Phorbol myristate acetate stimulates the formation of 5-oxo-6,8,11,14-eicosatetraenoic acid by human neutrophils by activating NADPH oxidase.

We have shown previously that human neutrophil microsomes contain a highly specific dehydrogenase which, in the presence of NADP+, converts 5S-hydroxy-6,8,11,14-eicosatetraenoic acid (5S-HETE) to its 5-oxo metabolite, 5-oxo-ETE, a potent agonist of these cells. However, intact neutrophils convert 5S-HETE principally to its omega-oxidation product, 5,20-diHETE, and to only small amounts of 5-oxo-ETE. Phorbol myristate acetate (PMA) dramatically shifts the metabolism of 5S-HETE by intact cells so that 5-oxo-ETE is the major metabolite. The objective of this investigation was to determine the mechanism for the stimulatory effect of PMA on 5-oxo-ETE formation. The possibility that oxidants released in response to PMA nonenzymatically oxidized 5S-HETE was ruled out, since PMA did not appreciably stimulate the formation of 5-oxo-ETE from 5R-HETE. On the other hand, inhibition of NADPH oxidase either by diphenylene iodonium or by mild heating nearly completely prevented the stimulatory effect of PMA on the formation of 5-oxo-ETE. The possibility that this effect was mediated by superoxide seems unlikely, since it was still observed, although somewhat attenuated, in the presence of superoxide dismutase. Moreover, superoxide generated by another mechanism (xanthine/xanthine oxidase) did not appreciably affect the formation of 5-oxo-ETE by neutrophils. However, phenazine methosulfate, which can nonenzymatically convert NADPH to NADP+, mimicked the effect of PMA on 5-oxo-ETE formation by intact neutrophils. It is concluded that PMA acts by activating NADPH oxidase, resulting in conversion of NADPH to NADP+, which enhances the formation of 5-oxo-ETE and reduces the formation of 5,20-diHETE. Serum-treated zymosan has an effect on the metabolism of 5S-HETE similar to that of PMA in that it also stimulates the formation of 5-oxo-ETE and inhibits that of 5,20-diHETE.

We have shown previously that human neutrophil microsomes contain a highly specific dehydrogenase which, in the presence of NADP', converts 5s-hydroxy-6,8,11,14-eicosatetraenoic acid (5S-HETE) to its 5-oxo metabolite, 5-OXO-Em, a potent agonist of these cells. However, intact neutrophils convert 5s-NETE principally to its w-oxidation product, 5~0-diNETE, and to only small amounts of 5-0xo-ETE. Phorbol myristate acetate (PMA) dramatically shifts the metabolism of 5s-HETE by intact cells so that 5-oxo-ETE is the major metabolite. The objective of this investigation was to determine the mechanism for the stimulatory effect of PMA on 5-oxo-ETE formation. The possibility that oxidants released i n response to PMA nonenzymatically oxidized 5s-WETE was ruled out, since PMA did not a p preciably stimulate the formation of 5-oxo-ETE from 5R-HETE. On the other hand, inhibition of NADPH oxidase either by diphenylene iodonium or by mild heating nearly completely prevented the stimulatory effect of PMA on the formation of 5-oxo-ETE. The possibility that this effect was mediated by superoxide seems unlikely, since it was still observed, although somewhat attenuated, in the presence of superoxide dismutase. Moreover, superoxide generated by another mechanism (xanthine/xanthine oxidase) did not appreciably affect the formation of 5-oxo-ETE by neutrophils. However, phenazine methosulfate, which can nonenzymatically convert NADPH to NADP' , mimicked the effect of PMA on 5-oxo-Em formation by intact neutrophils. It is concluded that PMA acts by activating NADPH oxidase, resulting in conversion of NADPH to NADP', which enhances the formation of 5-oxo-ETE and reduces the formation of 5,ZO-diHETE. Serum-treated zymosan has an effect on the metabolism of 5s-HETE similar to that of PMA in that it also stimulates the formation of 5-OXO-ETE and inhibits that of 5,20-diHETE.  (1,2). LTB, is a potent activator of neutrophils and induces a variety of responses by these cells, including elevated cytosolic calcium levels (31, chemotaxis (4,5), and adherence to endothelial cells (6). 5s-HETE also activates human neutrophils but only at relatively high and probably nonphysiolo~c concentrations (7,8).
