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Actions of vanadate of arachidonic acid metabolism by cells in culture
PROSTAGLANDINS
ACTIONS
OF VANADATE
Department
ON ARACHIDONIC ACID METABOLISM IN CULTURE L. Levine
BY CELLS
of Biochemietry, Brandeis Univereity, Waltham, Hasaachueetts 02254
Abstract Sodium vanadate (11 l.tM) amplified the PGI2 production of rat liver cells (the C-9 cell line) incubated with thrombin, platelet activating factor, lysine-vasopressin, the Ca2+-ionophore A-23 187, interleukin- 1B, 12-tetradecanoylphorbol- 13-acetate, teleocidin, epidermal growth factor, palytoxin, thapsigargin and colchicine but not that stimulated by exogenous arachidonic acid. Sodium vanadate (2.2 I.~M)also amplified PGF2, production of dog kidney cells (the MDCK cell line) incubated with norepinephrine and, at 0.4 pM, PGI2 production of bovine aorta smooth muscle cells stimulated by serotonin. Sodium vanadate (55 pM) did not affect production of PGE2 and PGF2o in rat basophil leukemia cells (the RBL-1 cell line) stimulated by the Ca2+ionophore A-23187, but did inhibit synthesis of peptide-containing leukotrienes and 12hydroxyeicosatetraenoic acid. When used with cultured cells at micromolar concentrations, vanadate is known to inhibit protein tyrosine-phosphate phosphatases. These results suggest that in some cells deesterification of lipids is positively regulated, at least in part, by phosphorylation of tyrosine whereas in leukocytes, lipoxygenase activities are negatively regulated, at least in part, by phosphorylation of tyrosine. Introduction Arachidonic acid metabolism by cells in culture is stimulated by a variety of compounds including growth factors, tumor promoters and physiologically active agonists (l-3). With several of these agonists, deesterification of cellular lipids also is enhanced. Many compounds that stimulate arachidonic acid metabolism also affect phosphorylations and dephosphorylations (2,3). Sodium vanadate inhibits a variety of phosphorylation and dephosphorylation reactions, the effective concentration depending on whether the activity being measured is present in intact tissues, cell-free systems, or cells. In general, millimolar concentrations are required to inhibit activities in intact tissues. In cell free systems, 40 nM vanadate effectively inhibits the Na+,K+-ATPase activity of dog kidney (4), but in intact cells, vanadate, even at 50 PM, does not inhibit Na+/K+ pump activity (5). Vanadate (1 PM) inhibits dephosphorylation of phosphotyrosine histones but not phosphoserine or phosphothreonine histones by A-431 membrane phosphatases (6) and, at 0.5 l.tM, inhibits phosphotyrosine histone dephosphorylation by a phosphatase in the plasma membranes of astrocytoma (7). The concentrations of vanadate required to inhibit different phosphotyrosine protein phosphatases may vary (reviewed in [S]).
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In intact cells, vanadate (37.5 pM) inhibited specifically protein-tyrosine phosphate dephosphorylation in NRK-1 cells (9). Micromolar concentrations of In human fibroblasts, 4 to 10 pM vanadate vanadate also affect cell functions. stimulated thymidine incorporation into DNA and enhanced, at 6 pM, the mitogenic activities of epidermal growth factor (EGF) or fibroblast growth factor (10). Vanadate, between 5 and 50 pM, was mitogenic for Swiss mouse 3T3 and 3T6 cells (5), and at 37.5 pM induced transformation of NRK-1, and at 19 FM, induced transformation of 10 tt/2, NIH6-Cl32 and mouse embryo fibroblasts (9). The results presented here show that vanadate, at micromolar concentrations, enhances production of PGI2, measured as 6-keto-PGFl,, by rat liver cells stimulated by several agonists but not the 6-keto-PGFto synthesized from exogenous arachidonic acid and enhances synthesis of prostaglandin F2o (PGF2o) by dog kidney cells when stimulated by norepinephrine or production of 6-keto-PGFl,, by bovine aorta smooth muscle cells when stimulated by