Distinct Roles in Signal Transduction for Each of the Phospholipase A 2 Enzymes Present in P388D 1 Macrophages*

Receptor-stimulated arachidonic acid (AA) mobiliza- tion in P388D 1 macrophages consists of a transient phase in which AA accumulates in the cell and a sus- tained phase in which AA accumulates in the incubation medium. We have shown previously that a secretory group II phospholipase A 2 (sPLA 2 ) is the enzyme responsible for most of the AA released to the incubation me- dium. By using selective inhibitors for each of the PLA 2 s present in P388D 1 macrophages, we demonstrate herein that the cytosolic group IV PLA 2 (cPLA 2 ) mediates accumulation of cell-associated AA during the early steps of P388D 1 cell activation. The contribution of both cPLA 2 and sPLA 2 to AA release can be distinguished on the basis of the different spatial and temporal characteristics of activation and substrate preferences of the two phospholipase A 2 s (PLA 2 s). Furthermore, the results suggest the possibility that a functionally active cPLA 2 may be necessary for sPLA 2 to act. cPLA 2 action pre- cedes that of sPLA 2 , and overcoming cPLA 2 inhibition by artificially increasing intracellular free AA levels re-stores extracellular AA release. Although this suggests cross-talk between cPLA 2 and sPLA 2 , selective inhibition of one other PLA 2 present in these cells, namely the Ca 2 (cid:49) -independent PLA 2 , does not block, but instead en- hances receptor-coupled AA release. These data indicate that Ca 2 (cid:49) -independent PLA 2 does not mediate AA mobilization in P388D 1 macrophages. Collectively, the results of this work suggest that sn -glycero-3-phosphoethanol-amine; arachidonyl fluorophosphonate; choline-con- taining glycerophospholipids; glycerophospholipids;

Receptor-stimulated arachidonic acid (AA) mobilization in P388D 1 macrophages consists of a transient phase in which AA accumulates in the cell and a sustained phase in which AA accumulates in the incubation medium. We have shown previously that a secretory group II phospholipase A 2 (sPLA 2 ) is the enzyme responsible for most of the AA released to the incubation medium. By using selective inhibitors for each of the PLA 2 s present in P388D 1 macrophages, we demonstrate herein that the cytosolic group IV PLA 2 (cPLA 2 ) mediates accumulation of cell-associated AA during the early steps of P388D 1 cell activation. The contribution of both cPLA 2 and sPLA 2 to AA release can be distinguished on the basis of the different spatial and temporal characteristics of activation and substrate preferences of the two phospholipase A 2 s (PLA 2 s). Furthermore, the results suggest the possibility that a functionally active cPLA 2 may be necessary for sPLA 2 to act. cPLA 2 action precedes that of sPLA 2 , and overcoming cPLA 2 inhibition by artificially increasing intracellular free AA levels restores extracellular AA release. Although this suggests cross-talk between cPLA 2 and sPLA 2 , selective inhibition of one other PLA 2 present in these cells, namely the Ca 2؉ -independent PLA 2 , does not block, but instead enhances receptor-coupled AA release. These data indicate that Ca 2؉ -independent PLA 2 does not mediate AA mobilization in P388D 1 macrophages. Collectively, the results of this work suggest that each of the PLA 2 s present in P388D 1 macrophages serves a distinct role in cell activation and signal transduction.
Phospholipase A 2 (PLA 2 ) 1 enzymes play a fundamental role in numerous cellular processes by generating an array of metabolites with various biological functions. PLA 2 -mediated hydrolysis of glycerophospholipids results in the release of arachi-donic acid (AA) and lysophospholipids, which may either exert direct effects or serve as substrates for the generation of other lipid messengers such as the eicosanoids or platelet-activating factor (PAF) (1).
Mammalian cells contain multiple PLA 2 forms (1), and there is considerable interest in determining the role that each PLA 2 plays in mediating cellular functions. At least three different cellular PLA 2 s have been proposed to play a role in the mobilization of AA from phospholipids. These are the cytosolic group IV PLA 2 (cPLA 2 ) (2-4), the secretory group II PLA 2 (sPLA 2 ) (5-7), and a cytosolic Ca 2ϩ -independent PLA 2 (iPLA 2 ) (8,9). Involvement of one or another PLA 2 form appears to depend on the cell type and agonist involved.