We have shown recently that neutrophils convert 5s-HETE to a &oxo metabolite, 5-oxo-ETE (91, which, like LTB,, is a potent stimulus of cytosolic calcium levels and chemotaxis in these cells (10). 5-Oxo-ETE has also been shown to stimulate degranulation of neutrophils, which was enhanced by priming with tumor necrosis factor-a (11). The actions of 5-oxo-ETE are clearly independent of the LTB, receptor, since they are not prevented by prior treatment with either LTB, (Le. no crossdesensitization) or the LTB, antagonist LYE5283 (10,11). Although not quite as potent as LTB, in stimulating neutrophils, 5-oxo-ETE is much more potent than LTB, as a chemotactic agent for eosinophils (12) and in stimulating changes in cell volume in guinea pig intestinal epithelial cells? LTB, is rapidly biologically inactivated in human neutrophils by conversion to %O-hydroxy-LTB, and w-carboxyl-LTB, by the w-oxidation pathway (13, 14). 5s-HETE is also metabolized by this pathway to 5,20-diHETE (15) and can be converted to other dihydroxy metabolites by 12-lipoxygenase (16) and 15lipoxygenase (17) as shown in Fig. 1. The distribution of dihydroxy products formed is dependent on the enzymes that are present as well as whether or not the cells have been activated (18). In addition, 5-HETE can be esterified into triglycerides and phospholipids in neutrophils (19, 20) as well as in other cells (21,22). As noted above, 5-HETE is also converted to 5-oxo-ETE by a microsomal dehydrogenase, which requires NADP+ as a cofactor and is highly specific for a substrate with a 5-hydroxyl group in the S c o~l~r a t i o n followed by a 6-trans double bond (9). Thus the three diHETE metabolites of 5-HETE shown in Fig. 1 are all converted to the corresponding 5-oxo derivatives by this pathway (9). The oxidation of 5s-HETE by microsomes i s reversible, since 5-oxo-ETE is stereospecifically reduced to the former product in the presence of NADPH (9). It is not clear whether the reverse reaction is also catalyzed by 5-hydroxyeicosanoid dehydrogenase or whether this is accomplished by a distinct reductase in neutrophil microsomes.

5,20-diHETE 5-0XO-ETE
has a dramatic effect on the metabolism of 5s-HETE, strongly stimulating the formation of 5-oxo-ETE and inhibiting the formation of 5,20-diHETE (23). The effects of PMA were completely prevented by the addition of staurosporine, suggesting that they were mediated by protein kinase-dependent protein phosphorylation 123). It was not clear from our previous study (23) whether the effects of PMA on the metabolism of 5s-HETE were due to direct effects on the enzymes metabolizing 5S-HETE or whether they were mediated indirectly by the activation of other cellular processes, such as release of myeloperoxidase or activation of the respiratory burst. The objective of the current study was to determine the mechanism for the effects of PMA on the metabolism of 5s-HETE by human neutrophils.
