THE JOURNAL OF BIOLOGICAL CHEMISTRY

Printed in U. S.A. Vol. 257, No. 24, Issue of December 25, pp. 14937-14943,1982

Role of Glutathione Peroxidase and Hexose Monophosphate Shunt in the Platelet Lipoxygenase Pathway*

(Received for publication, June 21, 1982)

Robert W. Bryant, Theodore C. Simon, and J. Martyn Bailey From the George Washington University Medical School, Washington, D. C. 20037

The primary lipoxygenase products of arachidonate metabolism are hydroperoxyeicosatetraenoic acids (HPETEs). The major route for HPETE metabolism in most cells is by enzymatic reduction to hydroxyeicos- atetraenoic acids (HETEs), utilizing an external reduc- tant. In this paper the nature of the external reductive step in the human platelet lipoxygenase pathway is analyzed and the role of the enzyme glutathione per- oxidase (glutathione:Hz02 oxidoreductase, EC 1.11.1.9) (GSH peroxidase) in the formation of 12-HETE is char- acterized.

Arachidonate (33 m ~ ) stimulated l4COZ release and glucose metabolism 7- to 10-fold above control values in human platelets incubated with [l-’4C]glucose. Stim- ulation was dependent upon arachidonate metabolism since l4COZ production was inhibited 90% by 5,8,11,14- eicosatetraynoic acid (10 PM), an inhibitor of both the platelet lipoxygenase and cyclooxygenase enzymes. Glucose metabolism was not stimulated by 11,14-eico- sadienoic acid, which i s a poor substrate for the platelet oxygenases. Arachidonate-dependent I4CO2 release was inhibited 40-70% by 15-HETE (30 p ~ ) , a selective platelet lipoxygenase inhibitor. Approximately 2 mol of 12-[14C]HETE were formed per mol of 14C02 pro- duced. This stoichiometry is in accord with the forma- tion via the hexose monophosphate shunt of 2 mol of NADPH, which are then utilized via GSH reductase and GSH peroxidase to reduce 2 mol of 12-HPETE. In accordance with this scheme, platelets depleted

70-80% in GSH by pretreatment with azodicarboxylic acid bis(dimethy1amide) (Diamide) (0.3-1.0 m ~ ) or di- ethyl maleate (1.0-3.0 m ~ ) released substantial amounts of the un reduced product, 12-HPETE. Pro- duction of 12-HPETE from arachidonate was barely detectable in untreated platelets. In addition, treated platelets accumulated considerable amounts of the 12- HPETE rearrangement products 8,9,12- and 8,11,12- trihydroxyeicosatrienoic acids and 10-hydroxy-11,12- epoxy-5,8,14-eicosatrienoic acid.

These studies indicate that metabolism of arachi- donic acid by the platelet lipoxygenase pathway is tightly coupled through GSH peroxidase to glucose metabolism via the hexose monophosphate shunt. Dis- tribution of products from lipoxygenase derived HPETEs between hydroperoxy rearrangement or re- ductive pathways can thus be regulated by the availa-

* This work was supported by United States Public Health Service, National Institutes of Health Grant HL 25645 and United States Department of Agriculture Grant 59-2113-0-1-487-0. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer-

this fact. tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate

This paper is dedicated to Professor Samuel M. Rapoport of the Humboldt University, Berlin, GDR, on the occasion of his seventieth birthday.

bility of reducing equivalents from the hexose mono- phosphate shunt.

Lipoxygenases which convert arachidonic acid to various isomeric hydroperoxyeicosatetraenoic acids have been iden- tified in a number of cells. In general HPETEs’ do not normally seem to be released as such by cells but are further metabolized to products such as trihydroxyeicosatrienoic acids (1,2), epoxyhydroxyeicosatrienoic acids (3,4), and the bioac- tive leukotrienes (5, 6). Another major route of HPETE metahohm is reduction to hydroxyeicosatetraenoic acids. The nature of this peroxidase step in intact cells has not previously been elucidated, although indirect evidence has suggested a role for glutathione peroxidase (EC 1.11.1.9) in this step (2,4, 7). Glutathione peroxidase has been known for some time to be capable of reducing organic and fatty acid hydroperoxides, as well as hydrogen peroxide, at the expense of reduced glutathione cosubstrate (8, 9). Despite the suggestion in 1973 by Nugteren and Hazelhof (10) that GSH peroxidase might play a role in conversion of PGGz to PGHz, direct experimental evidence supporting this hypothesis has not yet been pre- sented. GSH peroxidase would appear to be weLl suited for the reduction of hydroperoxides of arachidonic acid on theo- retical grounds since the enzyme is assured of an adequate supply of reduced glutathione via the efficient, concerted operation of glutathione reductase and enzymes of the HMP shunt. Coupling of GSH peroxidase and the HMP shunt for reduction of hydroperoxides is well established in erythrocytes (11) and liver cells (12). This paper presents data which demonstrates that glutathione peroxidase and the supporting hexose monophosphate shunt are directly involved in the reduction of 12-HPETE to 12-HETE in the platelet lipoxy- genase pathway. Furthermore, impairment of this lipoxygen- ase-glutathione peroxidase couple in platelets can result in the formation of additional lipoxygenase metabolites of arachi- donic acid.

EXPERIMENTAL PROCEDURES

Materials [l-’4C]Arachidonic acid (58 mCi/mmol), [l-’4C]glucose (60 mCi/

mmol), and NaHI4CO3 (0.1 mCi/mmol) were obtained from Amer- sham Cop. [6-“C]Glucose (56 mCi/mmol) was obtained from New

The abbreviations used are: HPETE, hydroperoxyeicosatetrae- noic acid; HETE, hydroxyeicosatetroinoic acid; a-HEPA, hydroxye- poxyeicosatrienoic acid; THETE, trihydroxyeicosatrienoic acid; GSH, reduced glutathione; GSSG, oxidized glutathione; HMP. hexose monophosphate; ETYA, 5,8,11,14-eicosatetraynoic acid; ICm, concen- tration of 50% inhibition; AA, arachidonic acid; HHT, 12-hydroxy- 5,8,lO-heptadecatrienoic acid; TXB2, thromboxane B2; HPHT, 12- hydroperoxy-5,8,10-heptadecatrienoic acid; HPLC, high performance liquid chromatography; GC-MS, gas chromatogaphy-mass spectrom- etry; ME, methyl ester; Measi, trimethylsilyl.