serotonin. On the other hand, vanadate inhibits synthesis of lipoxygenase metabolites, but not cyclooxygenase products, of two leukocyte cell lines when stimulated by the Ca2+-ionophore, A-23187. Materials and Methods Mate ‘als. Thrombin, 600 NM units per mg protein, platelet activating factor (PAF), A-y3187 12-0-tetradecanoylphorbol-13-acetate (TPA) sodium vanadate (NajVOd), colch&ine and arachidonic acid were purchased from Sigma Chemical Co. (St. Louis, MO). Teleocidin and palytoxin were gifts from Dr. H. Fujiki, National Cancer Center, Tokyo, Japan. Lysine-vasopressin (Lys-ADH) was purchased from Vega Biotechnologies, Inc. (Tuscan, AR). Thapsigargin was purchased from LCServices Co., Woburn, MA; epidermal growth factor (EGF) was purchased from Collaborative Research, Lexington, MA; and recombinant interleukin- lp (IL-1j3) was a gift from Dr. Dagmar Ringe, Department of Biochemistry, Brandeis University, Waltham, MA. 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 (11). Cells were seeded at 0.4 x 106 tells/35-mm tissue culture dishes (P60; Falcon Plastics Co., Oxnard, CA) in 1 or 2 ml of the serumsupplemented medium. After 1 day of growth, the medium was removed and the cells were washed twice with MEM and then treated as described under design of cell culture experiments with rat liver cells. Bovine aorta smooth muscle cells (SMC) were prepared from a primary culture of freshly obtained calf aorta (12). Cultures were maintained in serum-supplemented MEM. For assays of the effects of agonists on prostaglandin production, cells were plated at a density of 105 tells/35-mm dish and incubated overnight at 37” in serum supplemented MEM. The medium was removed, and the cells were washed twice and
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were then incubated at 37°C in an atmosphere of 95% 02-5% CO2 in MEM with penicillin and streptomycin containing various concentrations of agonists. Rat basophil leukemia (RBL-1) cells and 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 (13). 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 incubated in 1 ml of MEM or MEM containing vanadate in the presence and absence of A-23 187. Exponentially growing dog kidney (MDCK) cells were seeded at 2 x l@ cells per 35-mm Falcon tissue culture dish in 1 or 2 ml of MEM containing 2 mM L-glutamate and 10% (v/v) fetal bovine serum and incubated for 20 hr. In the experiments described, the cells were washed twice and incubated with 1 ml of the MEM or MEM containing vanadate in the presence and absence of norepinephrine (14).
es D in f 11 Is, The experimental design was as follows: The cells, in the P 35-mm dishes, were preincubated for 60 min with either non-serum supplemented MEM or non-serum supplemented MEM containing Na3V04, after which they were washed twice with 1 ml of MEM and incubated with 1 ml of various agonists diluted in MEM or, for the NagVO4 preincubated cells, with the agonists diluted in MEM containing Na3V04. Experiments were performed with cells prepared for that day. The number of cells as well as unknown factors probably account for some small differences in absolute levels of 6keto-PGF1, sometimes found in different experiments. At various times, the culture fluids were collected and stored at -20°C until assayed by radioimmunoassay (RIA) for cyclooxygenase or lipoxygenase products. RTA. Culture fluids were assayed for 6-keto-PGFla (C-9 and SMC cells), PGF2o (MDCK cells), peptide-containing leukotrienes, 12-hydroxyeicosatetraenoic acids, PGF2, and PGE:! (RBL-1 and WEHI- cells) with previously characterized antisera. The serologic specificities of these immune systems have been described (15). 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 with different rat liver cell populations, i.e. cells preincubated with MEM compared to cells preincubated with MEM containing vanadate, statistical significance was evaluated by the unpaired Student’s t-Test. A P value of ~0.05 was considered significant.
