Vol. 178, No. 2, 1991 July 31, 1991

REGULATION

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 641-647

OF ARACHIDONIC BY PHENYLARSINE

ACID METABOLISM OXIDE

Lawrence Levine Department of Biochemistry, Brandeis University, Waltham, MA 02254 Received June 3, 1991 SUMMARY: Preincubation of rat liver cells (the C-9 cell line) for 25 min with phenylarsine oxide at levels ranging from 0.06 to 0.6 l.tM amplifies prostaglandin 12 production when subsequently stimulated by platelet activating factor, lysine vasopressin, bradykinin, thapsigargin, and the Ca2+ ionophore, A-23187, but not that stimulated by exogenous arachidonic acid. The amplification is decreased after preincubation for 25 min with 1.8 pM phenylarsine oxide. Preincubation of mouse lymphoma cells (the WEI-B-3 cell line) with phenylarsine oxide at levels ranging from 0.06 to 1.8 @l for 60 min does not affect prostaglandin E2 levels but inhibits leukotriene B4 and C4 production stimulated by the Ca2+-’tonophore, A-23 187. Amplification of prostaglandin production by phenylarsine oxide is reversed 100 times more effectively by 2,3dimercaptopropanol than by 2-mercaptoethanol. Deesterification of lipids appears to be regulated positively in rat liver cells and leukotriene production negatively in mouse lymphoma cells by phosphorylation of tyrosine. 0 1991Academic Press, Inc. Sodium vanadate amplifies production of cyclooxygenase products of arachidonic acid metabolism in: 0 rat liver cells stimulated by a variety of agonists including the Ca2+ ionophore A-23 187 but not exogenous arachidonic acid, @ bovine aorta smooth muscle cells stimulated by serotonin, and @ dog kidney cells stimulated by norepinephrine (1). On the other hand sodium vanadate does not affect the synthesis of prostaglandin E2 but inhibits production of lipoxygenase products in rat basophilic leukemia cells and mouse lymphoma cells stimulated by the Ca2+ ionophore A-23 187 (1). Another protein-tyrosine phosphate phosphatase inhibitor, phenylarsine oxide (PAO), has been described (2-4). Its inhibition depends on formation of a complex with vicinal dithiols (5). PA0 treatment of 3T3-LI, 3T3-HIR 3.5 and 2B4 T cells leads to an accumulation of phosphoproteins, most phosphorylated on tyrosine residues (2-4). PA0 treatment of rat liver cells or mouse lymphoma cells affects arachidonic acid metabolism in a manner similar to that of sodium vanadate. It amplifies prostaglandin 12 production by rat liver cells stimulated by several agonists including the Ca2+-ionophore, A-23187, but not that by exogenous arachidonic acid and inhibits synthesis of leukotrienes B4 and C4 but not prostaglandin E2 in mouse lymphoma cells stimulated by the Ca2+ ionophore, A-23187. MATERIALS

AND METHODS

Mate& Phenylarsine oxide (PAO), platelet activating factor (PAF), A-23187, bradykinin and arachidonic acid were purchased from Sigma Chemical Co. (St. Louis, MO).

641

0006-291X/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

178,

No.

2, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Lysine-vasopressin (Lys*ADH) was purchased from Vega Biotechnologies, Inc. (Tuscan, AR). 2,3-Dimercapto-1-propanol and 2-mercaptoethanol were purchased from Aldrich Chemical Co, Milwaukee, WI. Cell Culture, The rat liver cells (the C-9 cell line) were grown as monolayers using minimal essential media (MEM) containing 2 mM L-glutamine and supplemented with 10% (v/v) fetal bovine serum (6). Cells were seeded at 0.4 x 106 cells/35mm tissue culture dishes (Falcon Plastics Co., Oxnard, CA) in 1 or 2 ml of the serum-supplemented medium. After 1 day of growth, the medium was removed and the cells were washed twice with MEM and then treated as described. Mouse lymphoma cells (the WEHI- cell line) were cultured as monolayers using MEM containing 2 mM L-glutamine and supplemented with 10% (v/v) fetal bovine serum (7). Exponentially growing cells were treated with 1 ml of trypsin-EDTA solution and plated at a density of approximately 5 x 10s cells per 35 mm culture dish in 1 or 2 ml of MEM containing 10% fetal bovine serum and 2 mM L-glutamine. The cells were washed twice with MEM and then treated as described. Design of Cell Culture Exoeriments, The experimental design was as follows: The cells, usually in triplicate P 35mm dishes, were preincubated for 25 min (C-9) or 60 min (WEI-II) with either non-serum supplemented MEM or non-serum supplemented MEM containing PAO, after which they were washed twice with 1 ml of MEM and incubated with 1 ml of various agonists diluted in MEM. The number of cells as well as unknown factors probably account for some small differences in absolute levels of arachidonic acid metabolites sometimes found in different experiments. At various times, the culture fluids were collected and stored at -2O’C until assayed by radioimmunoassay (RIA) for cyclooxygenase or lipoxygenase products. RIA. Culture fluids were assayed for 6 keto-prostaglandin Fl, (6-keto-PGFl,) peptidecontaining leukotriene C4 (LTC4), LTB4, PGE2 and PGF2o with previously characterized antisera (8). The radiolabeled ligands for the RIAs were purchased from New England Nuclear Corp. (Boston, MA). Statistical Methods.. If data were obtained from at least 5 separate experiments, i.e. cells preincubated with MEM compared to cells preincubated with MEM containing phenylarsine oxide, statistical significance was evaluated by the unpaired Student’s r-Test. A P value of co.05 was considered significant. RESULTS Preincubation