14937

14938 Lipoxygenase:Glutathione Peroxidase Couple

England Nuclear. Antimycin AI, Diamide (azodicarboxylic acid biddimethylamide)), diethyl maleate, glutathione reductase (yeast), and NADPH were purchased from Sigma. 5,5"Dithionitrobenzoic acid was supplied by Fisher. Ethyl ether and methylene chloride were of American Chemical Society certified grade (Fisher). Ethyl ether was redistilled immediately before use. 15-HPETE, 15-HETE, and 12-HPETE were prepared as described (13).

Methods Metabolism of [ll-14C]Glucose by Washed Platelets-Platelets

from healthy volunteers were obtained and washed according to Holmsen and Robkin (14). Washed platelets were resuspended at a final concentration of 3-4 X lo* platelets/ml in a modified Krebs- Henseleit buffer without calcium (15) containing 1 m glucose and antimycin AI (1 pg/ml). Following addition of [l-14C]glucose (0.5 pCi/ ml), platelet suspension aliquots (0.75 ml) were placed in 10-ml siliconized Erlenmeyer flasks fitted with rubber septa closures and hanging well cups (Kontes, Vineland, NJ). The flasks were gassed with 5% CO, in air, closed, and placed in a shaking water bath (120 strokes/min, 37 "C).

The I4COp released via metabolism of [l-14C]glucose was trapped by adding 0.06 ml of 1 M citric acid to the suspension (pH 3.4-3.6) and 0.3 ml of Protosol (New England Nuclear) to the center well. The flasks were further incubated (37 "C 120 strokes/min) for 90 min. The 14C0, trapped in the center well cup was determined in Econofluor (New England Nuclear) scintillant using glass vials. Recovery of NaH14C03 from buffer in this procedure was at least 95%. The metabolism of [l-14C]glucose by unstimulated platelets, measured by release of I4CO2 was constant over an incubation period of 50 min and had a rate of approximately 650 dpm/l0 min/3 X 10' platelets. Stimulants of glucose metabolism such as t-butyl hydroperoxide or arachidonic acid were added in ethanol after the platelets had been incubated at 37 "C as described above for approximately 30 min. Ethanol at a final concentration less than 0.35% did not affect release of I4CO2 from [l-14C]glucose. All assays were conducted in triplicate.

Isolation of [l-'4CjArachidonic Acid Metabolites from Incuba- tions with [l-14C]Glucose-For the simultaneous measurement of [l- 14C]glucose and [l-14C]arachidonic acid, platelets were incubated and acidified to trap I4CO2 as described above. The incubation solutions were then extracted with 6 volumes of a chloroform:methanol mixture (2:1, v/v). The chloroform layer was dried with anhydrous magnesium sulfate and treated with ethereal diazomethane to convert fatty acid metabolites to methyl esters. The methylated extract was chromato- graphed on 250 nm Silica Gel G hard layer plates (Analtech, Newark, DE) f i s t in the solvent ethyl acetate:isooctane:water (7575:100, upper phase) to a distance of about 15 cm, then redeveloped in water- saturated ethyl acetate to a distance of 6 cm. This double development procedure allowed complete separation of AA-ME (RF 0.73), 12HETE-ME (RF -0.64), HHT-ME (RF 0.59), and TXBz-ME ( R F 0.13). "C-labeled metabolites were located by autoradiography and quantitated by liquid scintillation counting (16). The recovery of these metabolites through the complete incubation and extraction proce- dure was greater than 90%. Cross-contamination of the I4CO2 assay by [l-'4C]arachidonic acid and the I4C-metabolites assay by [1-I4C] glucose was negligible.

Isolation of 12[14C]HPETE and Further Lipoxygenase Metabo- lites from PZatelets-Washed platelets resuspended in buffer contain- ing 5 mM glucose were treated with Diamide according to Kosower et al. (17) with modifications. Platelet suspensions were incubated at 0 "C for 5 to 10 min with Diamide (0.01 to 1 mM) then incubated further for 10 min at 37 "C. Platelet suspensions were incubated with diethyl maleate (0.03 to 3 m for 30 min at 37 "C (18)). Platelets so treated were then incubated with [l-14C]arachidonic acid at 37 "C. Reactions were stopped by addition of 3 volumes of the cold (-20 "C) solvent mixture ethyl ether:methanol:l M citric acid (135:15:1) which contained 5 ppm of butylated hydroxytoluene (19). After removal of the organic layer, the aqueous layer was re-extracted with 2 volumes of ethyl ether (-20 "C). At this point 15-HPETE, 15-HETE, and AA (10 pg each) were added to the combined ether extract to serve as internal standards. The extracts were back washed one time with water, dried with anhydrous sodium sulfate, and treated with ethereal diazomethane. Alternatively the platelet incubations were extracted with 6 volumes of cold chloroform:methanol (2:l). The chloroform extract was dried (Na2S04), evaporated, and methylated. The samples were reduced in volume to approximately 50 to 100 pl, applied to cellulose powder preadsorbent Silica Gel G thin layer (250 nm) plates, and developed in the solvent system hexane:ethyl ether (7030). With this system AA-ME (RF 0.82). 12- and 15-HPETE-ME ( R ~ 0 . 4 6 ) , and

12- and 15-HETE-ME ( RF 0.28) were well separated. High perform- ance liquid chromatography for identification of 12-HPETE and its metabolites was conducted as previously described (13). For GC-MS identification of these compounds in HPLC fractions, the fractions were evaporated and the metabolites were redissolved in methanol (0.04 ml) containing NaBK (1 mg/ml) and kept at 0 "C for 15 min in order to reduce any hydroperoxides present to the respective alcohols. The methanolic solutions were diluted with water (0.4 ml), acidified with formic acid, and the metabolites were extracted with ethyl acetate. The metabolites were analyzed by GC-MS as methyl ester, trimethyl silyl ether derivatives on a 1% SE-30 column packing as previous described (20).

