Nitric Oxide Activates the Glucose-dependent Mobilization of Arachidonic Acid in a Macrophage-like Cell Line (RAW 264.7) That Is Largely Mediated by Calcium-independent Phospholipase A2
1995; Elsevier BV; Volume: 270; Issue: 25 Linguagem: Inglês
10.1074/jbc.270.25.14855
ISSN1083-351X
AutoresRichard W. Gross, Amy E. Rudolph, Jian Wang, Cynthia D. Sommers, Matthew J. Wolf,
Tópico(s)Peroxisome Proliferator-Activated Receptors
ResumoHerein, we demonstrate that nitric oxide is a potent (>20% release) and highly selective inducer of [3H]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 [3H]arachidonic acid release into the medium (IC50 20% release) and highly selective inducer of [3H]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 [3H]arachidonic acid release into the medium (IC50 <0.5 μM) and the concomitant inhibition of in vitro measurable calcium-independent phospholipase A2 activity (92% inhibition) without demonstrable effects on calcium-dependent phospholipase A2 activity. Since nitric oxide is a potent stimulator of glycolysis (and therefore glycolytically derived ATP) and since cytosolic calcium-independent phospholipase A2 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 [3H]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, [3H]arachidonic acid release is coupled to cellular glucose metabolism through alterations in the activity of calcium-independent phospholipase A2. 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. INTRODUCTIONRecent 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(4Moncada S. Palmer R.M. Higgs E.A. Pharmacol. Rev. 1991; 43: 109-142PubMed Google Scholar)), 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)(7Albina J.E. Mastrofrancesco B. Am. J. Physiol. 1993; 264: C1594-C1599Crossref PubMed Google Scholar, 8Leslie C.C. Detty D.M. Biochem. J. 1986; 236: 251-259Crossref PubMed Scopus (21) Google Scholar). Macrophages contain both calcium-dependent (9Leslie C.C. Voelker D.R. Channon J.Y. Wall M.M. Zelarney P.T. Biochim. Biophys. Acta. 1988; 963: 476-492Crossref PubMed Scopus (182) Google Scholar, 10Channon J.Y. Leslie C.C. J. Biol. Chem. 1990; 265: 5409-5413Abstract Full Text PDF PubMed Google Scholar) and calcium-independent phospholipase A2(11Ulevitch R.J. Watanabe Y. Sano M. Lister M.D. Deems R.A. Dennis E.A. J. Biol. Chem. 1988; 263: 3079-3085Abstract Full Text PDF PubMed Google Scholar) 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 A2 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 A2 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(16Hazen S.L. Gross R.W. J. Biol. Chem. 1993; 268: 9892-9900Abstract Full Text PDF PubMed Google Scholar). These findings led us to suggest that glycolysis and phospholipolysis are interwoven metabolic pathways coupled through the interaction of the calcium-independent phospholipase A2 catalytic complex with glycolytically derived ATP(16Hazen S.L. Gross R.W. J. Biol. Chem. 1993; 268: 9892-9900Abstract Full Text PDF PubMed Google Scholar). Since nitric oxide is a potent stimulator of macrophage glucose uptake and glycolytic flux(7Albina J.E. Mastrofrancesco B. Am. J. Physiol. 1993; 264: C1594-C1599Crossref PubMed Google Scholar, 17Lander H.M. Sehajpal P. Levine D.M. Novogrodsky A. J. Immunol. 1993; 150: 1509-1516PubMed Google Scholar), we hypothesized that some of the effects of nitric oxide on macrophage phospholipid catabolism and physiologic function could result from the calcium-independent phospholipase A2-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 A2; and 3) nitric oxide-mediated arachidonic acid mobilization by calcium-independent phospholipase A2 possesses an obligatory dependence on glucose uptake and metabolism.EXPERIMENTAL PROCEDURESMaterialsRAW 264.7 cells were obtained from American Type Tissue Culture Collection. 1-O-(Z)-Hexadec-1′-enyl-2-[9,10-3H]octadec-9′-enoyl-sn-glycero-3-phosphocholine-(plasmenylcholine) and (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one (BEL)1( 1The abbreviations used are: BEL(E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-oneDMEMDulbecco's modified Eagle's mediumFBSfetal bovine serumLPSlipopolysaccharideSNPsodium nitroprusside.) were synthesized and purified by established methods(18Han X. Zupan L.A. Hazen S.L. Gross R.W. Anal. Biochem. 1992; 200: 119-124Crossref PubMed Scopus (45) Google Scholar, 19Krafft G.A. Katzenellenbogen J.A. J. Am. Chem. Soc. 1981; 103: 5459-5466Crossref Scopus (159) Google Scholar, 20Zupan L.A. Weiss R.H. Hazen S.L. Parnas B.L. Aston K.W. Lennon R.J. Getman D.P. Gross R.W. J. Med. Chem. 1993; 36: 95-100Crossref PubMed Scopus (55) Google Scholar, 21Hazen S.L. Loeb L.A. Gross R.W. Methods Enzymol. 