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Results Vanadate (11 l&i) does not affect the viability of rat liver cells (the C-9 cell line) after incubation for 20 hr as measured by cell number and trypan blue exclusion. However, vanadate (11 PM) amplified 6-keto-PGFl, production stimulated by PAF (1.9 nM), teleocidin (23 nM) and Lys-ADH (10 nM) after incubation with the agonists for 20 hrs (see experimental design) (Table 1). Table 1. Effect of Vanadate (11pM) on the Levels of 6-keto-PGFl, in Culture Fluids of Rat Liver Cells (the C-9 Cell Line) Stimulated by PAF, Teleccidin and Lys-ADH* Vanadate (11 l.W Agonist None PAF Teleocidin Lys-ADH
Concentration
1.9 nM 23 nM 1onM
P value Unpaired r-Test t
ng/ml ** 0.08 0.16 0.19 0.12
* f * f
0.004 0.018 0.010 0.012
(5) (5) (5) (5)
0.06 0.29 0.25 0.23
f f f +
0.005 0.020 0.012 0.012
(5) (5) (5) (5)
0.072 0.001 0.006
* The C-9 cells were preincubated in MEM or vanadate for 60 min, at which time they were washed with MEM and incubated in MEM or vanadate or the agonist diluted in MEM or the agonist diluted in MEM containing vanadate for 20 hr. ** Mean + SEM. ( ) represents number of dishes. t Statistical significance between 6-keto-PGF1, produced in the presence and absence of vanadate. The data in Table 1 represent one experiment in which 5 dishes were analyzed. The data obtained in five separate treatments with cells (triplicate dishes) treated with PAF (1.9 nM), thrombin (0.1 unit/ml), Lys-ADH (10 nM), the Ca2+-ionophore A23187 (0.1 PM), TPA (17 nM), IL-lj3 (1.1 PM), teleocidin (23 nM), palytoxin (20 PM), EGF (17 nM), thapsigargin (23 nM), colchicine (250 nM) and arachidonic acid (10 PM) for 20 hr in the presence and absence of vanadate (11 l&l) are shown in Table 2. Only when the cells were treated with exogenous arachidonic acid was amplification m seen. The combination of vanadate and the agonists also did not affect the viability of the cells as measured by cell number and trypan blue exclusion after the 20 hr of incubation. The effects of vanadate on the time of appearance of 6-keto-PGFlo stimulated by PAF (1.9 nM), arachidonic acid (10 PM), the CaZ+-ionophore A-23187 (0.1 PM) and thrombin (0.1 units/ml); palytoxin (20 PM) and teleocidin (23 nM) are shown in Fig. 1 and 2. Again, the amplification of 6-keto-PGFlo synthesis after preincubation of the
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Table 2. Effect of Vanadate (11 pM) on the Production of 6-keto-PGFto by Rat Liver Cells (the C-9 Cell Line) in the Presence of Various Agonists *
Na3V04 Agonist
6-keto-PGFlo ng/ml t
Concentration
0.07 + 0.006 (15)
None
P Value Unpaired r-Test $
0.10 * 0.010 (15)
0.071
Arachidonic acid Thrombin
1OpM 0.1 units/ml
1.45 zb 0.123 (6)
1.49 + 0.010 (6)
0.12 f 0.018 (5)
0.23 f 0.025 (5)
0.872 0.007
PAF
1.9 nM
0.17 + 0.010 (5)
0.42 + 0.072 (5)
0.010
LysADH
1onM 0.1 pM
0.18 + 0.017 (10)
0.50 f 0.036 (10)
A-23187
0.23 f 0.041 (5)
0.42 f 0.066 (5)
0.044
IL-@
1.1 pM
0.06 zb 0.005 (3)
0.14 + 0.02 (3)
TPA
17nM
0.12 + 0.016 (5)
0.22 zb 0.022 (5)
Teleocidin
23 nM
0.18 & 0.015 (4)
0.26 zb 0.013 (4)
EGF Palytoxin
17nM 20 pM
0.10 f. 0.003 (5) 0.12 f 0.013 (3)
0.36 f 0.083 (3)
Thapsigargin
23 nM
0.37 + 0.026 (5)
3.30 f 0.205 (5)
Colchicine
250 nM
0.14 zb 0.008 (4)
0.28 + 0.030 (4)
0.14 + 0.008 (5)
0.006 0.007
* Cells were preincubated in MEM or vanadate (11 pM) for 60 min. They then were washed 2 times with MEM and incubated for 20 hr in the presence of the agonists diluted in MEM or agonists diluted in MEM containing Na3V04 (11 PM). t Mean +_ SEM ( ) = Number of experiments. Each experiment was done with triplicate dishes. The values within the triplicate dishes agreed within 20%. $ Statistical significance between 6-keto-PGF1, of vanadate.