Our laboratory has been examining the molecular mechanisms involved in AA mobilization in murine P388D 1 macrophage-like cells (6, 10 -12). Stimulation of these cells with nanomolar quantities of the receptor agonist PAF results in a very modest mobilization of free AA. However, preincubation of the cells with bacterial lipopolysaccharide (LPS) prior to stimulation with PAF increases the release of AA by these cells by about 2-3-fold (10). Recently, we have demonstrated that AA mobilization in response to LPS/PAF involves participation of a sPLA 2 localized at the outer surface of the cell and that this enzyme accounts for the majority of the AA released to the extracellular medium (6,12). In the current study, we have obtained further evidence using chemical inhibitors for the role of sPLA 2 and have aimed at defining the roles played by the other two PLA 2 s present in P388D 1 macrophages, namely cPLA 2 and iPLA 2 .
Cell Culture and Labeling Conditions-P388D 1 cells were maintained at 37°C in a humidified atmosphere at 90% air and 10% CO 2 in * This work was supported by National Institutes of Health Grants HD 26171 and GM 20501. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
‡ Recipient of a Postdoctoral Fellowship from the American Heart Association, California Affiliate.
Iscove's modified Dulbecco's medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units/ml penicillin, 100 g/ml streptomycin, and nonessential amino acids. Cells were plated at 10 6 per well, allowed to adhere overnight, and used for experiments the following day. All experiments were conducted in serum-free Iscove's modified Dulbecco's medium. Radiolabeling of the cells with [ 3 H]AA was achieved by including 0.5 Ci/ml [ 3 H]AA during the overnight adherence period (20 h) (12). When double-labeled cells were used, the cells were first labeled with [ 3 H]AA for 20 h as described above and then were incubated in serum-free medium with [ 14 C]AA (0.1 Ci/ml) for 10 min. Labeled AA that had not been incorporated into cellular lipids was removed by washing the cells four times with serum-free medium containing 5 mg/ml albumin.
Stimulation of P388D 1 Cells-The standard regimen for activating cells with LPS and PAF has been described previously (12). Briefly, P388D 1 cells were placed in serum-free medium containing 1 mg/ml bovine serum albumin for 30 -60 min before the addition of LPS (200 ng/ml) for 1 h. After the LPS incubation, cells were overlaid with serum-free medium containing 1 mg/ml albumin for 5-15 min, after which they were challenged with 100 nM PAF for the time indicated. More than 99% of released radioactive material remains as unmetabolized AA under these experimental conditions (12).
Measurement of Extracellular AA Release and Cell-associated Free AA-LPS-treated cells labeled with either [ 3 H]AA alone or [ 3 H]AA plus [ 14 C]AA were stimulated with 100 nM PAF for the times indicated. The supernatants were removed, cleared of detached cells by centrifugation, and assayed for radioactivity by liquid scintillation counting. For analysis of cell-associated, free AA, the cell monolayers were scraped in 1 ml of 0.5% Triton X-100. Lipids were extracted according to Bligh and Dyer (15) and separated by thin-layer chromatography using n-hexane/diethyl ether/acetic acid (70:30:1, v/v/v) as a solvent system. Authentic AA was co-chromatographed and visualized by exposing the plates to iodine vapors. Areas containing AA were scraped into scintillation vials, and the amount of radioactivity was measured by liquid scintillation counting.
Measurement of lyso-PC Levels-For the measurement of lyso-PC, cells were labeled with 0.5 Ci/ml [ 3 H]choline for 3 days. The cells were activated with LPS and PAF as described above. After the indicated times, supernatants were discarded, and the cell monolayers were scraped in 1 ml of 0.5% Triton X-100. Lipids were extracted with ice-cold n-butanol and separated by thin-layer chromatography, using chloroform/methanol/acetic acid/water (50:40:6:0.6, v/v/v/v) as a solvent system (16). Spots corresponding to lyso-PC were scraped into scintillation vials, and the amount of radioactivity was measured by liquid scintillation counting.
