From the
Herein, we demonstrate that nitric oxide is a potent (>20%
release) and highly selective inducer of
[
Recent studies have underscored the unanticipated ubiquity,
potency, and diversity of nitric oxide-mediated cellular activation (cf. Refs. 1-3). Although many mechanisms have been
proposed to explain the sequence of molecular transformations that
mediate the biologic sequelae of nitric oxide stimulation and some have
received substantial experimental support (e.g. cGMP-mediated
vascular relaxation(4) ), the detailed chemical mechanisms
underlying many of the effects of this gaseous second messenger remain
enigmatic (e.g. Refs. 5 and 6). For example, macrophages
undergo a dramatic biochemical and functional metamorphosis after
nitric oxide stimulation and yet the chemical mechanisms mediating the
majority of nitric oxide-induced alterations in macrophage chemistry
and biology remain unknown.
Macrophage activation induced by a
variety of different ligands (e.g. lipopolysaccharide,
zymosan, etc.) results in increased cellular glycolytic flux,
accelerated phospholipid catabolism, and stimulation of many macrophage
physiologic functions (e.g. phagocytosis and
chemotaxis)(7, 8) . Macrophages contain both
calcium-dependent (9, 10) and calcium-independent
phospholipase A
The present study demonstrates that: 1) nitric oxide is a
potent activator of arachidonic acid mobilization in mammalian cells;
2) nitric oxide-induced arachidonic acid mobilization is predominantly
mediated by a calcium-independent phospholipase A
Recent studies have recognized the
preponderance of calcium-independent phospholipase A
H]arachidonic acid mobilization in the
macrophage-like cell line RAW 264.7. Treatment of RAW 264.7 cells with (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahy-dropyran-2-one
resulted in the inhibition of the large majority (86%) of nitric
oxide-induced [
H]arachidonic acid release into
the medium (IC
<0.5 µM) and the
concomitant inhibition of in vitro measurable
calcium-independent phospholipase A
activity (92%
inhibition) without demonstrable effects on calcium-dependent
phospholipase A
activity. Since nitric oxide is a potent
stimulator of glycolysis (and therefore glycolytically derived ATP) and
since cytosolic calcium-independent phospholipase A
exists
as a catalytic complex comprised of ATP-modulated
phosphofructokinase-like regulatory polypeptides and a catalytic
subunit, we examined the role of glucose in facilitating nitric
oxide-mediated arachidonic acid release. Nitric oxide-induced release
of [
H]arachidonic acid possessed an obligatory
requirement for glucose, was highly correlated with the concentration
of glucose in the medium, and was dependent on the metabolism of
glucose. Thus, [
H]arachidonic acid release is
coupled to cellular glucose metabolism through alterations in the
activity of calcium-independent phospholipase A
.
Collectively, these results identify a unifying metabolic paradigm in
which the generation of lipid second messengers is coordinately linked
to the signalstimulated acceleration of glycolytic flux, thereby
facilitating integrated metabolic responses to cellular stimuli.
(11) activities that are catalyzed
by separate and distinct classes of polypeptides (e.g. Refs.
12 and 13). We have previously demonstrated that native
calcium-independent phospholipase A
in several cell types
exists as a high molecular weight catalytic complex (400 kDa) comprised
of regulatory polypeptides highly homologous with, or identical to,
phosphofructokinase and a 40-kDa catalytic entity (e.g. Refs.
14 and 16). Calcium-independent phospholipase A
activity is
modulated by physiologic alterations in ATP concentration through the
interaction of ATP with phosphofructokinase-like regulatory
polypeptides, which are tightly associated with, and functionally
coupled to, the 40-kDa catalytic subunit(16) . These findings
led us to suggest that glycolysis and phospholipolysis are interwoven
metabolic pathways coupled through the interaction of the
calcium-independent phospholipase A
catalytic complex with
glycolytically derived ATP(16) . Since nitric oxide is a potent
stimulator of macrophage glucose uptake and glycolytic
flux(7, 17) , we hypothesized that some of the effects
of nitric oxide on macrophage phospholipid catabolism and physiologic
function could result from the calcium-independent phospholipase
A
-mediated mobilization of arachidonic acid in response to
the nitric oxide-induced accelerated production of glycolytically
derived ATP. We now report that: 1) nitric oxide is a potent activator
of arachidonic acid mobilization in macrophages; 2) the highly
selective mobilization of arachidonic acid by nitric oxide is
predominantly mediated by calcium-independent phospholipase
A
; and 3) nitric oxide-mediated arachidonic acid
mobilization by calcium-independent phospholipase A
possesses an obligatory dependence on glucose uptake and
metabolism.