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produced in the presence and absence
11
PRMTAGLANDINS
PAF b 0.4
0.3
0.2
0.1
C E 3l C II?
0
5
B
0
15
Arachidonic Acid
B
d
10
0.3
2.0
3
t
10
Teleocidin
; A
1.5
2
0.2
I=1.0 t
0.1
o.3L 0
3
10
0
13
I
I
10
20
Hours
Fig. 1, Effect of time on 6-keto-PGFf, production by rat liver cells stimulated by PAF, arachidonic acid, palytoxin or teleocidin in the presence and absence of vanadate (11 PM). The cells were preincubated with MEM or vanadate for 60 min, and then incubated with: PAF, 1.9 nM; PAF in MEM (D ), PAF in vanadate ( b). Palytoxin, 20 PM; palytoxin in h4EM ( q ), palytoxin in vanadate ( 0 ). Arachidonic acid, 10 pM; arachidonic acid in MEM (A), arachidonic acid in vanadate (A). Teleocidin, 23 nM; teleocidin in MJ3M ( O), teleocidin in vanadate ( 0). Cells preincubated with MEM and treated with MEM ( 0) and cells preincubated with vanadate and treated with vanadate (W).
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Thrombin
0
_
r 10
20
Hours
Fig. 2, Effect of time on 6-keto-PGFt, production by rat liver cells stimulated by A23 187 or thrombin in the presence and absence of vanadate (11 @VI). The cells were preincubated with MEM or vanadate for 60 min, and then incubated with: A-23187, 0.1 l.tM; A-23187 in MEM (V), A-23187 in vanadate (‘I). Thrombin, 0.1 unit/ml; thrombin in MEM (a), thrombin in vanadate ( 4). Cells preincubated with MEM and treated with MEM ( 0) and cells preincubated with vanadate and treated with vanadate (H). cells with vanadate was found with all agonists except when exogenous arachidonic acid was used as a substrate for the cyclooxygenase. The MDCK cells are about 5 times more sensitive with respect to toxic effects of NajVOq than the C-9 cells. Vanadate (2.2 ltM> amplified PGF2o production by MDCK cells incubated with norepinephrine and, at 0.4 FM, amplified 6-keto-PGFlo production by SMC cells stimulated by serotonin (Table 3). In these.experiments toxicity as measured by cell number and trypan blue exclusion was not determined; however, the treatments did not obviously affect the morphology or the number of cells as judged microscopically. RBL-1 and WEHI- cells, when stimulated by the Ca2+-ionophore, A-23187, metabolize arachidonic acid via cyclooxygenase and lipoxygenase pathways (13). When stimulated by the Ca2+-ionophore, A-23187, the production of both
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Table 3. Effect of Vanadate on the Levels of PGF2o in Culture Fluids of MDCK or 6-keto-PGF1, in Culture Fluids of SMC Incubated with Norepinephrine and Serotonin, Respectively * In absence of
In presence of
Ligand
Cell
J.&&**
MDCK
Noreninenhrine
Noreoinenhrine PGF2a
1.45 + 0.097 (11) Norepinephrine (59 pM) Vanadate (2.2 ttM) t
1.56 f 0.108 (3) 7.50 _+0.350 (3)
1.33 i 0.030 (3) Serotonin
SMC
Serotonin 6-keto-PGF1o
0.42 f 0.031 (8) Serotonin (5.7 PM) Vanadate (0.4 t&I) t
1.63 f 0.190 (8) 5.10 f 0.472 (4)
0.44 + 0.008 (4)
* The cells were incubated in presence and absence of norepinephrine or serotonin for 20 hrs. ** Mean + SEM. ( ) = number of dishes. t Higher doses of vanadate (11 l.tM, MDCK; 2.2 l,tM, SMC) were toxic as judged microscopically.
Table 4. Effect of Vanadate on Arachidonic Acid Metabolism of RRL-1 Cells Stimulated by the Ca2+-Ionophore A-23 187
Arachidonic acid metabolite
A-23 187 (0.4 w) and Vanadate (55 ClM) *
A-23187 (0.4 PM)
Q&Lo PGE2 PGFza LTC4 12-HETE
0.90 1.02 12.98 13.43
* f f f
0.150 0.037 0.654 0.511
(8) (8) (8) (8)
1.14 0.91 2.09 3.15
f f f f
0.085 0.020 0.199 0.349
(4) (4) (4) (4)
* Vanadate at levels < 55 pM were not tested. t In MEM alone, ~0.2 ng of the cyclooxygenase and lipoxygenase products were present. Vanadate (55 JIM) did not affect these basal levels. Mean f SEM. ( ) = number of dishes.