Data Presentation-Except for the data shown in Fig. 1, agoniststimulated AA release is expressed by subtracting the basal rate observed in the absence of agonist and inhibitor. These background values were in the range of 2000 -3000 cpm for extracellular [ 3 H]AA and 500-1000 cpm for cell-associated free [ 3 H]AA. Assays were carried out in duplicate or triplicate. Each set of experiments was repeated at least three times with similar results. Unless otherwise indicated, the data presented are from representative experiments.

RESULTS
Two Different AA Pools Contribute to AA Release during Activation of P388D 1 Cells-Our previous work established that AA release in LPS-primed, PAF-stimulated P388D 1 cells is composed of two events: a transient phase in which AA accumulates in the cell and a sustained phase in which the fatty acid accumulates in the extracellular medium (12). We began the current study by determining whether the fatty acid liberated during these two phases arises from separate phospholipid pools. To this end, we used the methodology described by Fonteh and Chilton (17) to selectively label the AA-containing phospholipids. The cells were first labeled with [ 3 H]AA for 20 h, a time frame long enough to allow [ 3 H]AA to equilibrate among phospholipids (12). Under these conditions, PE accounts for the majority of esterified [ 3 H]AA in phospholipids, the remainder being esterified in PC and PI/phosphatidylserine (12). After the 20-h incubation with [ 3 H]AA, the cells were primed with LPS for 1 h, washed, and pulse-labeled with [ 14 C]AA for 10 min. At these short labeling times, the distribution of [ 14 C]AA esterified in phospholipids dramatically differs from that seen at long incubation times in that PC, not PE, is the phospholipid that incorporates most of the radiolabeled AA (12). Treating the cells with LPS alone did not raise the levels of extracellular or cell-associated free AA.
Subsequent to the labeling, the cells were activated with PAF, and the [ 14 C]/[ 3 H] ratios were determined in the phospholipid classes as well as in the AA liberated at the two different locations. Cell-associated free AA and extracellular free AA had very different [ 14 C]/[ 3 H] ratios, indicating that the AA released at these two locations was derived from different pools (Fig. 1). The [ 14 C]/[ 3 H] ratio for extracellular free AA had a ratio well below those of PC and PI/phosphatidylserine, but close to that of PE ( Fig. 1). This suggested that PE may be a major source for the AA released to the extracellular medium. Consistent with this view, the 14 C/ 3 H ratio for extracellular free AA in unstimulated cells was 0.7 Ϯ 0.1, that is, slightly higher than that observed in PAF-activated cells (0.4 Ϯ 0.1). In contrast, the [ 14 C]/[ 3 H] ratio for cell-associated free AA was intermediate between that of PE and those of PC and PI/ phosphatidylserine ( Fig. 1), suggesting that cell-associated free AA has been derived from all of these phospholipid classes. Within error, there was no difference between the 14 C/ 3 H ratio for cell-associated AA in unstimulated cells (1.0 Ϯ 0.2) versus PAF-stimulated cells (0.8 Ϯ 0.1), suggesting that intracellular resting levels of AA may also derive from all major phospholipid classes.
PLA 2 Inhibition Studies-A useful approach to study the involvement of distinct PLA 2 s in AA mobilization is the use of selective inhibitors for each of these enzymes. Because each PLA 2 group exhibits different catalytic properties and substrate preferences (1), inhibitors based on these characteristics should allow one to distinguish among the different cellular PLA 2 s. The PLA 2 inhibitors used in this work are all based on the aforementioned properties.