Determination of Platelet Glutathione Content-Platelet glutathi- one content was measured by the glutathione reductase-5,5"dithio- nitrobenzoic acid cycling method of Tietz (21) for total glutathione, i.e. oxidized plus reduced forms, and by the method of Griffith (22) for oxidized glutathione. For glutathione determinations, platelet suspensions (1 ml) were extracted by addition of 1 ml of methylene chloride and 0.2 ml of 0.2 M acetic acid to achieve pH 5.0. The samples were vortexed and centrifuged. The upper aqueous layer was removed and assayed for total and oxidized glutathione. The addition of methylene chloride served to lyse the platelets and remove the sulfhydryl reagents Diamide or diethyl maleate from the platelet glutathione extract. These reagents otherwise interfered with the glutathione assay. When diamide and diethyl maleate were incubated with platelets as described above, generally greater than 90% of the measured total glutathione was present as the reduced form.

RESULTS

Arachidonic Acid Stimulation of the HMP Shunt-Addi- tion of t-butyl hydroperoxide to platelets incubated in the presence of [l-'4C]glucose greatly stimulated 14C02 release (Table I).' No stimulation of I4CO2 release was observed with t-butyl hydroperoxide when platelets were incubated in the presence of [6-'4C]glucose (data not shown). Thus the hydro- peroxide enhancement of glucose oxidation in platelets rep- resents classical glutathione peroxide-mediated stimulation of the hexose monophosphate shunt (11): It was further found that addition of arachidonic acid to platelets also stimulated glucose metabolism via the HMP shunt approximately 10-fold above resting levels (Table I). This stimulation was not due to a fatty acid or detergent effect since another polyunsatu- rated fatty acid, 11,14-eicosadienoic acid, did not stimulate the HMP shunt in platelets (data not shown). This fatty acid is a poor substrate for both the prostaglandin cyclooxygenase and platelet lipoxygenase enzymes (23, 24).

A significant portion of the arachidonate-mediated stimula- tion of the HMP shunt was dependent on its metabolism via the platelet lipoxygenase pathway. Table I1 shows the effect on arachidonate stimulation of the HMP shunt of several inhibitors of arachidonic acid metabolism. 5,8,11,14-Eicosa- tetrynoic acid, at a concentration in which both platelet lipoxygenase and cyclooxygenase activities are blocked (25), prevented arachidonate stimulation of the shunt by approxi- mately 90% while it had only a slight inhibitory effect on t- butyl hydroperoxide stimulation of the HMP shunt. 15-Hy- droxyeicosatetraenoic acid, a specific platelet lipoxygenase inhibitor (16) at a concentration which inhibited 12-HETE formation by more than 90%, reduced arachidonate stimula- tion of the HMP shunt by over 50%, on the average, without affecting t-butyl hydroperoxide stimulation of the HMP shunt. 15-HETE did, however, produce a modest but consistent inhibition (15%) of background, unstimulated, metabolism of glucose via the HMP shunt. Finally indomethacin, at a con- centration which typically reduced cyclooxygenase activity by

Note that resting glucose metabolism was suppressed in these experiments with the respiratory inhibitor antimycin AI.

A preliminary account of the stimulation of the platelet HMP shunt by t-butyl and other hydroperoxides has appeared (Holmsen, H., Robkin, L., and Driver, H. (1979) Fed. Proc. 38,826).

Lipoxygenase:Glutathione Peroxidase Couple 14939

TABLE 1 Stimulation ofplatelet hexose monophosphate shunt by t-butyl

hydroperoxide and arachidonic acid Washed platelet suspensions (2.3-3.0 X 10' platelets in 0.75 d)

were preincubated (in triplicate) a t 37 "C for 30 min in presence of [l-'4C]glucose (1 m ~ , 1 pCi/pmol) and antimycin A, (1 p g / d ) and for an additional 12 min with the stimulants (added in 1.5 pl of ethanol). 14C02 was trapped as described under "Experimental Procedures."

Stimulants Release of "CO? from [l-"C] glucose

dpm/l2 min/3 x 10Rplatelets Ethanol control 780 f 140" '(4) tButyl hydroperoxide (389 p ~ ) 39,600 f 11,400 (4) Arachidonic acid (33 @I) 7,100 f 1,550 (4)

a Mean f P.D. (number of experiments is in parentheses).

TABLE I1 Percentage of inhibition of arachidonic stimulation of HMP shunt

by platelet lipoxygenase inhibitors Washed platelets were incubated in the presence of [l-14C]glucose

and antimycin A as described in Table I. Platelets were further incubated for 15 min with either ethanol vehicle (1 pl) or the following inhibitors: ETYA (10 PM), 15-HETE (30 p ~ ) , and indomethacin (0.5 p ~ ) . Glucose metabolism was then stimulated by addition of either ethanol vehicle (unstimulated control), t-butyl hydroperoxide (150 p ~ ) , or arachidonic acid (32 p ~ ) as described in Table I. The data is expressed as percentage of inhibition of stimulated glucose metabo- lism with respect to stimulated incubations which were not treated with inhibitors.

