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Departments of
*
Pharmacology and Toxicology, and
Medicine and Institute for Environmental Toxicology, Michigan State University, East Lansing, MI 48824; and
Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of Michigan, Ann Arbor, MI 48109
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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An important pathway for the release of AA or other unsaturated fatty acids from phospholipid pools involves phospholipase A2 (PLA2)-dependent hydrolysis of sn-2-acyl ester bonds. Several different isoforms of PLA2 have been described that represent distinct gene products. These include two well-characterized, small-m.w., calcium-dependent enzymes that are not selective for AA, the14-kDa secretory PLA2 and pancreatic PLA2 (13, 14); an 85-kDa cytosolic PLA2 (cPLA2), which is calcium dependent and arachidonoyl selective (15, 16, 17); and two calcium-independent enzymes, one of which is selective for AA (18, 19, 20) and one that is not (21, 22, 23). Many cell types including neutrophils contain multiple isoforms of PLA2 (24, 25, 26); however, the functional significance of the presence of different isoforms within the cell is not well understood. It has been suggested that different PLA2s within the cell carry out distinct biologic functions (26).
In neutrophils, PLA2-derived AA is important as a substrate for eicosanoid production. AA has also been implicated as a second messenger for generation of O2-, which is essential for neutrophil-mediated killing of microbial pathogens (10, 11, 12); however, this function remains controversial (27). In support of a role for AA in generation of O2-, virtually all agents that stimulate O2- production in neutrophils also cause the release of AA (11, 12, 28). In addition, experiments with intact cells, as well as with reconstituted NADPH oxidase systems, suggest that AA, rather than its metabolites, regulates a hydrogen ion channel that is linked to NADPH oxidase activity (29, 30). It has been suggested that arachidonate plays an important role in assembly of oxidase components by exposing SH3 domains of p47phox, thus allowing interaction with cytochrome b558 and p67phox (31).
PLA2-mediated AA release appears to play an essential role in production of O2- by neutrophils upon exposure to the commercial mixture of polychlorinated biphenyls (PCBs), Aroclor 1242 (28). PCBs are environmental toxicants that cause diverse biologic effects, including modulation of inflammatory-mediated responses such as neutrophil-mediated liver injury (32). Aroclor 1242 consists of a complex mixture of coplanar and ortho-substituted, noncoplanar PCB congeners. Release of AA and production of O2- can be attributed to the ortho-substituted congeners within the mixture, since congeners such as 2,2',4,4'-tetrachlorobiphenyl (TCB) stimulate both the release of AA and the generation of O2-, whereas coplanar congeners such as 3,3',4,4'-TCB cause neither effect (28).
The magnitude of release of AA in response to Aroclor 1242 is similar to that observed from neutrophils stimulated with the calcium ionophore, A23187. However, the fate of released AA in response to these two stimuli may be different. AA released from A23187-stimulated neutrophils is metabolized extensively to eicosanoids (33); however, A23187 does not stimulate the production of O2- (34). In contrast, AA released in response to Aroclor 1242 appears to be linked to O2- generation (28). It is unknown whether AA mobilized by Aroclor 1242 results in eicosanoid production. One possible explanation for the different fates of AA released in response to these two stimuli may be that two discrete pools of AA exist, one destined to serve as a precursor for metabolism to eicosanoids and the other to serve as a second messenger for O2- generation.