Materials
RAW 264.7 cells were obtained from
American Type Tissue Culture Collection.
1-O-(Z)-Hexadec-1`-enyl-2-[9,10-H]octadec-9`-enoyl-sn-glycero-3-phosphocholine-(plasmenylcholine)
and (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one
(BEL)
(
)were synthesized and purified by
established methods(18, 19, 20, 21) .
Radiolabeling of RAW 264.7 Cells and Measurement of
Phospholipase A
RAW 264.7 cells were maintained in
culture at 37 °C in Dulbecco's modified Eagle's medium
(DMEM) containing 10% fetal bovine serum. Cells were seeded and
incubated for 6 h, and 0.5 ml of fresh DMEM containing 10% FBS and 0.25
µCi of either [Activity in the Cytosolic and
Microsomal Fractions
H]arachidonic acid (100
Ci/mmol) or [
H]oleic acid (10 Ci/mmol) was added
to each well. The media contained 6 µM arachidonic acid
and 7 µM oleic acid (as endogenous constituents in FBS)
rendering the final specific activity of these fatty acids to values of
183,330 and 157,140 dpm/nmol, respectively. After 16 h, media were
removed, and cells were washed (3 times) with phosphate-buffered saline
and incubated with either vehicle alone (EtOH, final concentration,
<0.1% v/v) or vehicle containing BEL (0.5-10 µM as indicated) for 15 min. After washing with phosphate-buffered
saline containing 0.25% bovine serum albumin (3 times), cells were
subsequently treated with either media containing albumin (0.25 mg/ml),
media containing the indicated concentration of SNP and albumin, or
media containing albumin previously bubbled with nitric oxide (the
final pH was adjusted with 1 N NaOH) for 60 min at 37 °C.
Fatty acids and phospholipids were extracted, separated, and quantified
as described previously(21) . Phospholipase A
activity in the cytosolic or microsomal fractions of control or
BEL-treated RAW 264.7 cells was measured by quantifying the release of
radiolabeled fatty acid from synthetically prepared plasmenylcholine or
purchased
phosphatidyl-choline(1-octadecanoyl-2-[5,6,8,9,11,12,14,15-
H]eicosa-5,8,11,14-tet-raenoyl-sn-glycero-3-phosphocholine)
as described previously(21) .
Nitric Oxide-induced Mobilization of
[
Incubation of
[H]Arachidonic Acid from Macrophage-derived RAW
264.7 Cellular Phospholipids
H]arachidonic acid-prelabeled RAW 264.7 cells
with sodium nitroprusside resulted in the dose-dependent mobilization
of [
H]arachidonic acid from endogenous cellular
phospholipid storage depots (Fig. 1A). The mobilization
of [
H]arachidonic acid from RAW 264.7 cells
induced by SNP-generated nitric oxide was indistinguishable from that
induced by bubbling nitric oxide gas through the media (Fig. 1B). The identity of the storage species of nitric
oxide in plasma as a thionitroso adduct of cysteine residues in
proteins has been well
documented(22, 23, 24, 25) . The
presence and persistence of the thionitroso adducts in the media after
nitric oxide (gas) bubbling was demonstrated by the appearance of new
uv peaks (
= 335 and 545 nm), which are
stable for hours under the conditions of the experiments (23). Since
BEL possesses over a 1,000-fold selectivity for inhibition of
calcium-independent phospholipase A
compared with
calcium-dependent phospholipase A
(20, 26) ,
we exploited the specificity inherent in mechanism-based inhibition to
identify the type of phospholipase A
activity mediating the
nitric oxide-induced release of arachidonic acid. The overwhelming
majority of arachidonic acid mobilization induced by nitric oxide was
inhibited by BEL with over 75% inhibition manifest even at the lowest
concentration of inhibitor employed (i.e. 0.5 µM BEL) (Fig. 1C).