Stimulant Inhibitor

ETYA 15-HETE Indomethacin

Ethanol control 7 r 7 (2)" 16 f 1 (2) -3 f 2 (2) t-Butyl hydroperox- 10 f 4 (2) 4 f 4 (4) 3 f 4 (4)

Arachidonic acid 89 r 3 (2) 54 f 18 (8) 13 f 10 (6) ide

Mean f S.D. (number of separate experiments is in parentheses).

greater than 60%, only slightly decreased arachidonate-stim- ulated HMP shunt metabolism of glucose.

In order to relate further the lipoxygenase pathway and arachidonic stimulation of glucose oxidation via the HMP shunt, platelet metabolism of [l-*4C]arachidonic acid and [l- 14C]glucose were simultaneously measured in the presence of various concentrations of 15-HETE. As shown in Fig. 1, inhi- bition of 12-HETE formation and glucose metabolism both occurred between 10 and 25 ~ L M in a near parallel fashion with concentrations for 50% inhibition of 20 p~ and 24 p ~ , respec- tively. In a second experiment inhibition of 12-HETE forma- tion and glucose metabolism occurred between 5 and 20 PM with ICs of 14 and 12 PM, respectively. The platelet cycloox- ygenase pathway was not inhibited by 15-HETE as previously reported (16). Thus there was a close association between the flux of arachidonate through the platelet lipoxygenase path- way and stimulation of glucose metabolism via the HMP shunt. In addition a stoichiometric relationship was observed between the amount of 12-HETE formed and the amount of glucose metabolized via the HMP shunt. Table I11 shows the results of four separate experiments comparing the amount of 12-HETE formed from added arachidonic acid and the amount of glucose metabolized as a result of arachidonate lipoxygenase metabolism. It should be noted that at present these ratios are estimates based upon the assumption that the added [l-'4C]arachidonate and [l-'4C]glucose were not signif- icantly diluted by unlabeled material in the cells. Dilution of exogenous arachidonate (26) via phospholipase activity or glucose via phosphorylase (27) may contribute to the variation of the 12-HETE to COZ ratio between the experiments. The average molar ratio of 12-HETE formation to Con released for the four experiments is 2.4 & 0.4. This ratio is in accord

with the conversion of 1 molecule of glucose 6-phosphate to ribulose 5-phosphate and COz with the concomitant produc- tion of 2 molecules of NADPH which in turn we propose are used for the reduction of 2 molecules of 12-HPETE to 12- HETE via glutathione reductase and glutathione peroxidase as indicated in Fig. 2.

Platelet Glutathione and 12-HPETE Release-The role of GSH peroxidase in platelet reduction of 12-HPETE to 12- HETE was further investigated with the agents Diamide and diethyl maleate, both of which have been reported to lower the level of reduced glutathione in cells (7, 18). Pretreatment

1 - I T T

-4 , I2 HETE

J 0 1 I I I I I

0 5 10 1 5 20 2 5

[15 HETElvM

FIG. 1. Dose-response curves for inhibition of arachidonate lipoxygenase metabolism and arachidonate-stimulated glu- cose metabolism via the HMP shunt. Washed platelets, suspended in buffer containing antimycin A1 (1 p g / d ) and [l-14C]glucose (1 mM, 0.5 pCi/pmol), were pretreated with various concentrations of 15- HETE for 15 min and then incubated for 15 min with [1-l4C]arachi- donic acid (33 p ~ , 6 pCi/pmol). I4CO2 and ["CI-arachidonate metab- olites were isolated and quantitatively measured as described under "Experimental Procedures." Vertical bars represent 1 standard de- viation.

TABLE 111 Stoichioemetric relationship between 12-HETE formation and

glucose metabolism via the HMP shunt - Experiment 12-HETE formed CO, released from Molar ratio of 12-

from AA" ehcoseb HETE to CO,, ~- nmol/incubation

1 6.11 3.00 2.03 2 8.27 3.04 2.72 3 13.16 4.60 2.87 4 3.64 1.74 2.09

Calculated from the difference in 12-[14C]HETE formed from [I4C]AA in the presence and absence of the lipoxygenase inhibitor 15- HETE.

*Calculated from the difference in 14C02 released from [1-'4C] glucose in the presence and absence of 15-HETE.

/"" % GLUCOSE-6-POa LIPOXYGENASE

i NADP \ /2 GSH, f 12-HPETE

HMP SHUNT GLUTATHIONE GLUTATHIONE

REDUCTASE PEROXIDASE

Yz RIBULOSE-5-POa I

% CO? 1 2-HETE

FIG. 2. The 1ipoxygenase:glutathione peroxidose couple. This diagram illustrates the dependence of the platelet lipoxygenase pathway on glutathione peroxidase and the hexose monophosphate shunt for enzymatic reduction of 12-HPETE to 12-HETE.

14940 Lipoxygenase:Glutathione Peroxidase Couple

of washed platelets with 0.3 m~ Diamide followed by incu- bation with AA resulted in the accumulation of 12-HPETE (Fig. 3). This metabolite was positively identified as 12- HPETE based upon the facts that it co-migrated on HPLC with authentic 12-HPETE (13) and was chemically reduced to 12-HETE (see below). Platelets not treated with Diamide or those treated with only 0.1 m~ Diamide (Fig. 3) did not produce detectable amounts of 12-HPETE. The virtual ab- sence of 12-HPETE in untreated control platelets was also observed in short term incubations (15-90 s) where a transient accumulation of 12-HPETE might have been expected. With 0.3 mM Diamide pretreatment, there was a nearly linear increase in 12-HPETE accumulation over a 5-min incubation period following arachidonate addition (data not shown).