The purpose of the present study was to characterize the PLA2s activated upon stimulation of rat neutrophils with A23187 and Aroclor 1242. The results of this study indicate that A23187 and Aroclor 1242 activate different PLA2 isoforms. In addition, the data suggest that, in neutrophils, activation of distinct PLA2 isoforms results in AA hydrolysis that subserves distinct biologic functions.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Aroclor 1242 was obtained from ChemService (West Chester, PA). Cytochrome c, superoxide dismutase (SOD), xanthine, xanthine oxidase, and A23187 were obtained from Sigma Chemical Company (St. Louis, MO). [3H][5,6,8,9,11,12,14,15]AA (3H-AA; 180–240 Ci/mmol) was purchased from DuPont NEN (Boston, MA). [3H][9,10,12,13]linoleic acid (3H-LA; 60–120 Ci/mmol), 1-palmitoyl-2-arachidonoyl [arachidonoyl-1-14C]phosphatidylcholine (14C-AA-PC; sp. act. = 55 mCi/mmol), and 1-palmitoyl-2-linoleoyl [linoeloyl-1-14C]phosphatidylcholine (14C-LA-PC; sp. act. = 55 mCi/mmol) were obtained from American Radiolabeled Chemicals (St. Louis, MO). 1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetra-(acetoxymethyl)-ester (BAPTA-AM) was from Calbiochem (San Diego, CA). E-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)2H-pyran-2-one (BEL) was purchased from Biomol (Plymouth Meeting, PA). Sep-pak cartridges were obtained from Waters (Milford, MA). For all experiments, Aroclor 1242 was dissolved in methanol. Neutrophils received 1 µl of methanol. A23187 and BEL were dissolved in DMSO and diluted so that the final concentration of DMSO was < 1%.
Neutrophil isolation
Glycogen-elicited neutrophils were obtained from the peritoneal cavities of male Sprague-Dawley rats (Charles River Laboratories, Portage, MI). Rats were anesthetized with diethyl ether, and 30 ml of 1% glycogen were injected i.p. The rats were anesthetized again 4 h later and killed by decapitation. The peritoneal cavity was washed with 30 ml of heparinized (1 U/ml) 0.1 M PBS and the peritoneal fluid collected, filtered through gauze, and centrifuged at 500 x g for 7 min. Contaminating RBCs were lysed with 15 ml of 0.15 M NH4Cl, and neutrophils were suspended to a final volume of 50 ml with PBS and centrifuged for 7 min at 300 x g. Cells were washed once with PBS and suspended in HBSS of the following composition: 4.5 mM KCl, 0.6 mM Na2HPO4, 0.62 mM KH2PO4, 120 mM NaCl, 23 mM Tris, 1.6 mM CaCl2, 0.68 mM MgCl2, 10 mM glucose, and 14 mM NaHCO3. The percentage of neutrophils in this preparation is routinely >95% (28).
Labeling of neutrophils with 3H-fatty acids
Neutrophils (107/ml) were suspended in Mg2+- and Ca2+-free HBSS containing 0.1% BSA and incubated in the presence of 0.5 µCi/ml [3H]AA or [3H]linoleic acid for 90 min, gently shaking at 37°C. At the end of the incubation period, neutrophils were washed two times with Mg2+- and Ca2+-free HBSS. The cell count was adjusted so that the final concentration of neutrophils was 2 x 106/ml. The incorporation of 3H-AA was approximately 60% and that of [3H]linoleic acid was approximately 70% of the total radioactivity added to the cells.
Determination of fatty acid release from prelabeled neutrophils
Prelabeled neutrophils were suspended in HBSS containing 0.1% BSA. In these experiments BSA was used to trap released fatty acids, thus inhibiting subsequent metabolism and reacylation: as a result, the radioactivity in the supernatant reflects cumulative deacylation of [3H]fatty acid from phospholipid pools. Release of [3H]fatty acid was determined in the presence and absence of the inhibitor of the calcium-independent PLA2, BEL. Neutrophils were incubated for 15 min with BEL before addition of stimuli. To determine the role of extracellular Ca2+ in stimulated release of AA, incubations were conducted in the presence or absence of extracellular Ca2+. To determine the role of intracellular Ca2+ in release of AA, prelabeled neutrophils were loaded for 1 h with the cell-permeant calcium chelator BAPTA-AM. Release of [3H]fatty acids from prelabeled cells was measured in neutrophils stimulated for 20 min at 37°C with Aroclor 1242, A23187, or the appropriate vehicle as described previously (28).