Figure 1:
Nitric oxide induces the mobilization
of [H]arachidonic acid from RAW 264.7 cell
phospholipids, which is inhibited by the mechanism-based inhibitor BEL. A, RAW 264.7 cells were prelabeled for 16 h with
[
H]arachidonic acid, unincorporated
[
H]arachidonic acid was removed and cells were
incubated with the indicated concentrations of sodium nitroprusside for
60 min at 37 °C. Released fatty acids in the media (or
phospholipids in cells) were purified by TLC and quantified by
scintillation spectrometry as described under ``Materials and
Methods.'' Released [
H]arachidonic acid is
expressed as the percent of [
H]arachidonic acid
in the media divided by the total incorporated
[
H]arachidonate in RAW 264.7 cells. Results
represent the mean of at least six independent determinations. B, RAW 264.7 cells prelabeled for 16 h with
[
H]arachidonic acid were incubated with buffer
alone (control), buffer containing sodium nitroprusside (100
µM), or buffer previously bubbled for 10 min with nitric
oxide gas (the pH was subsequently adjusted to pH 7.4 with 2 N NaOH) for 60 min at 37 °C. Fatty acids released into the media
were quantified as described above. C, RAW 264.7 cells were
prelabeled for 16 h with [
H]arachidonic acid,
preincubated with the indicated concentrations of BEL, and subsequently
incubated for 60 min at 37 °C with either buffer alone (
) or
with buffer previously bubbled with NO gas (
).
[
H]Arachidonate release into the media was
quantified as described above.
Nitric Oxide Selectively Releases Arachidonic Acid from
Endogenous RAW 264.7 Cell Phospholipid Storage Pools
The nitric
oxide-induced mobilization of fatty acids from endogenous RAW 264.7
phospholipid storage depots mediated by SNP was highly selective for
arachidonic acid in comparison with oleic acid (Fig. 2).
Furthermore, SNP-mediated mobilization of
[H]arachidonic acid was greater (>20% release)
than that manifested by saturating concentrations of LPS or zymosan (Fig. 2). Over 95% of radiolabeled products released by SNP, LPS,
or zymosan in RAW 264.7 cells were identified as bona fide arachidonic
acid and not oxygenated eicosanoid metabolites (Fig. 2). The
large majority of LPS and zymosan-induced
[
H]arachidonic acid mobilization in macrophages
was inhibited by BEL demonstrating that the release of
[
H]arachidonic acid after stimulation by these
effectors largely results from hydrolysis mediated by
calcium-independent phospholipase A
.
Figure 2:
Fatty acyl selectivity of nitric
oxide-induced phospholipid hydrolysis and comparisons with
lipopolysaccharide and zymosan-mediated RAW 264.7 cell stimulation. A and B, RAW 264.7 cells were prelabeled for 16 h
with either [H]arachidonic acid (A) or
[
H]oleic acid (B). Cells were either
pretreated with buffer alone (control) or with BEL (10 µM)
for 15 min. Subsequently, cells were incubated with either buffer alone
(control), sodium nitroprusside (100 µM),
lipopolysaccharide (5 µg/ml) or zymosan (100 µg/ml) alone, or
the indicated combinations of agonists. Fatty acid released into the
media was quantified as described in Fig. 1. C, RAW 264.7
cells were labeled for 16 h with [
H]arachidonic
acid (AA) and incubated for 15 min with either buffer alone or
with BEL (10 µM). Subsequently, cells were stimulated for
60 min with either buffer alone (control), sodium nitroprusside (100
µM), lipopolysaccharide (5 µg/ml), or zymosan (Zym, 100 µg/ml), either alone or in the indicated
combinations. The media were extracted by the Bligh and Dyer technique
and applied to a Whatman Silica Gel 60A TLC plate, and metabolites were
resolved utilizing a mobile phase comprised of
chloroform/methanol/acetic acid/H
O (90:8:1:0.8, v/v).
Measurement of RAW 264.7 Cell Phospholipase A
Measurement of RAW 264.7 cell phospholipase
A Activities and [
H]Arachidonic Acid
Reacylation Rates
activity in vitro demonstrated the presence of
both calcium-dependent and -independent phospholipase A
activities (Fig. 3A). Calcium-dependent
phospholipase A
activity was predominant in the cytosolic
fraction, while calcium-independent phospholipase A
predominated in the microsomal fraction. Stimulation of RAW 264.7
cells with SNP did not result in substantial alterations of in
vitro measurable calcium-dependent or -independent phospholipase
A
activities in either the cytosolic or microsomal
fractions utilizing plasmenylcholine substrate (plasmenylcholine is not
susceptible to hydrolysis by phospholipase A
) (Fig. 3A) or phosphatidylcholine substrate containing
[
H]arachidonic acid at the sn-2 position
(data not shown). Treatment of RAW 264.7 cells with BEL (10
µM) resulted in the inhibition of 92% of
calcium-independent phospholipase A
activity without
inhibition of calcium-dependent phospholipase A
activity.