Treatment of platelets with Diamide as described under "Experimental Procedures" resulted in a dose-responsive de- crease in platelet total glutathione content (oxidized plus reduced) as shown in Fig. 4A. Treatment of platelets with diethyl maleate also resulted in a dose-responsive decline in platelet glutathione (Fig. 4 B ) . Depletion of platelet glutathi- one with either Diamide or diethyl maleate had a complex effect on lipoxygenase metabolism of added arachidonic acid as also shown in Fig. 4. At lower concentrations of the sulfhy- dryl reagents, the initial decline of platelet glutathione was associated with an increased conversion of added arachidonic acid to 12-HETE. At higher concentrations of either Diamide (1 mM) or diethyl maleate (3 mM) synthesis of 12-HETE was decreased by greater than 50%. The most striking effect of glutathione depletion on the lipoxygenase pathway, however, was the formation of significant amounts of 12-HPETE. This occurred when the platelet glutathione pool was reduced to a level which was 10-30% of control levels. That 12-HPETE was in fact released by intact, although glutathione-deficient, platelets was shown by experiments measuring both platelet lysis by release of lactate dehydrogenase and distribution of 12-HPETE in the incubations after centrifugation. Incubation of platelets with Diamide (0.3 mM) and arachidonic acid (33 PM), resulted in approximately the same, rather minimal de- gree of cell lysis (5-lo%), as occurred in incubations with arachidonic acid alone (four experiments). The 12-HPETE formed in those incubations was found primarily in the super- natant fraction (60-80% of total 12-HPETE recovered). These

DIAMIDE CONC (mM)

0 0 0 1 0 3

SOLVENT FRONT-

12-HPETE-ME - 12.HETE-ME-

HHT.ME - \

- onlol\l-

f- "" 7 - _e. . .. - FIG. 3. Stimulation of 12-HPEI'E formation from added ar-

achidonic acid by Diamide. Washed platelets were treated with Diamide for 5 min at 0 "C and then maintained at 37 "C for 5 min. Platelets were incubated with ['4C]arachidonate (33 VM) for 5 min and the metabolites were extracted, methylated, and separated by thin layer chromatography (petroleum ether:ethyl ether, 7030) as de- scribed under "Experimental Procedures."

A T T

\ \

\

GSH " '0

I I 1 1 0 0.1 0.5 1 .o

[DIAMIDE] rnM

0 0 3 1 2 3 [DIETHYLMALEATE] mM

FIG. 4. Stimulation of 12-HPETE formation associated with diminished glutathione levels in platelets treated with either Diamide or diethyl maleate. A, platelets were treated in duplicate with various concentrations of Diamide as describe in Fig. 3. Portions of each sample were either assayed for glutathione as described under "Experimental Procedures" of incubated with [l-"C] arachidonic acid (33 p ~ , 6 pCi/pmol) as described for Fig. 3. B, platelets were pretreated with various concentrations of diethyl maleate for 30 min and portions were either assayed for glutathione or incubated with [I-''Clarachi- donate (33 PM, 6 pCi/pmol) for 5 min.

results thus indicate that glutathione is required for conver- sion of 12-HPETE to 12-HETE in platelets and furthermore that depletion of glutathione results in release of 12-HPETE in platelets incubated with arachidonic acid.

Platelets depleted of glutathione with either Diamide or diethyl maleate accumulated small amounts of other arachi- donate metabolites in addition to 12-HPETE, 12-HETE, HHT, and TXB2. Fig. 5 shows that HPLC isolation of ["C] arachidonate metabolites from a control incubation (Fig. 5A) and an incubation with Diamide-treated platelets. The pres- ence of 12-HETE and HHT in the control incubations was confirmed by GC-MS4 The compounds isolated in the Diam-

' TXBP eluted in this HPLC system at approximately 60-70 min and is not shown in Fig. 5.

Lip0xygenase:Glutathione Peroxidase Couple 14941

ide-treated incubations were as follows. Fraction 1 was iden- tified as 12-HPETE. The major A 2 3 5 peak in Fraction 1 had the same retention time (11.6 min) as authentic 12-HPETE. Chemical reduction (NaBH4) of Fraction 1 yielded a com-

25- A 12-HETE

i

10 20 30 40

12-HETE

{ A

- I I I I I 10 20 30 40

FIG. 5. HPLC isolation of arachidonate metabolites from platelets pretreated with Diamide. Suspensions (5 ml) of washed human platelets were pretreated with either buffer (A) or with Diamide (0.3 m) ( B ) as described under "Experimental Procedures" and then incubated with [l-'4C]arachidonic acid (50 pg, 0.5 pCi) for 5 min at 37 "C. The metabolites were extracted as described under "Experimental Procedures." The metabolites, as free acids, were chromatographed on a silicic acid column (Si-100,5-10 p particle size, packed by Alltech Associates, Deerfield, IL, 1.0 cm inner diameter X 25 cm). Solvent A was hexane:acetic acid (999:l) and Solvent B was hexane:2-propanolacetic acid (899100:1). The flow rate was 4 ml/min and the absorbance at 254 nm and 235 nm was continuously monitored (this data not shown). Fractions (2.6 ml) were collected for measure- ment of radioactivity (0.20-ml aliquots taken) and metabolite identi- fication by GC-MS as described under "Experimental Procedures."

