Modulation of alveolar macrophage-derived 5-lipoxygenase products by the sulfhydryl reactant, N-ethylmaleimide.

M Peters-Golden,C Shelly

doi:10.1016/s0021-9258(18)61004-x

Abstract

The sulfhydryl reactant N-ethylmaleimide (NEM) stimulates the release and cyclooxygenase metabolism of arachidonic acid in rat alveolar macrophages. Because both 5-lipoxygenation and leukotriene (LT) C4 synthesis represent sulfhydryl-dependent steps in the 5-lipoxygenase pathway, we examined the effect of NEM on 5-lipoxygenase, as well as cyclooxygenase, metabolism in resting and agonist-stimulated cells by reverse-phase high performance liquid chromatography and radioimmunoassay. NEM at 5-10 microM stimulated the synthesis of thromboxane, but not prostaglandin E2 or the 5-lipoxygenase products LTC4, LTB4, or 5-hydroxyeicosatetraenoic acid from endogenously released arachidonate. In the presence of exogenous fatty acid, however, NEM stimulated the synthesis of large quantities of LTB4. The effect of NEM on arachidonate metabolism stimulated by the calcium ionophore A23187 and the particulate zymosan was also investigated. NEM augmented arachidonate release and thromboxane synthesis stimulated by A23187 but inhibited A23187-induced LTC4 synthesis with an IC50 of approximately 4.3 microM. This inhibitory effect closely paralleled the ability of NEM to deplete intracellular glutathione (IC50 approximately 4.3 microM). Preincubation with the intracellular cysteine delivery agent L-2-oxothiazolidine-4-carboxylate augmented intracellular glutathione concentration and A23187-stimulated LTC4 synthesis and attenuated the capacity of NEM to deplete glutathione and inhibit LTC4 synthesis. While LTB4 and 5-hydroxyeicosatetraenoic synthesis were unaffected at these low NEM concentrations, LTB4 synthesis was inhibited at high concentrations (IC50 approximately 210 microM). Zymosan-induced eicosanoid synthesis was modulated by NEM in a similar fashion. Thus, NEM is an agonist of arachidonate metabolism with the capacity to modulate the spectrum of macrophage-derived eicosanoids by virtue of specific biochemical interactions with substrates and enzymes of the 5-lipoxygenase pathway.

Highlights

NEM on 5-lipoxygenaseYas well as cyclooxygenase, examine SH reactant-induced arachidonate metabolism bemetabolism in resting and agonist-stimulated cells by cause, as the major resident effector cell in the distal lung[4], rpld5leae-hiatnvhyeseydeErdadsrznetoodh-axrperyrhaaetacdhishsyiceeoion5dist-hmohlaiintgempaeshootuitexsfnrp.Iytaoenhgearrefnsoonostmhraaimysecpbae.roaceNixnpdsacrEenoenMfedrcl,ouieqamcobuttfsui5deLet-xnTc1ondCh0goor,egt,oMneLmopnMTuaroBostsuots,fsi,gtmalayrrtgoauetly---ran-dosictifoezmnieastsgeoeolxnnapnstroigtrsrseetaesltdaqet(udti6voa)ena.irtItniaonthcidehaoesliteedhdodoef,nretGioiicncarxoucfilsinnadasdmn,froeemiilssdeatsaatesoninerndryairccnecehdsolepl-msdwoan(eor5itsarnae)kcb,ehaotrionlsdidsoa(mn3sviyc)ainnhraitaehcrvtiaeyedtacid, NEM stimulated the synthesisof large AMs stimulated with acrolein, a toxic component of smoke, quantities of LTB4
Failure of NEM to stimulate the synthesis of 5-lipoxygenase products was further demonstrated by high performance liquid chromatography (HPLC) assessment of Eicosanoid Synthesis Induced by NEM-Preliminary experiments indicated that arachidonate release and TxBz synthesis by AMs were reliably seen at NEM concentrations 25 p ~co,nfirming published observations [3]
+ Zymosan 10 p~ NEM 970 f 190 340 2 76 4 f 3 101f 22 one Levels and LTC, Synthesis-The above experiments investigating the effect of NEMonglutathionecontentand LTC, synthesis were performed using simultaneous incubations of cultures withA23187 and NEM.T o examine the time iments showing thatdespitethe presence of NEM,OTC course and reversibility of its effects, AMs were pretreated pretreatment abrogated the ability of NEM to deplete glutawith 10 p~ NEM for 5 or 15 min, the media removed, and thione and inhibitLTC, synthesis

Summary

MATERIAL SA N DM ETHODS

Wistar rats were obtained from Charles River Breeding Laboratories, Inc, (Wilmington, MA) and housed under specific pathogen-free conditions. The specificities of the LTB, and LTC, RIAs were validated in our laboratory by measuring immunoreactive LTB, and LTC, in HPLC eluate fractions corresponding to the solvent front, each cyclooxygenase and lipoxygenase metabolite, and free arachidonate followingthe injection of a mixture of all authentic standards. This analysis revealed that 95% of the immunoreactive LTC, measurable in all fractions was found in the LTC, plus LTD, fractions, and 100% of the immunoreactive LTB, measured in all fractions was found in the LTB, fraction.

RESULTS

Capacity of NEM to Interfere with LT Detection in Culture

Immunoreactive eicosanoid productwn stimulated by NEM

Findings

DISCUSSION