Determination of PLA2 activity
PLA2 activity was measured using whole cell sonicates of fresh neutrophils (7 x 107 cells/ml) according to the method of Smith and Waite (35). Neutrophils were isolated as described above, washed once in Ca2+-free PBS containing 5 mM EDTA and 1 mM PMSF, resuspended in cold homogenizing buffer (deionized water, 5 mM EDTA, and 1 mM PMSF), placed on ice, and sonicated two times (80% duty) for 30 s. Light microscopy was used to verify that cells were broken. The substrates, 14C-AA-PC and 14C-LA-PC, were dried under nitrogen and resuspended by sonication (90% duty for 5 min) in assay buffer containing 120 mM NaCl and 40 mM Tris-HCl (pH 9) to a final concentration of 3 µM radiolabeled substrate/test tube. To determine the role of calcium in activation of PLA2, assays were performed in the presence of either 5 mM CaCl2 or 5 mM EGTA. To determine the effect of PCBs on PLA2 activity, whole cell sonicates were incubated in the presence or absence of Ca2+ with 10 µg/ml of Aroclor 1242 or vehicle for 2 min before addition of substrate. In addition, sonicates were preincubated in the presence or absence of Ca2+ with 10 µM BEL or vehicle for 2 min to determine the effect of this inhibitor on calcium-dependent and calcium-independent PLA2 activities. Optimal conditions for PLA2 were determined in preliminary experiments by measuring activity in the presence of various concentrations of substrate and protein. These optimal conditions, i.e., 3 µM substrate and 60 µg protein/250 µl assay buffer, were used thereafter. Reactions were initiated by addition of either 3 µM 14C-AA-PC or 14C-LA-PC to whole cell sonicates that were then incubated for 30 min at 37°C in a shaking water bath. Reactions were terminated by addition of 1.2 ml of chloroform-methanol, 2:1 (v/v). The chloroform layer was extracted, dried under nitrogen, resuspended in 60 µl of chloroform, and spotted on silica gel thin layer chromatography plates. The plates were chromatographed in a neutral lipid solvent system containing hexane, diethyl ether, and glacial acetic acid (70:30:2, by volume). The lipids were visualized with I2 vapor, and the zones corresponding to fatty acid and phospholipid were cut out and radioactivity determined by scintillation counting. Results were expressed as percent of total radioactivity present as free fatty acid.
Determination of metabolism of released AA by neutrophils
For determination of metabolic products of AA, neutrophils prelabeled with 0.5 µCi/ml [3H]AA were stimulated for 20 min at 37°C with Aroclor 1242, A23187, or the appropriate vehicle in Ca2+- and Mg2+-containing HBSS. For determination of metabolic products of AA, experiments were performed in the absence of albumin. Eicosanoids were extracted from cell-free supernatant fluids with C18 Sep-pak cartridges, separated by reverse-phase HPLC, and identified by coelution with known standards (36). Products were quantified via an on-line radioactivity detector. To verify that biosynthesis of radiolabeled eicosanoids in prelabeled cells reflected the total mass of eicosanoids from endogenous stores of AA, selected eicosanoids released into the extracellular medium from unlabeled neutrophils were measured by enzyme immunoassay (Cayman Chemical, Ann Arbor, MI). For each sample, the average of duplicate determinations was used. Experimental incubations were the same as those described above.
Generation and detection of O2-
O2- production was measured in neutrophils stimulated for 20 min at 37°C with Aroclor 1242, A23187, or the appropriate vehicle. Previous studies have demonstrated that A23187 does not cause significant production of O2- in rat neutrophils (34). To determine whether the failure of A23187 to generate O2- was related to the extensive metabolism of AA by cyclooxygenase and 5-lipoxygenase pathways in A23187-stimulated cells, O2- production was determined in neutrophils pretreated with the cyclooxygenase inhibitor, aspirin (100 µM; 37) and the 5-lipoxygenase inhibitor, zileuton (10 µM; 38). Neutrophils were preincubated for 30 min with aspirin and for 15 min with zileuton before stimulation with A23187. To determine the role of calcium-independent PLA2 in Aroclor 1242-induced neutrophil activation, O2- production was measured in the presence and absence of the calcium-independent PLA2 inhibitor, BEL. Neutrophils were preincubated with BEL or vehicle for 15 min before stimulation with Aroclor 1242. Cumulative O2- production was measured by the SOD-sensitive reduction of cytochrome c as described previously (28).