ELUTIONTIME (MINI

pound identified by GC-MS (methyl ester MesSi ether deriv- ative, equivalent chain length 21.4) as 12-HETE. Fraction 2 was tentatively identified as 12-hydroperoxy-8,10,14-hepta- decatrienoic acid based upon its absorbance at 235 nm and its reduction to a compound identified by GC-MS as HHT (chain length 19.4). The formation of HPHT was blocked by indo- methacin and is thus derived via the cyclooxygenase pathway. Fraction C was identified as a mixture (approximately 21) of HHT and 10-hydroxy-11,12-epoxy-5,8,14-eicosatrienoic acid (a-HEPA, an a-hydroxyepoxy acid). The latter lipoxygenase metabolite gave a mass spectrum (methyl ester, trimethylsilyl ether derivative, chain length 22.0) with characteristic ions (M/z , relative abundance and probable mode of origin in parentheses ) at 407 (0.2, "15, loss of CH3), 391 (0.5, "31, loss of -OCH3), 332 (0.1, "90, loss of MeaSiOH), 311 (0.8, "111, loss of CH~-(CHZ)~-CH=CH-CH~*), 282 (2.4, M- 140, loss Of CHr-(CH2)-CH=CH-CH2CHO), 269 (16.7 . +(Me~Si-O-CH(CH2-CH=CH)~-(CH~)~-COOCH3), 221 (6.5), 203 (4.4), and 73 (loo), in agreement with previous data (3, 4). The presence of a l0,ll-epoxy group in a-HEPA was c o n f i e d by opening the epoxide ring and subsequently forming a trihydroxy fatty acid. a-HEPA was f is t treated with glacial acetic acid at 120 "C for 2 h (28). The resultant adduct, an acetyldihydroxy fatty acid (not isolated) was then hydrolyzed in 0.5 M NaOH to yield a product identified as 10,11,12-trihydroxy-5,8,14-eicosatrienate. The mass spectrum of this trihydroxy fatty acid (methyl ester tris-Me3Si deriva- tive, chain length 23.9) (Fig. 6) had prominent ions at 494 ( M -

CH~-(CHZ)~-CH=CH-CHZ-CHO, by rearrangement),

(CHZ)~CH=CH-CHZ-CH-OS~M~~.), 315 ("269, loss of Me&3i-O-CH-(CH2-CH=CH)2-(CH2)3-COOCH3-),

SiMea)), 129 (base ion), and 73. Fraction D was identified predominantly as a mixture approximately 1:l of the lipoxy- genase-derived isomeric trihydroxy fatty acids 8,9(11),124rih- ydroxy-5,10(9),14-eicosatrenoic acid (8,9,12-THETE and 8,- 11,12-THETE). The methyl ester tris-Me3Si derivatives of these metabolites also co-eluted on GC (C23.8) and gave a m a s spectrum very similar to that obtained with authentic material (1,2). In particular, characteristic ions were observed at 251 and 243 for the 8,9,12-THETE derivative and at 281 and 213 for the 8,11,12-THETE derivative (1).

go), 479 ("(15 + go)), 463 ("(31 + go), 444 ("140, loss of

383 ("(31 + 180)), 371 ("213, loss of CH3-

269,225 (315-go), 213 (+(C&-(CH2)4-CH=CH-CH-O-

DISCUSSION

Oxygen-oxygen cleavage of the hydroperoxy group of HPETEs is a key initial event in the formation of the lipoxy- genase products such as leukotrienes (5) and trihydroxyeicos- atrienoic acids (2). Since the corresponding HETEs do not appear to be converted to these products, conversion of

100 129 31 5

73 2 1 3 4 3 7 1 y269 COOCH,

w u

FIG. 6 Mass spectrum of 10,11,12- $ trihydroxy-5,8,14- eicosatrienoic 2 acid (methyl ester tris-MesSi ether derivative) obtained from 10-hy- droxy-11,12-epoxy-5,8,14- eicosatri- 2 enoic acid as described in the text.

TMS

463 ("31 -90) 44 ' (f 479,M-15-90)

,494(M-90)

I T 1 , i , ) , I ! c, , , , , , , , , 450 500 550 600

MASSIAMU)

14942 Lipoxygenase:Ghtathione Peroxidase Couple

HPETEs to HETEs would appear to be one mechanism by which cells regulated the level of HPETE and thus the syn- thesis of its more highly oxygenated derivatives. The peroxi- dase activity responsible in cells for the reduction of HPETEs to HETEs has not been previously identified. Several groups working with purified preparations of the hemeprotein, pros- taglandin cyclooxygenase have identifed an associated per- oxidase activity capable of reducing either the 15-hydroperoxy version of PGEz to PGEz or 15-HPETE to 15-HETE (29,30). However, as other hemeproteins and hematin itself also ex- hibit peroxidase activity with hydroperoxy fatty acid sub- strates (31), it is unclear as to the importance relative to in vivo HPETE reduction of such general peroxidase activity. Furthermore, heme peroxidase reduction of hydroperoxides is frequently accompanied by free radical, activated oxygen side products (30).

Our results show that addition of arachidonic acid to plate- lets stimulates glucose metabolism via the HMP shunt and furthermore, that a major portion of this HMP shunt stimula- tion is dependent upon lipoxygenase metabolism of arachi- donic acid. This observation is based upon the fact that AA stimulation of the HMP shunt was strongly suppressed by the lipoxygenase inhibitors ETYA and 15-HETE. The possibility that the AA-stimulated response of the HMP shunt was due to platelet aggregation seems unlikely in view of our finding that in most subjects studied, 15-HETE (10-30 p ~ ) does not significantly inhibit aggregation of washed platelets induced by AA.5

The most reasonable explanation for these observations is that lipoxygenase formation of 12-HPETE from AA stimu- lates the shunt to restore reduced glutathione levels depleted during the GSH peroxidase-catalyzed reduction of the 12- HPETE to 12-HETE (Fig. 2). Indeed, Pescarmona et al. have shown a transient decrease in platelet GSH and NADPH levels immediately following addition of AA to platelets (32). The necessity of generating reduced glutathione pools via the HMP shunt can be appreciated when it is recognized that in incubations of platelets with moderate amounts of arachidonic acid, e.g. 30 PM, the amount of 12-HPETE formed and then reduced to 12-HETE is typically 10 times greater than avail- able reduced glutathione pool were it not regenerated.

This coupling of lipoxygenase metabolism and the HMP shunt via glutathione peroxidase reduction of 12-HPETE was also supported by the observation of a stoichiometric relation- ship of approximately 2 mol of 12-HETE formed/mol of COZ released by the HMP shunt (cfi Fig. 2).