Collectively, these results demonstrate that: 1) nitric oxide-mediated
stimulation of RAW 264.7 cells does not result in stimulation of
measurable phospholipase A
activity in in vitro homogenates; 2) BEL specifically inhibits calcium-independent
phospholipase A
(and not calcium-dependent phospholipase
A
) when administered to intact RAW 264.7 cells; and 3) BEL
ablates the mobilization of [
H]arachidonic acid
in RAW 264.7 cells under conditions in which calcium-independent
phospholipase A
activity is inhibited and calcium-dependent
phospholipase A
activity is fully active.
Figure 3:
Measurement
of phospholipase A activities in RAW 264.7 cells after
nitric oxide stimulation or inhibitor treatment and examination of
deacylation-reacylation cycling rates. A, phospholipase
A
activity in the cytosolic or microsomal fractions was
quantified by determining the initial rate of fatty acid release from
1-O-(Z)-hexadec-1`-enyl-2-[9,10-
H]-octadec-9`-enoyl-sn-glycero-3-phosphocholine
substrate as described under ``Materials and Methods.''
Phospholipase A
activity was assessed in in vitro homogenates after incubation of cells with either buffer alone
(control) or sodium nitroprusside (100 µM) following a
15-min preincubation period with either buffer (control) or BEL (10
µM). The data are presented in pmol/mg
min. B, RAW 264.7 cells were incubated with
[
H]arachidonic acid and either buffer (lane1), sodium nitroprusside (100 µM) (lane2), sodium nitroprusside (100 µM) and BEL (lane3), or BEL alone (10 µM) (lane4) for 60 min at 37 °C. Cells were drenched in
MeOH/H
O (1:1, v/v), and lipids were extracted by the Bligh
and Dyer procedure, resolved by TLC (chloroform/methanol/ammonium
hydroxide (65:25:5, v/v)) and visualized by autoradiography. PE, phosphatidylethanolamine; PC,
phosphatidylcholine; PI,
phosphatidylinositol.
Accumulation
of [H]arachidonic acid in the culture medium
could reflect either increased release of
[
H]arachidonic acid from endogenous phospholipid
storage depots or decreased reincorporation of released
[
H]arachidonic acid into cellular phospholipids.
Concomitant incubation of cells with
[
H]arachidonic acid and either SNP or BEL (or the
combination of SNP and BEL) did not substantially alter the rate of
incorporation of [
H]arachidonic acid into
cellular phosphatidylethanolamine, phosphatidylcholine, or
phosphatidylinositol in comparison with control (Fig. 3). Thus,
nitric oxide-induced accumulation of
[
H]arachidonic acid in the medium was the result
of accelerated phospholipid catabolism and not decreased reacylation of
released [
H]arachidonic acid into endogenous
phospholipid storage depots.
The Coupling of Nitric oxide-induced Mobilization of
[
Nitric oxide-induced mobilization of
[H]Arachidonic Acid with Cellular Glucose
Metabolism
H]arachidonic acid required the presence of
glucose in the media, and the magnitude of nitric oxide-induced
[
H]arachidonic acid release was highly correlated
with the concentration of glucose present in the media (Fig. 4A). As anticipated from prior studies(7) ,
the magnitude of nitric oxide-stimulated glucose oxidation was also
highly correlated with the concentration of glucose in the media (data
not shown). Furthermore, nitric oxide-induced
[
H]arachidonic acid mobilization required the
metabolism of glucose since neither 2-deoxyglucose nor
3-O-methylglucose (nonmetabolizable glucose analogs) could
facilitate the nitric oxide-induced mobilization of
[
H]arachidonic acid (data not shown).