Using antisense RNA technology, we have previously demonstrated that group II sPLA 2 is responsible for at least 60 -70% of the AA released to the incubation medium but is not involved in raising cellular AA levels shortly after cell activation with PAF (12). In the current study, pharmacological inhibition of sPLA 2 was accomplished by incubating the cells either with the water-soluble phospholipid analog, diC 6 SNPE (14), or the indole derivative LY311727, which is an indomethacin analogue (18). diC 6 SNPE inhibits human synovial group II PLA 2 with an IC 50 of 27 M when assayed in a spectrophotometric assay with 2 mM substrate. 2 At concentrations up to 100 M, diC 6 SNPE has no effect on pure human group IV cPLA 2 , 2 S. C. Boegeman, and E. A. Dennis, unpublished data. nor does it affect PLA 2 activity from P388D 1 cell homogenates as measured toward 1-palmitoyl-2-arachidonoyl-sn-glycero-3phosphocholine vesicles in the presence of Ca 2ϩ and ␤-mercaptoethanol. 3 In addition, diC 6 SNPE does not inhibit pure Ca 2ϩindependent PLA 2 from P388D 1 cells (13). The properties of the indole derivative LY311727 as a potent and selective inhibitor of sPLA 2 have recently been reported (18). sPLA 2 inhibition by either diC 6 SNPE or LY311727 markedly decreased the extracellular release of [ 3 H]AA from prelabeled P388D 1 cells ( Figs. 2A and 3A). No effect of these inhibitors was detected on the accumulation of cell-associated free [ 3 H]AA (Figs. 2B and 3B). These data are fully consistent with our previous data using antisense RNA technology to block sPLA 2 activity (12).
Involvement of group IV cPLA 2 was initially investigated by using MAFP (19). This compound is an irreversible inhibitor of the cPLA 2 and has no effect on the sPLA 2 (19). We have confirmed in our laboratory these findings and in addition have found that MAFP does not appreciably affect arachidonoyl-CoA synthetase, lysophosphatidylcholine:arachidonoyl-CoA acyltransferase, or CoA-independent transacylase activities in homogenates from MAFP-treated cells. Fig. 4 shows that MAFP strongly inhibited AA mobilization in PAF-activated cells. Whereas MAFP inhibited the extracellular release of [ 3 H]AA from prelabeled cells by about 75% (Fig. 4A), the PAF-induced accumulation of cellular [ 3 H]AA was almost completely blocked by the inhibitor (Fig. 4B).
P388D 1 macrophages possess a third PLA 2 enzyme, namely a cytosolic iPLA 2 that shows no preference for AA-containing phospholipids; in fact, it prefers palmitoyl over arachidonoyl residues (20). Recent evidence from our laboratory indicates that MAFP also inhibits pure iPLA 2 from P388D 1 cells. 4 Therefore, at least part of the MAFP-sensitive AA mobilization could be mediated by the iPLA 2 in addition to the cPLA 2 . The iPLA 2 from P388D 1 macrophages is potently and irreversibly inhibited by the mechanism-based inhibitor (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (bromoenol lactone, BEL) (13). This compound manifests over a 1000-fold selectivity for inhibition of the iPLA 2 s versus the Ca 2ϩ -dependent sPLA 2 s (21) and has previously been used to investigate the role of iPLA 2 in AA release in certain cell types (8,9). In our laboratory, we have found that BEL is a poor inhibitor of pure cPLA 2 . 5 This inhibitor does not affect any of the following activities, measured in homogenates from BEL-treated cells: cPLA 2 , sPLA 2 , arachidonoyl-CoA synthetase, lysophosphatidylcholine:arachidonoyl-CoA acyltransferase, and CoA-independent transacylase (22).
The effect of BEL on PAF-induced AA mobilization from LPS-primed P388D 1 cells is shown in Fig. 5. At concentrations up to 50 M, which totally block cellular iPLA 2 (22), BEL was ineffective in inhibiting either the extracellular release of [ 3 H]AA (Fig. 5A) or the accumulation of cellular free fatty acid (Fig. 5B). Instead, BEL enhanced both basal and PAF-stimulated [ 3 H]AA mobilization, although the ratio of stimulated versus unstimulated release remained the same at all BEL concentrations. The enhancing effect of BEL on P388D 1 macrophage AA release is probably related to its inhibitory action on cellular fatty acid incorporation into phospholipid (22). The lack of any inhibitory effect of BEL on [ 3 H]AA release demonstrates that the iPLA 2 does not significantly contribute to this release. Therefore, the MAFP-sensitive [ 3 H]AA release should be ascribed to the cPLA 2 .