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cyclooxygenase and lipoxygenase products is increased (13). In the presence of vanadate (55 PM), only the levels of lipoxygenase products were affected - their production by RBL-1 cells was inhibited (Table 4). Neither the morphology nor the number of the leukocytes was affected by these treatments. Arachidonic acid metabolism by WEHI- cells stimulated by the Ca2+- ionophore A-23 187 was affected by vanadate (55 PM) similarly, i.e. the levels of the peptide-containing leukotrienes were inhibited while those of cyclooxygenase products, PGE2 and TxB2, were not (data not shown). Discussion Sodium vanadate amplified 6-keto-PGFlo production by rat liver cells that had been incubated with several agonists, PGF2o production by MDCK cells that had been incubated with norepinephrine, and 6-keto-PGFld synthesis by SMC that had been stimulated by serotonin. The production of PGE2, PGF2d and 6-keto-PGFlo by rat mesangeal cells (16) and 6-keto-PGFld by glomerular epithelial cells (17) stimulated by Lys-ADH also was amplified by vanadate (11 @) (unpublished data). Vanadate, however, inhibited production of peptide-containing leukotrienes and 12hydroxyeicosatetraenoic acid by RBL- 1 cells, and peptide-containing leukotrienes by WEHI- cells stimulated by the Ca2+-’ionophore A-23187 - the levels of cyclooxygenase products of the two leukocyte cell lines stimulated by A-23187 were not affected. At none of these levels of vanadate used with these cells was a toxic response observed, at least as measured by the number of the cells and trypan blue exclusion or by microscopic examination after a 20-hour incubation. The amplification of PGF2, (MDCK) and 6-keto-PGFld (SMC and C-9) production by vanadate probably reflects regulation at a step distal to specific receptor binding as it was effective (at least with C9 cells) with several agonists that stimulate arachidonic acid metabolism by non-receptor mediated mechanisms, e.g. colchicine, A-23187 and thapsigargin (thapsigargin mobilizes intracellular Caz+ [ 191). With rat liver cells the regulation by vanadate is proximal to cyclooxygenase activity - no amplification was found when exogenous arachidonic acid was used as substrate. This is not surprising since several of the agonists used in this study (Table 2) stimulate arachidonic acid metabolism by increasing deesterification of phospholipids (l-3). Vanadate has been reported to affect several enzymes. Vanadate decreases Ca2+ATPase (20), ribonuclease (21), alkaline phosphatase (22) and acid phosphatase (23) activities - the effective concentrations used for some of these inhibitions are 2 to 3 orders of magnitude higher than those used in the present study. In studies with enzymes in a cell free system, concentrations of vanadate even less than those used in the present study were effective, especially inhibition of Na+,K+-ATPase (4). In intact cells, however, even 50 pM vanadate did not inhibit the Na+/K+-ATPase (5). At micromolar levels comparable to those used in this study, vanadate has been shown to be a specific inhibitor of protein-tyrosine-phosphate phosphatase -not an inhibitor of protein-serine (threonine)-phosphate phosphatase. The content of phosphotyrosine in
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normal rat kidney cells (NRK-1) treated with vanadate (37.5 PM) for 3 days and incubated with 5 mCi phosphate for 24 hr was increased as much as 36 fold, but levels of protein-serine(threonine)-phosphate did not change (9). Vanadate is found in the cytoplasm of human red blood cells as the vanadyl ion associated with hemoglobin (18) - the vanadyl ion is much less effective than vanadate at inhibiting Na+/K+-ATPase (18). If vanadate is acting by inhibiting protein-tyrosine phosphatase, the relative effectiveness of the vanadyl ion would be of interest. If vanadate is reduced to the vanadyl ion in the cytoplasmic compartments of C-9, SMC, kidney cells or leukocytes, as it is in human erythrocytes, and if vanadyl is much less effective at inhibiting phosphatases, then vanadate may be acting on non-cytoplasmic protein-tyrosine phosphatases. The most likely pathway being regulated in the rat liver, dog kidney and smooth muscle cells is deesterification of cellular lipids. Several enzyme reactions have been implicated in deesterification of cellular lipids, including coupled phospholipase Cdiacylglycerol