An important consequence of the coupling of the lipoxygen- ase pathway to glutathione peroxidase is that platelets de- pleted of glutathione with either Diamide or diethyl maleate converted substantial amounts of added arachidonic acid to 12-HPETE which was released from the platelets. Under normal circumstances, e.g. adequate glutathione levels, only the reduced product 12-HETE is released.

The association of glutathione depletion in platelets with greatly enhanced conversion of arachidonic acid to 12-hydro- peroxy-5,8,10-heptadecatrienoic acid, a cyclooxygenase me- tabolite previously isolated from the incubation of PGGz with a platelet thromboxane synthase preparation (19), raises the possibility that glutathione peroxidase may also serve to re- duce PGGz to PGHz in the platelet cyclooxygenase pathway. Further work is necessary to more firmly establish a cycloox- ygenase-glutathione peroxidase couple.

Formation of 12-HPETE has been reported in broken plate- let homogenates incubated with arachidonic acid (5). This accumulation of 12-HPETE may be due to a disruption of the

A. M. Makheja and J. M. Bailey, unpublished observations.

normal coupling between the platelet 12-lipoxygenase and the glutathione peroxidase system. It has recently been reported that addition of reduced glutathione to a platelet supernatant fraction incubated with arachidonic acid suppressed formation of 12-HPETE and its more polar breakdown products (33). There was also a resultant increase in 12-HETE formation. These results were taken as support for the role of glutathione peroxidase in 12-HPETE reduction. However, since cysteine and 2-mercaptoethanol were equally effective in preventing accumulation of 12-HPETE, and since these compounds are poorly utilized by glutathione peroxidase to reduce hydroper- oxides (34), these thiols, including GSH, may be functioning to decompose 12-HPETE to 12-HETE by acting as nucleo- philes (31). These studies with broken cells thus highlight some of the difficulties in examining lipoxygenase-glutathione peroxidase coupling in platelet homogenates or with partially purified enzyme preparations (35). Our studies with intact platelets utilizing the coupling of glutathione peroxidase to the HMP shunt and depletion of cellular glutathione would appear to be another approach to study the role of glutathione peroxidase in arachidonic acid metabolism without the com- plications of nonenzymatic reactions of thiol groups with arachidonate-derived hydroperoxides.

Release of 12-HPETE by platelets would appear to be deleterious to vascular tissue as both 15- and 12-HPETEs are potent inhibitors of vascular prostacyclin synthesis (36, 37). Thus, factors such as dietary selenium deficiency (7) or hered- itary deficiency of glucose-6-phosphate dehydrogenase (38) which may result in impaired reduction of 12-HPETE in platelets may adversely alter platelet-endothelial cell inter- actions.

Depletion of platelet glutathione also resulted in the con- version of arachidonic acid to the isomeric trihydroxy fatty acids 8,9(11),12-trihydroxyeicosatrienoic acids and 10-hy- droxy-ll,12-epoxyeicosatrienoic acid. These lipoxygenase products undoubtedly arise via rearrangement of 12-HPETE based upon their analogy to a-hydroxyepoxy and trihydroxy fatty acids which have been shown to arise via the rearrange- ment of 15-HPETE (39) and 13-hydroxyperoxy-9,1l-octade- cadienoic acid (40). The mechanism, Le. enzymatic uersus nonenzymatic pathways, for conversion of 12-HPETE to THETEs and HEPA in platelets is not clear. The formation of 8,11,12-THETE but not 8,9,12-THETE in rat lung homog- enates incubated with arachidonic acid suggests a role for some degree of enzymatic involvement in that system (41).

In any event, we propose that the glutathione peroxidase system plays a key role in THETE and a-HEPA formation in platelets as shown below:

GSH- Peroxidase , 2.HETE Lipoxygenase

A A 12-HPETE c+ GSH-Peroxidase THETE

Impaired

HEPA

Our observations suggest that these compounds form from 12- HPETE under conditions of impairment of the platelet glu- tathione peroxidase system as occurs when the glutathione pool is depleted with sulfhydryl reagents such as Diamide (Fig. 5). Another example of an association between increased THETE and a-HEPA formation and impairment of platelet glutathione peroxidase system includes our observation that platelets with diminished GSH peroxidase activity, obtained from selenium-deficient rats formed larger amounts of THETEs and HEPA' from added arachidonic acid than plate- lets from selenium-supplemented animals (7). Additionally, THETE and a-HEPA formation occurring in platelets incu-

R. W. Bryant, unpublished observations.

Lipoxygenase:Glutathione Peroxidase Couple 14943

bated in glucose-free buffer were greatly suppressed by addi- tion of glucose (1 mM) (4). This effect of glucose to inhibit THETE and a-HEPA formation is most probably mediated via the role of the HMP shunt in maintaining reduced gluta- thione levels for unimpaired 12-HPETE reduction.

Glutathione peroxidase may also be involved in the lipoxy- genase pathways of other cells. Parker et al. have shown that depletion of glutathione in polymorphonuclear leukocytes with either diethyl maleate or cyclohexene-2-one greatly in- creased the conversion of added arachidonic acid to a 5- HPETE-like product and decreased conversion to 5-HETE (18). Addition of arachidonic acid to leukocytes also stimulates glucose uptake (42). It is tempting to speculate that this uptake may be partly used via the HMP shunt and GSH peroxidase for 5-HPETE reduction. It would appear that the availability of reducing equivalents from the HMP shunt for reduction of HPETE via glutathione peroxidase may influence the intracellular level of HPETE and thus their availability to the enzymes which effect rearrangement of HPETE to epoxytrienes (i.e. leukotriene A) or hydroxyepoxyenes (HEPA).

lent technical assistance. Acknowledgment-We wish to thank Patricia So0 Chan for excel-

Note Added in Proof-The importance of glutathione peroxidase in modulating the release of 12-HPETE from platelets is suggested by the recent findings of Maclouf, J., Fruteau de Laclos, B., and Borgeat, P. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 6042-6046 that 12-HPETE, generated in situ by platelets, greatly enhances the conversion of arachidonic acid to leukotriene B4 by human blood leukocytes.