PMA, cytochrome c (horse heart), superoxide dismutase (bovine kidney, 4,00~10,000 unitdmg of protein), xanthine, xanthine oxidase ( B u t~r m i l~ 1.4 unitsfmg protein), prostaglandin B,, and phenazine methosulfate (PMS) were obtained from Sigma. Staurosporine and A23187 were purchased from Boehringer Mannheim and Calbiochem, Arbor, MI. respectively 5R-HETE was obtained from Cayman Chemical, Ann Preparation of Neutrophils-Human neutrophils were prepared by treatment of whole blood with dextran T-500 (Pharmacia Biotech Inc.) followed by centrifugation over Fico~l-Paque (Pharmacia) and lysis of remaining red blood cells with ammonium chloride (27). for 2 x 10 s at a setting of 40 cyclesh. The disruptate was centrifuged at 1,500 x g at 4 "C for 10 min to remove unbroken cells and nuclei. The postnuclear supernatant was centrifuged at 10,000 x g at 4 "C for 10 min, and the supernatant was centrifuged at 200,000 x g at 4 "C for 60 min. The pellet was resuspended in phosphate-buffered saline containing 1.8 m M CaCl, and 1 mnq MgCl, at a concentration equivalent to 20 x lo6 celldml, unless otherwise indicated. Analysis of 5-HETE Metabolites-Suspensions (1 ml) of neutrophils or neutrophil microsomes were incubated with 5R-HETE or 5s-HETE. The incubations were terminated by the addition of methanol (0.6 ml) the samples was adjusted to 15% by the addition of water, and 5-HETE and cooling to 0 "C. Prior to analysis, the concentration of methanol in high pressure liquid chromatography (RP-HPLC) (28) using a Waters metabolites were quantitated by precolumn e x~r a c~o~r e v e r s e~-p h a s e Millipore gradient controller, WISP automatic injector, WAVS auto-mated switching valve, model 991 diode array detector, and a model 600 solvent delivery system. The mobile phase was a linear gradient between a mixture of 80% solvent A (water/acetonitrile/acetic acid (8020:0.02) and 20% solvent B (acetonitrile/methanol/water/acetic acid (38.5:54:7.5:0.02) and a mixture of 5% solvent Aand 95% solvent B over 45 min unless otherwise indicated. The flow rate was 1 mlfmin. The stationary phase was a column of Novapak C,, (3.9 x 150 mm; Waters Millipore). Products were quantitated by comparing the areas of their peaks of W absorbance at their A,,, with that of the internal standard,

PMA Enhances the Enzymatic Oxidation of 5s-HETE to 5-Oxo-ETE by Intact Neutrophils
We have shown previously that PMA strongly stimulates the conversion of 5s-HETE to 5-oxo-ETE and inhibits the formation of 5,ZO-di~ETE by neutrophils. One possible explanation for these effects could be that PMA-induced degranulation results in the release of myeloperoxidase which, via the formation of hypochlorous acid, could nonenzymatically oxidize 5S-HETE. To determine whether this is the case, neutrophils were incubated with 5-HETE and PMA in the presence of sodium azide, which inhibits myloperoxidase, or methionine, which is a scavenger of hypoc~orous acid ( Table I). Although azide appeared to inhibit the formation of 5,20-diHETE somewhat, neither azide nor methionine was able to inhibit the stimulatory effect of PMA on the conversion of 5s-HETE to 5-oxo-ETE.
We have shown previously that 5R-HETE is not nearly as good a substrate for the microsomal 5-hydroxyeicosanoid dehydrogenase as 5s-HETE (23). If the oxidation of 5-HETE by intact cells stimulated with PMA is catalyzed by the same enzyme, then 5R-HETE should not be converted to appreciable amounts of 5-oxo-ETE under these conditions. Fig. 2 shows high pressure liquid chromatograms of the products obtained after incubation of 5s-HETE and 5R-HETE with intact neutrophils in the presence or absence of PMA (30 m). In the absence of PMA, 5s-HETE was converted principally to 5,20-diHETE, with only a small amount of 5-oxo-ETE being formed (Fig. 2 4 ) . However, in the presence of PMA, the situation was reversed, and the major product was 6-oxo-ETE (Fig. 2B). Like 5s-HETE, 5R-HETE was converted to substantial amounts of 5,20-diHETE in the absence of PMA (Fig. 2C). As in the case of 5S-HETE, PMA inhibited the o-oxidation of 5R-HETE to 5,20-d~E T E .
However, in contrast to 5S-HETE, 5R-HETE was converted to only very small amounts of 5-oxo-ETE in the presence of PMA (Fig. 20).