1991; 197: 400-411Crossref PubMed Scopus (14) Google Scholar).Radiolabeling of RAW 264.7 Cells and Measurement of Phospholipase A2Activity in the Cytosolic and Microsomal FractionsRAW 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 [3H]arachidonic acid (100 Ci/mmol) or [3H]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, 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 [3H]arachidonic acid mobilization in macrophages was inhibited by BEL demonstrating that the release of [3H]arachidonic acid after stimulation by these effectors largely results from hydrolysis mediated by calcium-independent phospholipase A2.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 [3H]arachidonic acid (A) or [3H]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 [3H]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/H2O (90:8:1:0.8, v/v).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Measurement of RAW 264.7 Cell Phospholipase A2 Activities and [3H]Arachidonic Acid Reacylation RatesMeasurement of RAW 264.7 cell phospholipase A2 activity in vitro demonstrated the presence of both calcium-dependent and -independent phospholipase A2 activities (Fig. 3A). Calcium-dependent phospholipase A2 activity was predominant in the cytosolic fraction, while calcium-independent phospholipase A2 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 A2 activities in either the cytosolic or microsomal fractions utilizing plasmenylcholine substrate (plasmenylcholine is not susceptible to hydrolysis by phospholipase A1) (Fig. 3A) or phosphatidylcholine substrate containing [3H]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 A2 activity without inhibition of calcium-dependent phospholipase A2 activity. Collectively, these results demonstrate that: 1) nitric oxide-mediated stimulation of RAW 264.7 cells does not result in stimulation of measurable phospholipase A2 activity in in vitro homogenates; 2) BEL specifically inhibits calcium-independent phospholipase A2 (and not calcium-dependent phospholipase A2) when administered to intact RAW 264.7 cells; and 3) BEL ablates the mobilization of [3H]arachidonic acid in RAW 264.7 cells under conditions in which calcium-independent phospholipase A2 activity is inhibited and calcium-dependent phospholipase A2 activity is fully active.Figure 3:Measurement of phospholipase A2 activities in RAW 264.7 cells after nitric oxide stimulation or inhibitor treatment and examination of deacylation-reacylation cycling rates. A, phospholipase A2 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-[9,10-3H]-octadec-9′-enoyl-sn-glycero-3-phosphocholine substrate as described under "Materials and Methods." Phospholipase A2 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 [3H]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/H2O (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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Accumulation of [3H]arachidonic acid in the culture medium could reflect either increased release of [3H]arachidonic acid from endogenous phospholipid storage depots or decreased reincorporation of released [3H]arachidonic acid into cellular phospholipids. Concomitant incubation of cells with [3H]arachidonic acid and either SNP or BEL (or the combination of SNP and BEL) did not substantially alter the rate of incorporation of [3H]arachidonic acid into cellular phosphatidylethanolamine, phosphatidylcholine, or phosphatidylinositol in comparison with control (Fig. 3). Thus, nitric oxide-induced accumulation of [3H]arachidonic acid in the medium was the result of accelerated phospholipid catabolism and not decreased reacylation of released [3H]arachidonic acid into endogenous phospholipid storage depots.The Coupling of Nitric oxide-induced Mobilization of [3H]Arachidonic Acid with Cellular Glucose MetabolismNitric oxide-induced mobilization of [3H]arachidonic acid required the presence of glucose in the media, and the magnitude of nitric oxide-induced [3H]arachidonic acid release was highly correlated with the concentration of glucose present in the media (Fig. 4A). As anticipated from prior studies(7Albina J.E. Mastrofrancesco B. Am. J. Physiol. 1993; 264: C1594-C1599Crossref PubMed Google Scholar), 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 [3H]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 [3H]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 [3H]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 [3H]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 [3H]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 [3H]arachidonic acid release from RAW 264.7 cell phospholipid storage depots is coupled to glucose utilization. A, RAW 264.7 cells were prelabeled with [3H]arachidonic acid for 16 h as described under "Materials and Methods." After exhaustive washing to remove unincorporated [3H]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. [3H]Arachidonate released into the media was quantified as described under "Materials and Methods." Results represent x ± S.E. of four determinations.