lipase reactions and phospholipase A2 activities (24). Vanadate amplifies arachidonic acid metabolism after incubation of cells with thrombin, PAF, Lys-ADH, A-23 187, IL- 1p, TPA, EGF, palytoxin, thapsigargin, colchicine, norepinephrine and serotonin. The mechanisms of stimulated arachidonic acid metabolism by such diverse agonists are likely to be very different. For example, serotonin acts by way of a type 2 receptor (2526) to liberate arachidonic acid from phospholipids by phospholipase A2 activity (26) while norepinephrine stimulates liberation of arachidonic acid by way of a (rl receptor and G-protein interaction to stimulate both phospholipase C and phospholipase A2 activity (27). EGF binds to its receptor to activate phospholipase C-y (28), although whether this activated phospholipase C-y liberates arachidonic acid via coupled diacylglycerol lipase activity is not known. Colchicine’s stimulation of 6-ketoPGFt, production probably reflects perturbation of the cytoskeleton (29) and activation of phospholipase AZ. Most likely both pathways of deesterification are being regulated by tyrosine phosphorylation, but not necessarily by the same proteins. Such regulation may not be attributable directly to tyrosine phosphorylation but indirectly to other kinases activated in a cascade that phosphorylates serine or threonine. Within the limits of our analyses, the initial rates of 6-keto-PGFt, production by the rat liver cells after incubation with vanadate are not affected. The increased stimulation of arachidonic acid metabolism by vanadate appears to be occurring at a relatively late stage of deesterification. Therefore, the possibility that vanadate is affecting other metabolic reactions in addition to inhibition of protein-tyrosine phosphatase can not be ruled out. The lipoxygenase, not the cyclooxygenase, pathway of the RBL-1 (Table 4) and WEHI- cells is inhibited by vanadate (55 l.tM) after stimulation by the Ca2+-ionophore A-23187. Presumably, phosphorylation of an enzyme is negatively regulating production of peptide-containing leukotrienes and hydroxyeicosatetraenoic acids. Most likely it is the lipoxygenase molecules that are inhibited by phosphorylation, but regulation of enzymes distal to 5-lipoxygenase activity, e.g. the 5-hydroperoxyeicosatetraenoic acid dehydrase or the glutathione-S-transferase are also possible.
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Although the effects of vanadate at levels less than 55 @I on RBL-1 cells were not measured, the effects of a higher level (275 pM) were. The A-23187-stimulated levels of LTC4 and 1ZHETE were inhibited even more effectively at 275 @4 than at 55 PM but those of PGEz and PGF2o still were not altered. Vanadate, even at 275 @l, caused no obvious morphological changes in the cells. The reasons for the lack of effect of vanadate on the cyclooxygenase products of the RBL-1 cells at levels higher than those found to be effective in other cells are not known. Possibly different pathways of deesterification exist in the leukocytes (30) or if the same pathways do exist, their threshold of regulatory control is higher. In either case, an enzyme in the lipoxygenase pathway is sensitive to negative regulation. In rat liver cells, the increases of PGI;! produced over basal levels after stimulation by physiological agonists in the presence of vanadate ranged from 2-fold with EGF to 7-fold with vasopressin; in SMC, 12-fold with serotonin; and in the dog kidney cells, Sfold with norepinephrine. The physiologic consequence of these amplifications (or lipoxygenase inhibitions) is not known. In addition, at the non-toxic levels used in this study, it is possible that not all protein-tyrosine phosphatase activity is being inhibited - the amplification (or inhibition) could be much higher if protein-tyrosine phosphatase activities were eliminated. Sodium vanadate, however, is toxic to cells in culture - the toxicity varies among cells (55 ~,LMfor C-9; 11 pM for MDCK, 2.2 l.tM for SMC, and greater than 55 @4 for the leukocytes). Earlier studies (9) showed that 50 p.M vanadate affected survival of sparse cultures of NRK-1 cells. NM-6Cl-32 cells and 10 Tic! were even more sensitive to the toxic effects of vanadate. This toxicity to vanadate may be caused by sustaining an activity that is regulated by phosphorylated tyrosines. Each cell’s sensitivity would reflect that sustained activity.