of rat liver cells with PA0 for 25 min amplifies their PGI2 production

(measured as its stable hydrolytic product 6-keto-PGFlo) when stimulated by PAF or thapsigargin (Fig. 1). Amplification is maximum when the cells are preincubated with 0.6 PM PA0 -

it is reduced after preincubation with 1.8 PM PAO. The morphology of the cells when measured by phase contrast microscopy is not affected by the preincubation with PA0 at levels up to 0.6 @I, but after treatment with PA0 at 1.8 pM some of the rat liver cells appear swollen. Preincubation with PA0 (0.2 PM) for as little as 5 min amplifies PGI2 production stimulated by PAF - it is still increasing after 60 min (data not shown). In most of our studies with rat liver cells, a preincubation time of 25 min with 0.2 @f PA0 was used. With PAF’s stimulation, early events are being regulated by PAO’s reaction - amplified PGI;! production is apparent 2 min after PAF’s addition (Fig. 2). Pretreatment of the rat liver cells with PA0 amplifies basal PGI2 production and that stimulated by Ly s*ADH, bradykinin and A-23 187 in addition to PAF, but not exogenous arachidonic acid (Table 1). The major but low levels of PGF2o and PGE;! are produced stimulated by LysADH (co.012 rig/ml without pretreatment). PA0 forms complexes with vicinal

cyclooxygenase product of C-9 cells is PGI2 (6). PA0 also amplifies PGF2o production PA0 pretreatment; 0.023 rig/ml with PA0 diols (9), and when the cells are preincubated

with 0.2 @I PA0 for 25 min containing varying concentrations of 2,3-dimercaptopropanol 642

or 2-

Vol. 178. No. 2, 1991

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

1.5 c: 52 if b 3 &

1.0

0.5

0

1 0

1.0

0.1 Phenylarsine

Oxide

Minutes

(pM)

m Effect of preincubation of rat liver cellswith varying concentrations of (0.2@4) PA0 on the 6-keto-PGF1,producedafter incubationwith MEM ( O), 3.8 nM PAF (0) and 23 nM thapsigargin (A). Rat liver cells(5 x 1@)/35mmdishwerepreincubatedwith the PA0 for 25 min. The cellswerewashedandincubatedwith MEM, PAF or thapsigarginfor 60 min. The bracketsrepresent the SEMof RL4analyses of triplicatedishes. && Effect of timeon6-keto-PGFlaproductionstimulatedby 3.8nM PAF afterpreincubation with 0.2 pM PA0 for 25 min. Rat liver cells,5 x lOs/dish,were( 0) preincubated with MEM for 25 min and,after washing,incubatedwith MEM; ( l ) preincubated with MEM for 25 min andincubatedwith 3.8 nM PAF; (A) preincubatedwith 0.2 pM PA0 for 25 minandincubated with MEM, and( A) preincubated with 0.2 @I PA0 for 25 minandincubatedwith 3.8nM PAF. mercaptoethanol, 2,3-dimercaptopropanol is about 100 times more effective than 2-mercaptoethanol at preventing PAO’s amplification of PAF’s stimulated 6-keto-PGFla production (IQ-j:

0.75 p.M, 2,3-dimercaptopropanol; 64 J.LM, 2-mercaptoethanol). Treatment with PA0 for 25 min followed by 2 washes with MEM and incubation in MEM for 30 min decreases some amplification by PA0 - dissociationof the vicinal dithiol-PA0 complex is probably occurring.

Nevertheless, the dithiol, 2,3-dimercaptopropanol

is 100 times more effective than the mono-

Table1. Effect of PA0 on theStimulatedProductionof 6-ket&‘GFl, byRat Liver Cells(theC-9 CellLine) MEM* Agonist

Concentration

Bradykinin PAF LysADH A-23187 Arachidonicacid

0.1 pM 3.8nM 1onM 0.1 pM 6.6 j.tM

PA0 ** 6-keto-PGFIQ rig/mlt

0.04 zk0.004(10) 0.09 f 0.004(5) 0.12f 0.019(10) 0.09f 0.032(6) 0.28f 0.007(5) 1.39f 0.031(9)

0.08+ 0.009(10) 0.48rt 0.026(5) 1.0 f 0.207(10) 0.54f 0.164(6) 0.70f 0.024(5) 1.33f 0.058(9)

PValue Fold Unpaired r-Test Stimulation$

Regulation of arachidonic acid metabolism by phenylarsine oxide.

Preincubation of rat liver cells (the C-9 cell line) for 25 min with phenylarsine oxide at levels ranging from 0.06 to 0.6 microM amplifies prostaglan...
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