Certain PLA2 inhibitors have been shown to scavenge oxygen radicals (28). Therefore, to determine whether BEL inhibited O2- production by a mechanism related to this effect, O2- generated during oxidation of xanthine by xanthine oxidase was measured in the presence and absence of BEL as described previously (28). In addition, absorbance of reduced cytochrome c was measured in the presence and absence of BEL to ensure that apparent inhibition of neutrophil-generated O2- was not a result of quenching of absorbance by BEL. There were no differences in absorbance values in the presence and absence of BEL, thus indicating that BEL does not interfere with the cytochrome c assay.
Determination of cytotoxicity
To ensure that inhibition of O2- by BEL did not result from injury to the cells, cytotoxicity was determined in neutrophils exposed to BEL. Neutrophils were incubated with BEL as described above, and activity of the cytosolic enzyme, lactate dehydrogenase (LDH), was determined in the cell-free supernatant fluids as described previously (39, 40).
Statistical analysis
Results are expressed as mean ± SEM.
[3H]AA or [3H]linoleic acid results
are expressed as percent of total cellular radioactivity released into
the medium. Data were analyzed by ANOVA, and group means were compared
using Student-Newman-Keuls’ test. Appropriate transformations were
performed on all data that did not follow a normal distribution. If
transformation failed to normalize the data, then nonparametric
statistics were used (Mann-Whitney test). For all studies, the
criterion for statistical significance was p 
0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A minimal amount of [3H]AA was released into
the medium from unstimulated neutrophils (Fig. 1
, A and B).
The release of [3H]AA increased in a
concentration-dependent manner in neutrophils exposed to the calcium
ionophore A23187 (Fig. 1A) or Aroclor 1242 (Fig. 1B). The magnitude of release of [3H]AA
was not significantly different between neutrophils stimulated with
maximal concentrations of A23187 (20 µM; 22.8 ± 0.6%) or
Aroclor 1242 (10 µg/ml; 22.5 ± 4.7%).
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. Unstimulated neutrophils released
small amounts of radioactivity (435 dpm in the representative
experiment shown in Fig. 2) that coeluted exclusively with AA (Fig. 2A). Total radioactivity released into the medium by
A23187-treated neutrophils was approximately 30-fold higher than
control (14847 dpm). Only 14% of the released radioactivity remained
as free AA, while 86% was metabolized to eicosanoids. The
cyclooxygenase products that were detected included prostaglandin
E2, prostaglandin D2, thromboxane
B2, 12-hydroxyheptadecatrienoic acid, and the stable
metabolite of prostacyclin, 6-keto prostaglandin F1
.
Also detected were products of 5-lipoxygenase, leukotriene
B4 and 5-hydroxyeicosatetraenoic acid (HETE), and of
12-lipoxygenase, 12-HETE (Fig. 2B). Total
radioactivity released into the medium by neutrophils exposed to
Aroclor 1242 was 10-fold higher than that seen with unstimulated
neutrophils (5168 dpm; Fig. 2C). However, in contrast
to A23187, 82% of the radioactivity released in response to Aroclor
1242 remained as free AA while only 18% was metabolized to
eicosanoids, predominantly thromboxane B2,
12-hydroxyheptadecatrienoic acid, and 12-HETE (Fig. 2C). A similar lack of metabolism was observed with a
PCB congener, 2,2',4,4'-TCB, which stimulates O2-
production by neutrophils (data not shown).
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). Thromboxane B2 was chosen
as a cyclooxygenase product and leukotriene B4 as a
5-lipoxygenase product. Exposure to A23187 caused release of large
amounts of thromboxane B2 and leukotriene B4
from neutrophils; however, significantly less of both products was
released from neutrophils stimulated with Aroclor 1242. Thus, results
using unlabeled neutrophils were consistent with those obtained with
cells labeled with [3H]AA.
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No O2- was generated from unstimulated
neutrophils (Fig. 1
, A and B). Aroclor
1242 stimulated the production of O2- with a
concentration-response relation that was similar to the
[3H]AA response induced by this compound (Fig. 1B). A23187, however, did not stimulate neutrophils
to produce O2-, despite significant release of
[3H]AA. The failure of A23187 to stimulate production of
O2- in rat neutrophils might be due to extensive
metabolism of AA via the cyclooxygenase and 5-lipoxygenase pathways,
resulting in inadequate levels of free AA to activate the NADPH
oxidase. To test this hypothesis, O2- production was
determined in A23187-stimulated neutrophils in the presence and absence
of aspirin and zileuton. No O2- was produced by
neutrophils stimulated with A23187, and pretreatment with aspirin
and/or zileuton did not alter this response (Table II).