Priming of AA Release by Okadaic Acid-It is well established that cellular cPLA 2 activity is regulated by phosphorylation (2)(3)(4). Okadaic acid, a protein phosphatase inhibitor, also induces activation of cPLA 2 by preventing dephosphorylation of the enzyme (4). Therefore, this reagent was used to further evaluate the involvement of cPLA 2 in the PAF activation. As shown in Fig. 6A, pretreatment with okadaic acid resulted in the cells becoming sensitized for an enhanced [ 3 H]AA release in response to PAF. Importantly, when okadaic acid was added along with LPS during priming, a strong potentiation of the AA release response was observed. Okadaic acid not only increased both extracellular (Fig. 6B) and cell-associated [ 3 H]AA (Fig.  6C), but it also augmented cellular lyso-PC levels in cells prelabeled with [ 3 H]choline (Fig. 6D). These data stress the ability of okadaic acid to amplify LPS/PAF activation of AA mobilization, thus supporting the involvement of the phosphorylationregulated cPLA 2 in this process. Interestingly, when the experiments depicted in Fig. 6 were carried out in the presence of the sPLA 2 inhibitor diC 6 SNPE (50 M), extracellular [ 3 H]AA release was still inhibited by 45 Ϯ 11% (mean Ϯ S.E., n ϭ 3), indicating that augmentation of cPLA 2 activity by okadaic acid does not result in a full response unless a functionally active sPLA 2 is present. diC 6 SNPE did not affect cell-associated free AA levels in okadaic acid-treated cells. cPLA 2 Activation Precedes That of sPLA 2 -From our previous results using antisense RNA technology (6,12) as well as our current data using the sPLA 2 inhibitors diC 6 SNPE and LY311727, it is apparent that this enzyme accounts for a major portion of the [ 3 H]AA released into the incubation medium after 10 min of activation with PAF. However, our data using MAFP clearly show that selective inhibition of cPLA 2 results in at least 75% inhibition of extracellular [ 3 H]AA release (Fig.  4A). Inasmuch as MAFP does not directly inhibit sPLA 2 (see above), an explanation for these data could be that activation of cPLA 2 is necessary for sPLA 2 to act. Should this be the case, cPLA 2 activation must precede that of sPLA 2 . Evidence in support of this view was obtained by investigating the time course of total [ 3 H]AA mobilization (i.e. cell-associated plus extracellularly released [ 3 H]AA) in cells treated with either MAFP or diC 6 SNPE to selectively block cPLA 2 or sPLA 2 , respectively. As shown in Fig. 7, inhibition of [ 3 H]AA release by diC 6 SNPE was only apparent after 2-3 min of cell activation, a time frame at which cell-associated AA nearly drops to levels occurring in unstimulated cells (12). On the other hand, inhibition of AA release by MAFP was already observed at the earliest point measured, i.e. 1 min (Fig. 7), demonstrating that the action of cPLA 2 on cellular phospholipids precedes that of sPLA 2 .