of 5-Oxo-ETE Formation 25375
PMA Does Not Act by Directly Stimulating 5-Hydroxyeicosanoid Dehydrogenase As would be expected, the addition of PMA directly to microsomal fractions did not affect the rate of conversion of 5s-HETE to 5-oxo-ETE in the presence of NADP+ (data not shown). However, the possibility remained that PMA could stimulate the phosphorylation of the dehydrogenase in intact cells, thus increasing its activity. To determine whether this could be true, neutrophils were incubated with PMAfor 15 min at 37 "C in the absence of any substrate. The cells were then cooled to 0 "C in an ice-water bath, sonicated, and microsomal fractions prepared. The amount of 5-oxo-ETE formed by microsomes from PMA-treated neutrophils was slightly higher than the amount formed by control microsomes (p < 0.05) (Table 11). However, it would seem very unlikely that this modest 16% increase in enzyme activity could explain the dramatic effect of PMA on the formation of 5-oxo-ETE by intact cells. Pretreatment of neutrophils with PMA appeared to slightly reduce the amount of 5,20-diHETE formed by microsomes, but this difference was not significant (Table 11).  The NADPH Oxidase Inhibitor DPI Inhibits the Effects of PMAon 5-Oxo-ETE Formation-To test the hypothesis that the effects of PMA on the metabolism of 5-HETE are dependent on its stimulatory effect on NADPH oxidase, neutrophils were treated with various concentrations of DPI prior to the addition of 5-HETE. Fig. 3 shows concentration-response curves for the effects of DPI on the conversion of 5-HETE to its major metabolites in the absence and presence of PMA (30 nM). In the absence of PMA, DPI had a modest stimulatory effect on the formation of 5-oxo-ETE but strongly inhibited the formation of 5,20-diHETE (ICbo, about 100 nM) (Fig. 3A). In contrast, in the presence of PMA, DPI strongly inhibited the formation Of 5-0x0-ETE (IC5,,, 50 nM) (Fig. 3B). DPI also inhibited the formation of 5,20-diHETE in the presence of PMA but was not quite as potent as in its absence. In the absence of PMA, 80 nM DPI inhibited the formation of 5,20-diHETE by 44% ( p < 0.025; paired t test) (Fig. 3 A ) , whereas in the presence of PMA this concentration of DPI had no effect on 5,20-diHETE synthesis (Fig. 3B). Formation of the o-oxidation product of 5-oxo-ETE, 5-oxo-20-hydroxy-ETE was inhibited by DPI, both in the presence and absence of PMA.
Mild Heating of Neutrophils Inhibits N m P H Oxidase as Well as the Effects of PMA on the Metabolism of 5-HETE-It has been shown previously that heating of neutrophils at 46 "C prevents the activation of NADPH oxidase by PMA by inactivating p67-phoq one of the cytosolic components of this enzyme (30). To determine whether this treatment can also prevent the effects of PMA on the metabolism of 5-HETE, neutrophils were heated at 46 "C for 9 min and then cooled rapidly on ice. This treatment completely prevented the ability of PMA to stimulate superoxide production (Fig. 4A), consistent with the reported inhibitory effect of mild heating on NADPH oxidase as discussed above. Incubation of the heated cells with 5-HETE resulted in the formation of a single major metabolite, 5,20-di-HETE (data not shown), and only a small amount of 5-oxo-ETE, as was the case with control, nonheated cells (Fig. 4B). However, preincubation of the heated cells with PMA (30 nM) had no effect on the subsequent metabolism of 5-HETE, in contrast to the stimulatory and inhibitory effects of PMA on the synthesis  (Fig. 4B). To demonstrate that the inhibitory effect of heating on the synthesis of 5-oxo-ETE by PMA-stimulated neutrophils was not due to inactivation of 5-hydroxyeicosanoid dehydrogenase, the activities of this enzyme in microsomal fractions from heated and control neutrophils were compared. As shown in Fig. 4C, microsomal fractions from heated cells were just as active as the corresponding fractions from control cells in converting 5-HETE to 5-oxo-ETE.