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To examine whether the differences in the metabolism of AA in
neutrophils stimulated with A23187 or Aroclor 1242 resulted from the
activation of different isoforms of PLA2, experiments
were performed to determine the characteristics of the PLA2
activated by these stimuli. The preference for AA was investigated by
comparing release of [3H]AA and
[3H]linoleic acid (3H-LA) from prelabeled
neutrophils stimulated with A23187 or Aroclor 1242 (Fig. 3). Similar to results in Figure 1, significant release of [3H]AA was observed in neutrophils
stimulated with either A23187 (Fig. 3A) or Aroclor
1242 (Fig. 3B). Neither compound, however, caused
significant deacylation of [3H]LA (Fig. 3, A
and B).
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).
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.
[3H]AA release stimulated by Aroclor 1242 was inhibited
in a concentration-dependent manner by BEL; significant inhibition was
achieved with a concentration of BEL as low as 0.1 µM. In contrast,
the release of [3H]AA evoked by 20 µM A23187 was not
significantly altered by BEL at concentrations up to 10 µM (Fig. 5).
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) is consistent with the
hypothesis that AA release in response to Aroclor 1242 is due to
activation of a calcium-independent PLA2. To test whether
this enzyme is important in the mechanism of Aroclor 1242-induced
stimulation of neutrophils, the production of O2-
evoked by Aroclor 1242 was measured in the presence and absence of BEL.
Aroclor 1242-induced O2- generation decreased in a
concentration-dependent manner in neutrophils exposed to BEL (Fig. 6). BEL inhibited O2-
production (Fig. 6) with a concentration-response relation that was
similar to BEL-induced inhibition of [3H]AA release (Fig. 5). The inhibition of [3H]AA release and
O2- production by BEL was not a result of cytotoxicity
since LDH values were not different in cells preincubated with BEL at
maximal concentrations and stimulated with Aroclor 1242 (LDH =
6.7 ± 0.8%) when compared with neutrophils incubated with
Aroclor 1242 alone (6.7 ± 1.6%) or vehicle alone (4.9 ±
0.4%).
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To confirm results obtained with intact neutrophils, cell-free
PLA2 assays were performed. PLA2 activity
was present when assayed against either 14C-AA-PC or
14C-LA-PC, both in the presence and absence of calcium;
however, PLA2 activity was significantly greater when
assayed against 14C-AA-PC and in the presence of
Ca2+ (Table III).
Pretreatment of lysates with Aroclor 1242 significantly enhanced
calcium-independent PLA2 activity, whereas
calcium-dependent PLA2 activity was significantly
attenuated in the presence of Aroclor 1242. BEL (10 µM) inhibited
calcium-independent activity by >80%; however, calcium-dependent
activity was inhibited by <5% in the presence of BEL. Similar results
were obtained when 14C-LA-PC was used as substrate,
although less PLA2 activity was observed under these
conditions.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The hypothesis that Aroclor 1242 activates a calcium-independent
PLA2 is corroborated by results in a cell-free system. BEL
inhibited activity of this enzyme in cell lysates and also inhibited
Aroclor 1242-induced O2- production in whole cells,
further supporting the interpretation that activation of
calcium-independent PLA2 and O2-
generation by Aroclor 1242 are linked. The possibility cannot be
excluded, however, that BEL inhibited AA release and
O2- production by a mechanism that was unrelated to
inhibition of calcium-independent PLA2. BEL has been
reported to inhibit -chymotrypsin activity (41) as well as to
inhibit cytosolic, magnesium-dependent phosphatidic acid
phosphohydrolase activity (42). These effects of BEL are unlikely to be
involved in the inhibition of AA release by BEL in our study, given the
observation that it significantly inhibited and Aroclor 1242
significantly enhanced calcium-independent PLA2 activity
under cell-free conditions that were optimized specifically for this
enzyme (Table III). Moreover, there is no evidence that phosphatidic
acid phosphohydrolase plays any role in NADPH oxidase activation. These
results provide strong evidence that Aroclor 1242 stimulates AA release
by activating a calcium-independent PLA2.