Importantly, preincubation of cells with from 1 to 25 mM
glucose for 2 h followed by subsequent incubation with 25 mM glucose prior to nitric oxide stimulation resulted in similar
amounts of [
H]arachidonic acid release in
comparison with cells kept at 25 mM glucose during the entire
experimental interval (Fig. 4B). Thus, attenuation of
nitric oxide-induced [
H]arachidonic acid
mobilization by low glucose concentration was entirely reversible after
restoration of glucose to normal culture levels (i.e. 25
mM glucose). Cells treated with 1 mM glucose for 2 h
remained attached to the cell culture surface, excluded trypan blue,
grew normally for at least 48 h after the experimental interval, and
did not release LDH into the medium. Furthermore, cells incubated with
1 or 3 mM glucose in the presence of 10% FBS grew at rates
greater than 60% of the growth rate manifest at 25 mM glucose
for extended intervals (i.e. 16 h) and incorporated
[
H]thymidine over the 3-h experimental interval
at nearly identical rates (within 10%) at either 1, 3, 8, or 25 mM glucose.
Figure 4:
Nitric oxide-stimulated
[H]arachidonic acid release from RAW 264.7 cell
phospholipid storage depots is coupled to glucose utilization. A, RAW 264.7 cells were prelabeled with
[
H]arachidonic acid for 16 h as described under
``Materials and Methods.'' After exhaustive washing to remove
unincorporated [
H]arachidonic acid, cells were
incubated with the indicated concentrations of glucose for 2 h prior to
stimulation with sodium nitroprusside (100 µM) or
incubation with buffer alone for 60 min (control). B,
experiments performed in B were conducted similarly to those
in A, except that the cells were first preincubated with the
indicated concentrations of glucose for 2 h prior to a subsequent 2-h
incubation with 25 mM glucose for re-equilibration before
stimulation with sodium nitroprusside (100 µM) or
incubation with buffer alone (control) for 60 min.
[
H]Arachidonate released into the media was
quantified as described under ``Materials and Methods.''
Results represent x ± S.E. of four
determinations.
; 3) the
activation of calcium-independent phospholipase A
by nitric
oxide results in the highly selective release of arachidonic acid; 4)
the dose-response profile of SNP-mediated arachidonic acid mobilization
is similar to the dose-response profile of other SNP-mediated responses
in RAW 264.7 cells(27) ; and 5) the nitric oxide-induced release
of arachidonic acid is dependent on glucose uptake and metabolism.
Since arachidonic acid and its oxygenated eicosanoid metabolites have
specific effects on distinct cell types, these results demonstrate a
novel mechanism through which nitric oxide can mediate a complex
repertoire of biologic effects.
activity in multiple tissues (e.g. heart, smooth muscle,
brain, and
kidney(13, 15, 28, 29, 30) ) and
have demonstrated that calcium-independent phospholipase A
is responsible for arachidonic acid mobilization after either
ligand-receptor coupling or cellular stimulation in at least some cell
types(26, 30) . Calcium-independent phospholipase
A
exists as a catalytic complex comprised of regulatory and
catalytic constituents, whose activity is regulated by physiologic
alterations in ATP concentration(16, 31) . The prior
demonstration that the polypeptides mediating ATP-induced activation of
calcium-independent phospholipase A
catalytic activity were
isoforms of phosphofructokinase suggested that ATP-induced allosteric
regulation of phosphofructokinase could couple glycolytic metabolism to
arachidonic acid mobilization(16) . Based upon the known
interaction of phosphofructokinase with calcium-independent
phospholipase A
in purified systems, in conjunction with
the inhibition of arachidonic acid mobilization by BEL in intact cells,
the present results suggest that intracellular alterations in local ATP
concentrations could represent a key regulatory element modulating
phospholipid catabolism and the resultant generation of biologically
active lipid second messengers (e.g. eicosanoids and
lysolipids). Many cell types respond to a variety of different stimuli
by concomitant increases in glycolytic flux and the release of lipid
second messengers (e.g. Ref. 32). The translocation of
glycolytic complexes that can catalyze substrate-based ATP synthesis to
specific membrane compartments during cellular perturbation is well
known(33) . Accordingly, we propose that increased glycolytic
flux and alteration of cytosolic ATP concentration in critical
subcellular loci represent a master control switch, which couples
glucose metabolism with the generation of lipid second messengers
through the activation of Ca
-independent
phospholipase A
. Collectively, the present results provide
a unifying hypothesis integrating alterations in glycolytic flux with
the generation of lipid second messengers, thereby providing a
fundamental mechanism through which cellular activation and
intercellular communication can be mediated. The power inherent in the
coordinated regulation of complex cellular metabolic networks (e.g. endothelium, smooth muscle, and macrophages) employing interwoven
alterations in nitric oxide production, glycolytic flux, and eicosanoid
generation through the activation of calcium-independent phospholipase
A
is evident.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.