We next explored whether the addition of metabolites result-  (Fig. 8). If, however, P388D 1 cells were exposed to exogenous AA (1 M) for 1 min before PAF addition, the inhibitory effect of MAFP on extracellular [ 3 H]AA was greatly diminished (Fig. 8). Preincubating the cells with lysophospholipids (i.e. lyso-PC, lyso-PI, or lyso-PE) or other free fatty acids, whether saturated (i.e. palmitic, stearic, or arachidic acids) or unsaturated (oleic or linoleic acids) did not overcome the inhibitory effect of MAFP. Control experiments had shown that at the doses employed, none of the above mentioned fatty acids or lysophospholipids exerted cytotoxic effects or affected basal [ 3 H]AA release. On the other hand, preincubating the cells with exogenous AA prior to PAF addition did not overcome the inhibitory effect of diC 6 SNPE on extracellular [ 3 H]AA release (Fig. 8). 2 Enzymes-By using antisense RNA technology to block the expression of sPLA 2 , we previously demonstrated that an extracellular pool of this enzyme is responsible for at least 60 -70% of the AA released from P388D 1 macrophages after activation with LPS/PAF (6). By using an anti-sPLA 2 monoclonal antibody, Pfeilschifter et al. (7) have estimated a similar contribution of sPLA 2 to extracellular AA release during receptor stimulation of rat mesangial cells. In the current study, we have utilized a third and different strategy to block sPLA 2 activity, i.e. the use of diC 6 SNPE and LY311727, two selective and structurally unrelated sPLA 2 inhibitors, and have confirmed that this enzyme mediates a major portion of the AA released to the incubation medium. Although these three different strategies emphasize the very important role of sPLA 2 in AA release, they also stress that another effector enzyme is involved as well. Moreover, both antisense inhibition of sPLA 2 and pharmacological inhibition of the enzyme by diC 6 SNPE and LY311727 have highlighted the fact that sPLA 2 is not the enzyme that mediates the small burst of cell-associated free AA that occurs shortly after PAF stimulation. Use of multiple selective inhibitors in this study has provided evidence that the second effector enzyme in AA release in PAF-stimulated LPSprimed P388D 1 macrophages is the cPLA 2 . Thus, the current work, along with our previous data (6,12), establish that cPLA 2 , acting intracellularly, and sPLA 2 , acting on the outer surface of the cell, both mediate AA release in response to PAF receptor stimulation. It is important to note that inhibition of sPLA 2 by either antisense techniques (6,12) or chemical inhibitors (this study) does not result in complete inhibition of AA release to the extracellular medium. This suggests that under PAF activation conditions, a portion of the AA released intracellularly by the cPLA 2 may exit the cell and mix with the fatty acid liberated by the sPLA 2 .

Regulation of AA Mobilization by Two Different PLA
The notion that both cPLA 2 and sPLA 2 s mediate receptor activation of AA release may represent a signaling mechanism common to agonists that elicit short-term (i.e. PAF) or longterm responses (i.e. cytokines and growth factors). Work by Schalkwijk et al. (23,24) in cytokine-stimulated rat mesangial cells and by Murakami et al. (5) in cytokine-stimulated human endothelial cells has also suggested that both PLA 2 s may participate in regulating AA mobilization in these cells, their relative contribution being dependent on the agonist used. However, there are other cell systems such as platelets, in which a role for sPLA 2 in AA release could not be demonstrated (25). Moreover, AA release in thrombin-stimulated platelets could be completely blocked by inhibiting the cPLA 2 , suggesting that in this system, cPLA 2 is perhaps the only effector involved in AA release (25). Domin and Rozengurt (26) have recently demonstrated that AA mobilization in Swiss 3T3 cells treated with platelet-derived growth factor follows a bimodal kinetics. In this system, cPLA 2 activation appears to be responsible for the small burst of AA mobilization that occurs during the first 20 min following agonist stimulation. However, the major component of agonistinduced AA release was found to be due to another unidentified effector. Because these data are very reminiscent of the situation in PAF-stimulated LPS-primed P388D 1 cells, it is tempting to speculate that the second effector involved in the system studied by Domin and Rozengurt (26)  , or neither (q) for 15 min. Subsequently, the cells were incubated with 100 nM PAF for the times indicated, except for the control (É), which also lacked inhibitor. Afterwards, the supernatants were mixed with the cellular homogenates obtained from scraping the cell monolayers with 0.5% Triton X-100, and the resulting mix was subjected to lipid extraction. Free [ 3 H]AA was separated by thin-layer chromatography, and radioactivity was determined by scintillation counting. platelet-derived growth factor-stimulated Swiss 3T3 cells and in PAF-stimulated P388D 1 cells are clearly distinct, this is most likely due to cell type differences and especially to the very distinct nature of the agonists employed. As a matter of fact, when 3T3 cells are challenged with a short burst agonist such as bombesin, a rapid and transient increase in cell-associated free AA is observed shortly after receptor occupancy (27).