Heating at 46 "C had a slight inhibitory effect on the formation of 5,20-diHETE by unstimulated neutrophils (33% inhibit i o n ;~ < 0.02 (data not shown)). PMAinhibited the formation of this compound by heated cells by only 21% ( p < 0.05), in contrast to the 69% inhibition observed with control cells ( p < 0.001) (data not shown).    n 0 U n of superoxide produced by neutrophils under conditions identical to those used to study the metabolism of 5-HETE. Since the amount of cytochrome c in these incubations was limiting, we could only measure superoxide production for the first few minutes in the absence of superoxide dismutase. Superoxide dismutase strongly inhibited the accumulation of superoxide by neutrophils in response to PMA (30 n~) under the conditions of our assay. However, it did not alter the inhibitory effect of PMA on the formation of 5,20-diHETE from 5-HETE (Fig. 5B). PMA stimulated the formation of 5-oxo-ETE in the presence of superoxide dismutase by about %fold ( p < 0.01; paired t test), but the degree of stimulation was only about one-half that observed in the absence of this enzyme ( p < 0.02) (Fig. 4B).

Generation of Superoxide by XanthinelXanthine Oxidase Does Not Stimulate the Formation of 5-Oxo-ETE from
5-HETE-To determine whether superoxide generated from a source other than NADPH oxidase could mimic the effects of PMA, neutrophils were incubated with 5-HETE in the presence of a mixture of xanthine oxidase (10, 30, or 100 milliunits/ml) and xanthine (400 PM). The addition of the two higher concentrations of xanthine oxidase resulted in the generation of superoxide at a rate in excess of that observed with PMA (30 nM), whereas the addition of 10 milliunits of xanthine oxidase resulted in a slightly lower rate of formation of superoxide (Fig. 6, inset). As shown in Fig. 6, xanthine in the presence of xanthine oxidase (100 milliunits) had only a slight and nonsignificant stimulatory effect on the formation of 5-oxo-ETE from 5-HETE, in contrast to the strong stimulatory effect of PMA. The addition of xanthine/xanthine oxidase significantly inhibited the conversion of 5-HETE to 5,20-diHETE ( p < 0.051, but the magnitude of this effect was less than that of PMA ( p < 0.05). Similarly, lower concentrations of xanthine oxidase (10 and 30 milliunitdml) in the presence or absence of xanthine had no significant effects on the formation of 5-oxo-ETE by neutrophils (data not shown).

The Formation of 5-Oxo-ETE Is Stimulated by an Agent
That Increases Intracellular Levels of N m P PMS Stimulates the Formation of 5-Oxo-ETE-Another consequence of activation of NADPH oxidase in neutrophils would be an increase in the the ratio of NADP+ to NADPH. We attempted to mimic the effects of PMA by using an agent, PMS, which is known to promote the nonenzymatic conversion of NADPH to NADP+ (31,321. Low concentrations (10 VM) of PMS completely inhibited the w-oxidation of 5-HETE and stimulated the formation of 5-oxo-ETE by about 2.4 fold (Fig. 7A). The modest stimulatory effect of low concentrations of PMS on 5-oxo-ETE formation may have been due to its inhibitory effect on the w-oxidation of 5-oxo-ETE, combined with a diversion of substrate (i.e. 5-HETE) from the w-oxidation pathway to the dehydrogenase pathway. Higher concentrations of PMS (EC,, about 75 p~) had a much stronger stimulatory effect on the formation of 5-oxo-ETE, presumably due to conversion of intracellular NADPH to NADP".
The Effects of PMS Are Not Mediated by N a P H Oxidase-To determine whether the effects of PMS could have been mediated by stimulation of NADPH oxidase, similar experiments were conducted in the presence of the NADPH oxidase inhibitor DPI (400 nM) (Fig. 7B). This concentration of DPI strongly inhibited the PMA-stimulated conversion of 5-oxo-ETE but had no effect on the response to PMS.
The Effects of PMS on the Conversion of 5-HETE to 5 0 x 0 -ETE by Microsomes-To determine whether the effect of PMS could be due to a direct effect on 5-hydroxyeicosanoid dehydrogenase, its effects on the conversion of 5-HETE to 5-oxo-ETE by neutrophil microsomes were investigated (Fig. 7C). Neutrophil microsomes alone or in the presence of NADPH were capable of forming only very small amounts of 5-oxo-ETE, whereas in the presence of NADP', large amounts of the latter substance were formed. In the absence of any cofactors, PMS (100 VM) had a slight stimulatory effect on the formation of 5-oxo-ETE, but its effect was much less than that of NADP+. PMS had no effect on 5-oxo-ETE formation in the presence of NADP+ but strongly stimulated its formation in the presence of NADPH to levels comparable to those formed in the presence of NADP' (Fig. 7C).