A23187, which released similar amounts of AA as Aroclor 1242, failed to
cause the production of O2-. The failure of A23187 to
elicit O2- production was not related to insufficient
levels of AA due to extensive metabolism via the cyclooxygenase and
lipoxygenase pathways, since pretreatment with inhibitors of these
pathways did not unmask generation of O2- by
A23187-treated cells. These data are consistent with the hypothesis
that in neutrophils, activation of calcium-dependent PLA2
does not lead to generation of O2-. Inhibition of
calcium-dependent PLA2 by Aroclor 1242 in cell-free
experiments (Table III) further supports this hypothesis. If
calcium-dependent PLA2 was important in the production of
O2-, Aroclor 1242, an agent that stimulates
O2-, should not inhibit its activity.
Cell-free assays confirmed the presence of both calcium-dependent and
-independent PLA2 activities suggested by experiments in
intact cells. Our results are most consistent with the possibility that
the enzyme activated by Aroclor 1242 in rat neutrophils is the
calcium-independent PLA2 first identified in cardiac
myocytes by Wolf and Gross (18); Hazen, Stuppy, and Gross (19); and
Hazen et al. (20). The myocardial enzyme, like the neutrophil enzyme,
does not require calcium for activity and is inhibited by BEL (Fig. 4
and Table III; Refs. 19 and 20). In addition, the myocardial enzyme
displays a preference for hydrolysis of phospholipids that contain
arachidonate at the sn-2 position (19). [3H]LA was not
released from neutrophils in response to activation with Aroclor 1242,
whereas [3H]AA was (Fig. 3). Moreover,
calcium-independent PLA2 activity under cell-free
conditions was 10-fold greater when 14C-AA-PC was used as
substrate compared with the response obtained in the presence of
14C-LA-PC (Table III). These data are consistent with the
hypothesis that Aroclor 1242 activates a calcium-independent,
arachidonoyl-selective PLA2 in neutrophils (18, 19, 20, 21, 22). This
study is the first to identify and define a role for a
calcium-independent, arachidonoyl-selective PLA2 in rat
neutrophils. Smith and Waite described a calcium-independent
PLA2 in human neutrophils that was optimally active at pH
9; however, the function of this enzyme was not elucidated (35). The
calcium-independent PLA2 described here may be the same as
or similar to the enzyme described in human neutrophils.
The effect of Aroclor 1242 to activate the calcium-independent
PLA2 may occur, at least in part, by a direct effect of
Aroclor 1242 on the enzyme, the substrate, or both. Interestingly,
while calcium-independent activity was enhanced by PCBs,
calcium-dependent activity was significantly inhibited in the presence
of Aroclor 1242 (Table III). Aroclor 1242-induced inhibition of a
calcium-dependent PLA2 that is linked to eicosanoid
metabolism may be, at least in part, responsible for the low level of
eicosanoid production in PCB-treated neutrophils. Alternatively, the
failure of AA released by Aroclor 1242 to be converted to eicosanoids
may be due to inhibition of cyclooxygenase and/or lipoxygenase by
Aroclor 1242. This explanation, however, seems unlikely since PCB
congeners, as well as the structurally related compound
2,3,7,8-tetrachlorodibenzo-p-dioxin, have been shown to
stimulate rather than inhibit eicosanoid-synthesizing enzymes (43, 44).
Aroclor 1242 is a complex mixture of PCB congeners; therefore, it is
possible that different components of the mixture are responsible for
the diverse effects on the calcium-dependent and -independent isoforms
of PLA2. This possibility was not examined directly in
these studies. However, activation of calcium-independent
PLA2 by Aroclor 1242 is likely due to ortho-substituted
congeners contained within the mixture since 2,2',4,4'-TCB, an
ortho-substituted congener present in Aroclor 1242, caused the same
effects as Aroclor 1242, i.e., stimulation of O2-
generation and release of AA (28) that was not metabolized extensively
to eicosanoids.