Different Phospholipid Sources for the Two PLA 2 s-Given the fact that cPLA 2 and sPLA 2 mobilize AA from activated P388D 1 cells with different spatial and temporal characteristics, it would seem possible that the two enzymes utilize different AA pools. We explored this issue by selectively labeling the different AA-containing phospholipids with [ 3 H]AA and [ 14 C]AA. In P388D 1 cells, two of the major AA-containing phospholipids, namely PC and PI, are labeled with exogenous radioactive AA very rapidly (within minutes), whereas PE is labeled more slowly (12). This phenomenon, along with the nonuniform distribution of arachidonoyl moieties in different phospholipid classes, allowed us to label the phospholipids with AA to different [ 14  H] ratios in free AA with those in the phospholipids, we could delineate the origin of cell-associated AA and extracellular AA. Although the interpretation of these data may be complicated by the phenomenon of mixing AA pools as well as the molecular heterogeneity of each phospholipid class, some definite conclusions can be reached. The fact that the [ 14 C]/[ 3 H] ratio for extracellular AA is considerably lower than that of cell-associated AA suggests that most of the extracellular free AA arises from PE, but this is not the case for cell-associated AA. As a matter of fact, PE is the only phospholipid whose [ 14 C]/[ 3 H] ratio is comparable with that of extracellular AA. Following a similar rationale, it can be concluded that all major phospholipids contribute to the early burst in cell-associated AA, although their relative contribution cannot be estimated from our data. Because cPLA 2 is responsible for raising the levels of cell-associated free AA, involvement of all major phospholipid classes in this process is consistent with the notion that this enzyme does not distinguish among phospholipid head groups (28,29).
It is generally assumed that the phospholipids are asymmetrically distributed in cellular membranes, PC being localized primarily at the outer leaflet and PE at the inner leaflet of the plasma membrane (30). Thus the notion that the extracellular AA release arises primarily from PE would seem, at a first glance, unexpected. However, sPLA 2 , the enzyme primarily responsible for mobilizing AA to the extracellular medium, has been reported to prefer PE over any other phospholipid when these are presented in a natural membrane system (29,31). It is possible that the sPLA 2 preference for PE in these studies could be caused by a higher proportion of PE relative to other phospholipids in these membranes, because studies with vesicles containing various kinds of phospholipids have failed to detect any head group specificity (32). However, studies in platelets have demonstrated that during activation, a rapid translocation of AA-containing PE from the inner to the outer leaflet of the plasma membrane takes place (33). Such a translocation would permit the AA-containing PE to be readily accessible to the extracellular sPLA 2 . Interestingly, recent work by Fourcade et al. (34) has suggested that loss of membrane asymmetry resulting from movement of phospholipids from the inner to the outer leaflet of the membrane may play a key role in regulating the activity of extracellular sPLA 2 .
Cross-talk between the PLA 2 s?-The results of this study raise the possibility that cross-talk may exist between the mechanisms of activation of cPLA 2 and sPLA 2 . We have found that cPLA 2 becomes activated before sPLA 2 begins to act and that inhibition of cPLA 2 by MAFP leads to a very marked inhibition of total AA release induced by PAF, higher than is expected if one considers that sPLA 2 is responsible for at least two-thirds of the AA released to the extracellular medium (6,12). Thus the question arises as to whether cPLA 2 -mediated events are required for the action of sPLA 2 . We explored this issue by directly adding the PLA 2 by-products, namely free fatty acids and lysophospholipids, shortly before agonist addition to cells in which cPLA 2 had been inactivated by MAFP. Exogenous AA, but not other fatty acids or lysophospholipids, was able to restore the extracellular [ 3 H]AA release in response to PAF. Treating the cells with exogenous AA has the effect of increasing cell-associated free fatty acid levels well above those found in untreated cells, thereby mimicking cPLA 2 activation. No effect of was seen when exogenous AA was added in the absence of PAF or when a sPLA 2 inhibitor was used, suggesting that the effect may be specific.