~e r~r n -t r e~t e d ~y r n o s a n Stimulates the Conversion of
In addition to PMA, we also investigated the effects of serumtreated zymosan on the metabolism of 5-HETE. Neutrophils were preincubated for 10 min in the presence or absence of serum-treated zymosan and then incubated with 5-HETE for a further 20 min. As shown in Fig. 8, serum-treated zymosan had

TIME (mln)
Con PMA X 0 Con PMA X 0

5-OXO-ETE 5,2O-diHETE
xanthine oxidase) on the metabolism of 6-HETE by neutrophils. an effect very similar to that of PMA, although not quite as pronounced, on the metabolism of 5-HETE. Preincubation with zymosan inhibited the formation of 5,20-diHETE by about 70% and stimulated the formation o f 5-oxo-ETE by more than 5-fold ( p < 0.02).

DISCUSSION
Although neutrophil microsomes have a high capacity to convert 5-HETE to 5-oxo-ETE, intact polymorphonuclear leukocytes produce only relatively small amounts of the latter substance, but instead convert 5-HETE principally to its w-oxidation product, 5,20-diHETE (23). PMA induces a dramatic shift in the metabolism of 5-HETE, strongly stimulating the formation of 5-0XO-Em and inhibiting the f o~a t~o n of 5,20-diHETE by a protein kinase-dependent mechanism (23).
It is clear that oxidation of 5-HETE to 5-oxo-ETE by neutrophil microsomes is an enzymatic process. With intact cells the possibility remained that PMA-induced degranulation could cause nonenzymatic oxidation of 5-HETE due to the release of myeloperoxidase and formation of oxidants such as hypochlorous acid. This is unlikely, however, since azide, a myeloperoxidase inhibitor, and methionine, a scavenger of hypochlorous acid, did not prevent the stimulatory effect of PMA on 5-oxo-ETE formation. Moreover, the metabolism of 5-HETE by PMAstimulated intact neutrophils is stereospecific, since 5s-HETE, but not 5R-HETE, is a good substrate. Since nonenzymatic oxidation of 5-HETE would not be stereospecific, this indicates that the stimulation of B-oxo-Em formation by PMA is due to increased activity of 5-hydroxyeicosanoid dehydrogenase.
Another possible explanation for the stimulatory effect of PMA on 5-oxo-ETE formation could be that it stimulates phosphorylation of the dehydrogenase, thereby increasing its activity. Although we could not test this directly, since we have not yet succeeded in purifying the enzyme, this possibility also seems unlikely, since preincubation of intact cells with PMA had only a very small effect on microsomal enzyme activity.
The effects of PMA could also be mediated by its dramatic stimulatory effect on NADPH oxidase, as illustrated in Fig. 9. This enzyme complex is latent in unstimulated neutrophils, but becomes highly active in cells stimulated with activators of protein kinase C, such as PMA, due to phospho~lation of one of its cytosolic components (p47-phox) and translocation of p47phox and p67-phox to the plasma membrane (33). Once activated, the enzyme rapidly oxidizes cytosolic NADPH to NADP+ and transfers the electrons to molecular oxygen, resulting in the formation of large amounts of superoxide (33).
To determine whether activation of NADPH oxidase was required for the stimulatory effect of PMA on 5-oxo-ETE formation, neutrophils were incubated with an inhibitor of this enzyme, DPI (34, 351, prior to the addition of PMA. DPI slightly stimulated the production of 5-oxo-ETE by intact neutrophils in the absence any other stimuli, presumably by inhibiting its conversion to its *oxidation product, 5-oxo-20-hy~oxy-ETE. In contrast, DPI strongly inhibited the formation of 5-oxo-ETE by cells stimulated with PMA. These results suggest that DPI does not directly affect 5-hydroxyeicosanoid dehydrogenase, but only prevents the increased activity of this enzyme resulting from activation of NADPH oxidase. This was confirmed by the finding that DPI had no effect on the conversion of 5s-HETE to 5-oxo-ETE by neutrophil microsomes (data not shown) or by intact cells in the presence of PMS.