REFERENCES 1. Jones, R. L., Kerry, P. J., Poyser, N. L., Walder, I. C., and Wilson,

2. Bryant, R. W., and Bailey, J. M. (1979) Prostuglandins 17,9-18 3. Walker, I. C., Jones, R. L., and Wilson, N. H. (1979) Prostaglan-

4. Bryant, R. W., and Bailey, J. M. (1980) Adu. Prostaglandin

5. Borgeat, P., and Samuelsson, B. (1979) Proc. Natl. Acad. Sci. U.

6. Murphy, R., Hammarstrom, S., and Samuelsson, B. (1979) Proc.

7. Bryant, R. W., and Bailey, J. M. (1980) Biochem. Biophys. Res.

8. Little, C., and O'Brien, P. J . (1968) Biochem. Biophys. Res.

9. Christophersen, B. 0. (1969) Biochim. Biophys. Acta 176,463-470 10. Nugteren, D. H., and Hazelhof, E. (1973) Biochim. Biophys. Acta

11. Cohen, G., and Hochstein, P. (1963) Biochemistry 2, 1420-1428 12. Sies, H., Gerstenecker, G., Menzel, H., and Flohe, L. (1972) FEBS

N. H. (1978) Prostaglandins 16,583-592

dins 18, 173-178

Thromboxane Res. 6,95-99

S. A. 76,3213-3217

Natl. Acad. Sci. U. S. A. 76,4275-4279

Commun. 92,268-276

Commun. 31,145-150

326,448-461

Lett. 27, 171-175

13. Bryant, R. W., Bailey, J. M., Schewe, T., and Rapoport, S. M.

14. Holmsen, H., and Robkin, L. (1979) Thromb. Hazmostasis 42,

15. Hamberg, M., Svensson, J., Wakobayaski, T., and Samuelsson, B.

16. Vanderhoek, J . Y., Bryant, R. W., and Bailey, J. M. (1980) J.

17. Kosower, N. S., Kosower, E. M., Wertheim, B., and Correa, W. S.

18. Parker, C. W., Fischman, C. M., and Widner, H. J. (1980) Proc.

19. Hammarstrom, S. (1980) J. Biol. Chem. 255,518-521 20. Bryant, R. W., Feinmark, S. J., Makheja, A. N., and Bailey, J. M.

21. Tietz, F. (1969) Anal. Biochem. 27, 502-522 22. Griffth, 0. W. (1980) Anal. Biochem. 106,207-212 23. Hemler, M. E., Crawford, C. G., and Lands, W. E. M. (1978)

24. Nugteren, D. H. (1975) Biochim. Biophys. Acta 380,299-307 25. Hamberg, M., and Samuelsson, B. (1974) Proc. Natl. Acad. Sci.

26. Bailey, J. M., Feinmark, S. J., and Whiting, J. D. (1980) in Recent Developments in Mass Spectrometry in Biochemistry and Medicine (Frigerio, A,, and McCamish, M., eds) Vol. 6, pp. 77-81, Elsevier Scientific Publishing Co., Amsterdam

27. Borregaard, N., and Henning, J. (1981) Eur. J. Clin. Znuest. 11,

28. Hamberg, H., and Gotthammar, B. (1973) Lipids 8, 737-744 29. Ohki, S., Ogino, N., Yamamoto, S., and Hayaishi, 0. (1979) J.

30. Egan, R. W., Gale, P. H., and Kuehl, F. A., Jr. (1979) J. Biol.

31. OBrien, P. J. (1969) Can. J. Bzochem. 47,485-492 32. Pescarmona, G. P., Bosia, A., Hofmann, J., Losche, W., Arese, P.,

33. Chang, W., Nakao, J., Orimo, H., and Murata, S. (1982) Biochem.

34. Flohe, L. (1979) CZBA Found. Symp. 65.95-113 35. Siegel, M. I., McConnel, R. T., Porter, N. A., and Cuatrecasas, P.

36. Moncada, S., Gryglewski, R. J., Bunting, S., and Vane, J . R. (1976)

37. Turk, J., Wyche, A., and Needleman, P. (1980) Biochem. Biophys.

38. Hofmann, J., Losche, W., Till, U., Bosia, A,, Arese, P., Pescamona,

39. Bryant, R. W., and Bailey, J. M. (1981) Prog. Lipid Res. 20,

40. Gardner, H. W. (1980) in Autoxidation in Food and Biological Systems (Simic, M. G., and Karel, M., eds) Plenum Press, New York

41. Pace-Asciak, C. R., Mizuno, K., and Yamamoto, S. (1981) Biochim. Biophys. Acta 665, 352-354

42. Bass, D. A., O'Flaherty, J. T., Szejda, P., DeChatelet, L. R., and McCall, C. E. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 5125-5129

(1982) J. Biol. Chem. 257,6050-6055

1460-1472

(1974) Proc. Natl. Acad. Sci. U. S. A. 71,345-349

Biol. Chem. 255,5996-5998

(1969) Biochem. Biophys. Res. Commun. 37, 593-596

Natl. Acad. Sci. U. S. A. 77,6870-6873

(1978) J. Biol. Chem. 253,8134-8142

Biochemistry 17, 1772-1779

U. S. A. 71,3400-3404

257-263

Biol. Chem. 254,829-836

Chem. 254,3295-3302

and Till, U. (1981) Acta Biol. Med. Germ. 40, K7-KI4

J. 202, 771-776

(1980) Proc. Natl. Acad. Sci. U. S. A. 77,308-312

Prostaglandins 14, 1024-1034

Res. Commun. 95, 1628-1634

G. P., and Thielmann, K. (1980) Artery 8,431-436

279-281