Abstract
The lipoxins (LX) are autacoids that act within a local inflammatory milieu to dampen neutrophil recruitment and promote resolution. 15-Hydroxyprostaglandin dehydrogenase (15-PGDH) and 15-oxoprostaglandin 13-reductase, also termed leukotriene B(4) 12-hydroxydehydrogenase (PGR/LTB(4)DH), are two enzymatic activities appreciated for their roles in the metabolism of prostaglandins and LTB(4). Here, we determined whether these oxidoreductases also catalyze the conversion of lipoxin A(4) (LXA(4)) and assessed the activities of these LXA(4) metabolites. 15-Oxo-LXA(4) was generated by incubating LXA(4) with 15-PGDH and NAD(+) for studies of its further conversion. PGR/LTB(4)DH catalyzed the NADH-dependent reduction of 15-oxo-LXA(4) to yield 13,14-dihydro-15-oxo-LXA(4). With NADH as a cofactor, 15-PGDH acted as a 15-carbonyl reductase and catalyzed the conversion of 13,14-dihydro-15-oxo-LXA(4) to 13, 14-dihydro-LXA(4). Human polymorphonuclear leukocytes (PMN) exposed to native LXA(4), 15-oxo-LXA(4), or 13,14-dihydro-LXA(4) did not produce superoxide anions. At concentrations where LXA(4) and a metabolically stable LXA(4) analog potently inhibited leukotriene B(4)-induced superoxide anion generation, the further metabolites were devoid of activity. Neither 15-oxo-LXA(4) nor 13, 14-dihydro-LXA(4) effectively competed with (3)H-labeled LXA(4) for specific binding to recombinant LXA(4) receptor (ALXR). In addition, introducing recombinant PGR/LTB(4)DH into a murine exudative model of inflammation increased PMN number by approximately 2-fold, suggesting that this enzyme participates in the regulation of PMN trafficking. These results establish the structures of LXA(4) further metabolites and indicate that conversion of LXA(4) to oxo- and dihydro- products represents a mode of LXA(4) inactivation in inflammation. Moreover, they suggest that these eicosanoid oxidoreductases have multifaceted roles controlling the levels of specific eicosanoids involved in the regulation of inflammation.
Highlights
The lipoxins (LX) are autacoids that act within a local inflammatory milieu to dampen neutrophil recruitment and promote resolution. 15-Hydroxyprostaglandin dehydrogenase (15-PGDH) and 15-oxoprostaglandin 13-reductase, termed leukotriene B4 12-hydroxydehydrogenase (PGR/LTB4DH), are two enzymatic activities appreciated for their roles in the metabolism of prostaglandins and Leukotriene B4 (LTB4)
Consistent with earlier findings [5], LXA4 was converted to 15-oxo-LXA4 upon incubation with recombinant human 15-PGDH and NADϩ. 15-OxoLXA4 was produced to assess the role of oxidoreductases in its conversion to oxo- and dihydro-LXA4 products, and its mass spectra (MS)/MS spectrum is shown for direct comparison to those of the further metabolites (Fig. 2). 15-Oxo-LXA4 had a retention time of 11.6 min and a molecular anion of 349.5 atomic mass units ([M Ϫ H]Ϫ ϭ m/z 349.5) (Fig. 1)
The 2 atomic mass units increase in mass corresponded to the addition of hydrogen across the 13,14-double bond of 15-oxo-LXA4. 13,14-Dihydro-15-oxo-LXA4 was isolated and incubated with 15-PGDH, with NADH as cofactor
Summary
The lipoxins (LX) are autacoids that act within a local inflammatory milieu to dampen neutrophil recruitment and promote resolution. 15-Hydroxyprostaglandin dehydrogenase (15-PGDH) and 15-oxoprostaglandin 13-reductase, termed leukotriene B4 12-hydroxydehydrogenase (PGR/LTB4DH), are two enzymatic activities appreciated for their roles in the metabolism of prostaglandins and LTB4. To determine whether the metabolites of LXA4 induce superoxide anion generation, LTB4, LXA4, 15-oxo-LXA4, or 13,14dihydro-LXA4 (100 nM) was added to the cell suspensions, incubated for 10 min, stopped with placement into an ice bath, and the absorbance spectrum measured.
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