ signaling pattern (transient or sustained) and response in AA release (Table I)  induced Ca 2ϩ signaling pattern is responsible for the release of AA. Calcium mobilization from internal stores and subsequent Ca 2ϩ entry from the extracellular space are the two major components of Ca 2ϩ signaling following activation of cell-surface receptors. The inability of PAF to induce AA release in Ca 2ϩ -free medium (Fig. 4A) or in the presence of Ca 2ϩ channel blockers (Fig. 4B) indicates the critical role of Ca 2ϩ influx in AA release. This is consistent with the previous observations, in which AA release has been linked to Ca 2ϩ influx (21). Ca 2ϩ entry across the plasma membrane occurs via voltage-, store-, or receptor-operated channels. Although the primary route of Ca 2ϩ influx in CHO cells remains to be elucidated, 100 M nifedipine, an L-type voltage-dependent Ca 2ϩ channel blocker, had no apparent effects on the Ca 2ϩ influx induced by PAF or LTB 4 (data not shown).
Stimulation of PAF receptor exerts various responses, including inhibition of adenylyl cyclase, activations of MAPK, phosphatidylinositol 3-kinase, phospholipases C and D, and tyrosine kinases, as well as increase in [Ca 2ϩ ] i (24,32,34,35). Although MAPK was activated by PAF and LTB 4 to about the same extent in PAF-LT cells (data not shown), LTB 4 did not induce AA release (Table I), suggesting that MAPK activation with a transient [Ca 2ϩ ] i increase is insufficient to induce AA release. The experiments using ionomycin and EGTA confirmed that a sustained [Ca 2ϩ ] i increase is essential for AA release in CHO cells (Fig. 5D). The difference in the extent of AA release between PAF and ionomycin (Fig. 5, C and D) is probably due to phosphorylation of cPLA 2 by MAPK. Whereas PAF induced MAPK activation (34) and mobility shift of cPLA 2 in SDS-polyacrylamide gel electrophoresis (36), ionomycin did not activate MAPK or promote the gel shift of cPLA 2 (data not shown). These results are consistent with the previous reports that phosphorylation of cPLA 2 increases its intrinsic enzymatic activity (7, 37, 38) and augments Ca 2ϩ -induced AA release (26,39). It was recently reported that, in mouse peritoneal macrophages, the effects of phosphorylation of cPLA 2 on AA release differ depending on whether calcium response is transient or sustained (40). Further studies are necessary to identify fully the individual and combined roles of calcium and phosphorylation for the activation of cPLA 2 in various types of cells.
Calcium-mediated translocation of heterologously expressed cPLA 2 to the nuclear envelope and endoplasmic reticulum was shown previously in CHO cells using an anti-cPLA 2 antibody (14). Because immunohistochemical studies provide only a static picture and have a possible loss in signal linearity due to fixation, permeabilization, and staining of the cells, we took advantage of the GFP fusion protein and digital imaging technique to monitor cPLA 2 translocation in real time in living cells. Although the endoplasmic reticulum forms a fine reticular network throughout the cytoplasm and around the nuclear envelope in CHO cells (41), cPLA 2 -GFP was translocated upon [Ca 2ϩ ] i increase to a restricted space at the perinuclear region including the nuclear envelope (Figs. 6 and 7). This observation may be consistent with the study in rat alveolar epithelial cells, in which stimulation with a calcium ionophore A23187 induced translocation of cPLA 2 to the nuclear membrane fraction and preferential loss of AA from this membrane fraction (42). Therefore, functional activation of cPLA 2 takes place after translocation to the perinuclear region. Mechanisms involved in the preferential targeting of the enzyme to this site are under investigation.
Another important finding of the present study is that a brief [Ca 2ϩ ] i rise caused a transient translocation of cPLA 2 without AA release, whereas an increase in [Ca 2ϩ ] i of longer duration (ϳ5 min) induced a prolonged translocation of cPLA 2 and AA release even after [Ca 2ϩ ] i returned to the resting level (Figs. 5 and 7). To explain these observations, we propose a two-step mechanism for cPLA 2 activation. In the initial step, cPLA 2 is translocated to the perinuclear region upon increase in [Ca 2ϩ ] i and weakly binds to the membranes. The enzyme is not yet activated, and the translocation can be readily reversed upon decrease in [Ca 2ϩ ] i . In the second step, the binding of cPLA 2 to the membranes becomes stronger with a marked reduction in the dissociation rate. Concomitantly, the enzyme is activated. The transfer from the first step to the second requires a critical length of continuous [Ca 2ϩ ] i increase. The critical duration of [Ca 2ϩ ] i increase was ϳ2 min in PAF11 cells but may differ depending on the cell type and the level of cPLA 2 expression. Possible mechanisms by which cPLA 2 is transferred from the weak-binding state to the strong-binding state include conformational change of cPLA 2 by phosphorylation (7), dissociation of inhibitory proteins such as p11 (43), and interaction with anchoring or activating factors (44). The requirement of critical duration of [Ca 2ϩ ] i increase for the activation of cPLA 2 may serve as a safety mechanism to discriminate appropriate signals from transient fluctuations of [Ca 2ϩ ] i . Once the [Ca 2ϩ ] i transient exceeds the critical duration, the strong-binding state of cPLA 2 allows the cells to produce AA for prolonged periods even after [Ca 2ϩ ] i returns to the resting value.