Although the results obtained with DPI are strongly suggestive of a role for NADPH oxidase in the PMA-induced stimulation of 5-oxo-ETE formation, the possibility that it could be acting by another mechanism cannot be excluded. For example, the data in Fig. 3 show that DPI inhibits the w-oxidation of 5-HETE in unstimulated cells, indicating that this substance can inhibit enzymes other than NADPH oxidase. To attempt to confirm the involvement of NADPH oxidase in the stimulatory effect of PMA on 5-oxo-ETE formation we investigated the effects of inactivating this enzyme by another mechanism. Heating of neutrophils at 46 "C has been shown to result in inactivation of NADPH oxidase but retention of other cellular functions such as phagocytosis and chemotaxis (36-39). This is due to the selective inactivation of the p67-phox component of the enzyme complex (30). The results in Fig. 4 clearly show that heating neutrophils at 46 "C for 9 min blocked superoxide production in response to PMA and nearly completely prevented the stimulatory effect of PMA on the conversion of 5s-HETE to 5-oxo-ETE. The i n h i b i~~ effect of heating on 5-oxo-ETE formation was not due to inactivation of 5-hydroxyeicosanoid dehydrogenase, since the activity of this enzyme in microsomal fractions prepared from heated cells was nearly identical to that in control cells. Thus both heating and DPI, which inhibit NADPH oxidase by completely different mechanisms, prevent the stimulatory effect of PMA on 5-oxo-ETE formation by neutrophils. The inhibitory effects of both treatments were observed only in intact cells, since in both cases 5-hydroxyeicosanoid dehydrogenase activity in microsomal fractions from the treated cells was the same as in microsomes from control cells. These results thus provide strong evidence that PMA acts by stimulating NADPH oxidase.
One possible mechanism for the stimulatory effect of activation of NADPH oxidase on 5-oxo-ETE formation could be that  superoxide somehow enhanced the activity of 5-hydroxyeicosanoid dehydrogenase. However, superoxide dismutase did not prevent the effects of PMA on the metabolism of 5-HETE, although its stimulatory effect on dehydrogenase activity was attenuated. Since the experiment with superoxide dismutase did not completely rule out the possibility that superoxide itself contributes to the effect of PMAon the production of 5-oxo-ETE, NADPH complex, resulting in the conversion of NADPH to NADP and superoxide production. The increased levels of NADP' and decreased levels of NADPH result in increased conversion of 5-HETE to 5-oxo-ETE, reduced conversion of 5-oxo-ETE to 5-HETE. and reduced conversion of 5-HETE to 5.20-diHETE. The effects of PMA are prevented by the NADPH oxidase inhibitor DPI and by heat inactivation of NADPH oxidase, but are still observed in the presence of superoxide dismutase (SOD). Production of 5-oxo-ETE is enhanced by PMS, which nonenzymatically converts NADPH to NADP, but is not affected by the superoxide generating system xanthine/xanthine oxidase (XIXO).
the effects of a superoxide generating system were also investigated. In contrast to the strong stimulatory effect of PMA on the formation of 5-oxo-ETE, addition of a mixture of xanthine and xanthine oxidase had only a small and nonsignificant effect on the formation of this substance. Thus it would appear very unlikely that the stimulatory effect of PMA is mediated by superoxide.
Another consequence of activation of NADPH oxidase by PMA in neutrophils would be conversion of NADPH to NADP+. This could have several consequences on the metabolism of 5-HETE: (i) oxidation to 5-oxo-ETE requires NADP+ (9); (ii) o-hydroxylation to 5,20-diHETE requires NADPH (40); and (iii) reduction of 5-oxo-ETE to 5s-HETE requires NADPH (9). Thus an increase in the ratio of NADP+ to NADPH would be