A perturbation of the lipid bilayer or "membrane rearrangement," initiated by an agonist/receptor interaction, appears to be required to activate sPLA 2 at the outer surface of the cell (35) The data reported herein suggest that, in addition to phospholipid translocation (34), such a membrane rearrangement may involve a transient elevation of free AA mediated by receptor-activated cPLA 2 . Thus, our results appear to suggest a new role for free AA in cellular signaling, i.e. to help regulate the accessibility of sPLA 2 to its substrate in the membrane.
However, such an intracellular elevation of free AA is not itself sufficient to elicit the cellular response. Therefore, other additional signals that occur at the earliest stages of PAF activation, such as inositol phospholipid turnover, Ca 2ϩ mobilization, or protein phosphorylation (11) are also required for the AA release process to fully take place. When all of these signals are induced sufficiently, sPLA 2 begins to hydrolyze phospholipids at the outer surface of the cell, and this results in full AA mobilization. According to this model, augmentation of any of these early signals could result in an increased liberation of AA to the extracellular medium. This is what occurs in the experiments using okadaic acid, wherein augmentation of cPLA 2 activity and hence cell-associated free AA levels dramatically enhance the extracellular AA release, provided sPLA 2 is functional.
We should emphasize that the above model of cross-talk between cPLA 2 and sPLA 2 has to be regarded as a working model and not as an established one. Much of our evidence in favor of a causal relationship between cPLA 2 and sPLA 2 rests on the use of the phosphonylfluoride MAFP, a highly reactive compound. It cannot be ruled out at this time that MAFP is exerting some other undesired effects or that the sPLA 2 -activating effect of exogenous AA is unrelated to cPLA 2 . iPLA 2 Role in AA Mobilization-Another striking feature of the current work is the role of the one other PLA 2 present in P388D 1 cells, i.e. the iPLA 2 , during PAF activation. We have investigated this issue by conducting studies with BEL, a selective inhibitor of the iPLA 2 . Our results clearly show that BEL does not inhibit AA release, ruling out a significant role for the iPLA 2 in this release. In a previous report, we demonstrated that the steady-state level of lysophospholipids in BELtreated cells is decreased by about two-thirds and that this effect directly correlates with the inhibition of AA esterification into phospholipids as well as the inhibition of cellular iPLA 2 activity (22). Moreover, BEL does not have any effect on the AA reacylating enzymes arachidonoyl-CoA synthetase and lysophospholipid:arachidonoyl-CoA acyltransferase (22). Based on these previous data, our current finding that the unstimulated levels of [ 3 H]AA are increased in media from BEL-treated cells may be explained as the consequence of the diminished capac-ity of these cells to reacylate AA into membrane phospholipids. Thus, the basal level of AA, being produced by constitutively active enzymes different from the iPLA 2 , increases because the iPLA 2 is blocked, thereby lowering the availability of acceptor.
The fact that the potentiating effect of BEL on extracellular AA release is still observed in activated cells further indicates that the receptor-regulated PLA 2 s releasing AA during PAF stimulation are distinct from the BEL-sensitive iPLA 2 . This view lends support to a model whereby each of the distinct PLA 2 s present in P388D 1 cells may play different roles in regulating free AA availability during PAF-induced activation (Fig. 9). By interacting with its specific receptor at the plasma membrane, PAF initiates the stimulation process by increasing the intracellular Ca 2ϩ concentration (step 1). PAF also triggers a second as yet unknown signal (step 2) (11). These signals act in concert to initiate translational/post-translational events that result in the activation of cPLA 2 and sPLA 2 . The two enzymes, acting either intracellularly (cPLA 2 ) or extracellularly (sPLA 2 ), are responsible for mobilizing AA upon PAF receptor stimulation. On the other hand, the iPLA 2 allows reincorporation of part of the fatty acid previously liberated by its Ca 2ϩ -dependent counterparts and in this manner helps replenish cellular AA pools. If the iPLA 2 were responsible for generating a significant portion of lyso acceptors under activation conditions, this enzyme might also play a role in eicosanoid metabolism by limiting the amount of AA available for eicosanoid biosynthesis.