AA release in response to A23187 required the presence of calcium (Fig. 4). In addition, A23187 caused the preferential release of AA over
linoleic acid (Fig. 3). These data are consistent with the hypothesis
that A23187 mobilized AA in rat neutrophils by activating a
calcium-dependent, arachidonoyl-selective cPLA2. These
studies are in agreement with previous reports in other cell types that
have suggested that A23187 causes AA release by a mechanism involving
cPLA2 (45, 46, 47). In the present study, a role for secretory
PLA2 and pancreatic PLA2 in A23187-induced AA
release is less likely since these enzymes show no preference for AA
over other unsaturated fatty acids (13, 14). In addition, preliminary
studies (not shown) in our laboratory suggest that A23187-induced AA
release is inhibited by the trifluoromethyl ketone derivative of AA,
AACOCF3, an agent that inhibits cPLA2 but not
small-m.w., calcium-dependent PLA2.
The results of this study suggest that different PLA2 release AA that subserves distinct functions within the cell. Why might AA mobilized by different PLA2 have different biologic fates? One possibility is that subcellular compartmentalization of PLA2 determines whether released AA is coupled to eicosanoid synthesis or O2- production. A number of eicosanoid-synthesizing enzymes are located at the nuclear envelope (48, 49, 50, 51), and the activation-associated translocation of cPLA2 to the nuclear envelope (48, 52, 53) with concomitant nuclear membrane phospholipid hydrolysis (53) would facilitate efficient coupling of released AA to eicosanoid synthesis. On the other hand, the NADPH oxidase is located at the plasma membrane (54). It is tempting to speculate that the calcium-independent PLA2 translocates to this site upon cellular activation, so that plasma membrane-derived AA hydrolyzed by this enzyme is in closer proximity to the NADPH oxidase than to eicosanoid-forming enzymes. Thus, nuclear membrane AA and plasma membrane AA would represent discrete pools of AA destined to serve distinct functions. This speculation will require direct examination.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Patricia E. Ganey, Department of Pharmacology and Toxicology, B440 Life Sciences, Michigan State University, East Lansing, MI 48824.
3 Abbreviations used in this paper: AA, arachidonic acid; O2-, superoxide anion; PLA2, phospholipase A2; cPLA2, cytosolic PLA2; BEL, bromoenol lactone; LDH, lactate dehydrogenase; HETE, hydroxyeicosatetraenoic acid; PCB, polychlorinated biphenyl; TCB, tetrachlorobiphenyl; SOD, superoxide dismutase.
Received for publication November 26, 1997. Accepted for publication October 1, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chymotrypsin. J. Biol. Chem. 258:15046.This article has been cited by other articles:
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J. Marshall, E. Krump, T. Lindsay, G. Downey, D. A. Ford, P. Zhu, P. Walker, and B. Rubin Involvement of Cytosolic Phospholipase A2 and Secretory Phospholipase A2 in Arachidonic Acid Release from Human Neutrophils J. Immunol., February 15, 2000; 164(4): 2084 - 2091. [Abstract] [Full Text] [PDF]
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P. K. Tithof, J. Olivero, K. Ruehle, and P. E. Ganey Activation of Neutrophil Calcium-Dependent and -Independent Phospholipases A2 by Organochlorine Compounds Toxicol. Sci., January 1, 2000; 53(1): 40 - 47. [Abstract] [Full Text] [PDF]
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Z. Ma, S. Ramanadham, M. Wohltmann, A. Bohrer, F.-F. Hsu, and J. Turk Studies of Insulin Secretory Responses and of Arachidonic Acid Incorporation into Phospholipids of Stably Transfected Insulinoma Cells That Overexpress Group VIA Phospholipase A2 (iPLA2beta ) Indicate a Signaling Rather Than a Housekeeping Role for iPLA2beta J. Biol. Chem., April 13, 2001; 276(16): 13198 - 13208. [Abstract] [Full Text] [PDF]
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) were measured in
neutrophils stimulated for 20 min with the indicated concentrations of
A23187 (A) or Aroclor 1242
(B). O2- production was
measured as the SOD-sensitive reduction of cytochrome c, as
described in Materials and Methods.
Release of [3H]arachidonate into the extracellular medium
was measured in the presence of 0.1% albumin. Results are expressed as
the percent of total incorporated cellular radioactivity.
n = 5 to 7. a, Statistically different from
respective value obtained in the absence of stimulus.