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONThe 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 A2; 3) the activation of calcium-independent phospholipase A2 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(27Salvemini D. Misko T.P. Masferrer J.L. Siebert K. Currie M.G. Needleman P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7240-7244Crossref PubMed Scopus (1377) Google Scholar); 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.Recent studies have recognized the preponderance of calcium-independent phospholipase A2 activity in multiple tissues (e.g. heart, smooth muscle, brain, and kidney(13Wolf R.A. Gross R.W. J. Biol. Chem. 1985; 260: 7295-7303Abstract Full Text PDF PubMed Google Scholar, 15Gross R.W. Ramanadham S. Kruszka K.K. Han X. Turk J. Biochemistry. 1993; 32: 327-336Crossref PubMed Scopus (112) Google Scholar, 28Hirashima Y. Farooqui A.A. Mills J.S. Horrocks L.A. J. Neurochem. 1992; 59: 708-714Crossref PubMed Scopus (127) Google Scholar, 29Portilla D. Shah S.V. Lehman P.A. Creer M.H. J. Clin. Invest. 1994; 93: 1609-1615Crossref PubMed Scopus (77) Google Scholar, 30Ramanadham S. Gross R.W. Han X. Turk J. Biochemistry. 1993; 32: 337-346Crossref PubMed Scopus (123) Google Scholar)) and have demonstrated that calcium-independent phospholipase A2 is responsible for arachidonic acid mobilization after either ligand-receptor coupling or cellular stimulation in at least some cell types(26Lehman J.J. Brown K.A. Ramanadham S. Turk J. Gross R.W. J. Biol. Chem. 1993; 268: 20713-20716Abstract Full Text PDF PubMed Google Scholar, 30Ramanadham S. Gross R.W. Han X. Turk J. Biochemistry. 1993; 32: 337-346Crossref PubMed Scopus (123) Google Scholar). Calcium-independent phospholipase A2 exists as a catalytic complex comprised of regulatory and catalytic constituents, whose activity is regulated by physiologic alterations in ATP concentration(16Hazen S.L. Gross R.W. J. Biol. Chem. 1993; 268: 9892-9900Abstract Full Text PDF PubMed Google Scholar, 31Hazen S.L. Gross R.W. Biochem. J. 1991; 280: 581-587Crossref PubMed Scopus (51) Google Scholar). The prior demonstration that the polypeptides mediating ATP-induced activation of calcium-independent phospholipase A2 catalytic activity were isoforms of phosphofructokinase suggested that ATP-induced allosteric regulation of phosphofructokinase could couple glycolytic metabolism to arachidonic acid mobilization(16Hazen S.L. Gross R.W. J. Biol. Chem. 1993; 268: 9892-9900Abstract Full Text PDF PubMed Google Scholar). Based upon the known interaction of phosphofructokinase with calcium-independent phospholipase A2 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(33Brooks S.P.J. Storey K.B. Am. J. Physiol. 1988; 225: R289-R294Google Scholar). 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 Ca2+-independent phospholipase A2. 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 A2 is evident. INTRODUCTIONRecent 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(4Moncada S. Palmer R.M. Higgs E.A. Pharmacol. Rev. 1991; 43: 109-142PubMed Google Scholar)), 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)(7Albina J.E. Mastrofrancesco B. Am. J. Physiol. 1993; 264: C1594-C1599Crossref PubMed Google Scholar, 8Leslie C.C. Detty D.M. Biochem. J. 1986; 236: 251-259Crossref PubMed Scopus (21) Google Scholar). Macrophages contain both calcium-dependent (9Leslie C.C. Voelker D.R. Channon J.Y. Wall M.M. Zelarney P.T. Biochim. Biophys. Acta. 1988; 963: 476-492Crossref PubMed Scopus (182) Google Scholar, 10Channon J.Y. Leslie C.C. J. Biol. Chem. 1990; 265: 5409-5413Abstract Full Text PDF PubMed Google Scholar) and calcium-independent phospholipase A2(11Ulevitch R.J. Watanabe Y. Sano M. Lister M.D. Deems R.A. Dennis E.A. J. Biol. Chem. 1988; 263: 3079-3085Abstract Full Text PDF PubMed Google Scholar) 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 A2 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 A2 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(16Hazen S.L. Gross R.W. J. Biol. Chem. 1993; 268: 9892-9900Abstract Full Text PDF PubMed Google Scholar). These findings led us to suggest that glycolysis and phospholipolysis are interwoven metabolic pathways coupled through the interaction of the calcium-independent phospholipase A2 catalytic complex with glycolytically derived ATP(16Hazen S.L. Gross R.W. J. Biol. Chem. 1993; 268: 9892-9900Abstract Full Text PDF PubMed Google Scholar). Since nitric oxide is a potent stimulator of macrophage glucose uptake and glycolytic flux(7Albina J.E. Mastrofrancesco B. Am. J. Physiol. 1993; 264: C1594-C1599Crossref PubMed Google Scholar, 17Lander H.M. Sehajpal P. Levine D.M. Novogrodsky A. J. Immunol. 1993; 150: 1509-1516PubMed Google Scholar), we hypothesized that some of the effects of nitric oxide on macrophage phospholipid catabolism and physiologic function could result from the calcium-independent phospholipase A2-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 A2; and 3) nitric oxide-mediated arachidonic acid mobilization by calcium-independent phospholipase A2 possesses an obligatory dependence on glucose uptake and metabolism.
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