Acknowledgements This work was supported by Grant RDP- 12K from the American Cancer Society. I wish to thank Nancy Worth for technical assistance and Inez Zimmerman for preparation of the manuscript.
References 1, Curtis-Prior, P.B., ed. Prostaglandins: Biology and Chemistry of Prostaglandins and Related Eicosanoids. Churchill Livingstone, Edinburgh, 1988. 2. Levine, L. Tumor promoters, growth factors and arachidonic acid metabolism. In: Prostaglandins in Cancer Research (Garaci, E., Paoletti, R., and Santoro, M.G., eds.), Springer-Verlag, Berlin, 1987, pp. 62-73. 3. Levine, L. Tumor promoters and prostaglandin production. In: Eicosanoids, Lipid Peroxidation and Cancer (Nigam, S.K., McBrien, D.C.H., and Slater, T.F., eds.) Springer-Verlag, Berlin, 1988, pp. 11-21.
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5. 6.
7.
8. 9. 10. 11. 12.
13.
14.
15.
16.
17.
18, 19.
18
Jr., L.C., Josephson, L., Warner, R., Yanagisawa, M., Lechene, C., and Guidotti, G. Vanadate is a potent (Na,K)-ATPase inhibitor found in ATP derived from muscle. J. Biol. Chem. m: 7421-7423. 1977. Smith, J.B. Vanadium ions stimulate DNA synthesis in Swiss mouse 3T3 and 3T6 cells. Proc. Natl. Acad. Sci. USA BQ: 6162-6166. 1983. Swarup, G., Cohen, S,. and Garbers, D.L. Inhibition of membrane phosphotyrosyl-protein phosphatase activity by vanadate. Biochem. Biophys. Res. Commun. m: 1104-l 109. 1982. Leis, J.F. and Kaplan, N.O. An acid phosphatase in the plasma membranes of human astrocytoma showed marked specificity toward phosphotyrosine protein. Proc. Natl. Acad. Sci. USA B: 6507-6511. 1982. Lau, K.-H.W., Farley, J.R., and Baylink, D.J. Phosphotyrosyl protein phosphatases. Biochem. J. m: 23-36. 1989. Klarlund, J.K. Transformation of cells by an inhibitor of phosphatases acting on phosphotyrosine in proteins. Cell Q: 707-717. 1985. Carpenter, G. Vanadate, epidermal growth factor and the stimulation of DNA synthesis. Biochem. Biophys. Res. Commun. m: 1115-l 121. 1981. Rigas, A. and Levine, L. Arachidonic acid metabolism by rat liver cells (the C-9 cell line). J. Pharmacol. exp. Ther. m: 230-235. 1984. Coughlin, S.R., Moskowitz, M.A., Antoniades, H.N. and Levine, L. Serotonin receptor-mediated stimulation of bovine smooth muscle cell prostacyclin synthesis and its modulation by platelet-derived growth factor. Proc. Natl. Acad. Sci. USA 3: 7134-7138. 1981. Levine, L. Inhibition of the A-23187-stimulated leukotriene and prostaglandin biosynthesis of rat basophil leukemia (RBL-1) cells by non-steroidal antiinflammatory drugs, anti-oxidants and calcium channel blockers. Biochem. Pharmacol. z: 3023-3026. 1983. Levine, L. and Moskowitz, M.A. Alpha- and beta-adrenergic stimulation of arachidonic acid metabolism by cells in culture. Proc. Natl. Acad. Sci. 26: 66326636. 1979. Levine, L. Measurement of arachidonic acid metabolites by radioimmunoassay. In: Manual of Clinical Laboratory Immunology, Third Edition (Rose, N.R., Friedman, H., and Fahey, J.L., eds.), American Society for Microbiology, Washington D.C., 1986, pp. 685-691. Uglesity, A., Kreisberg, J.I., and Levine, L. Stimulation of arachidonic acid metabolism in rat kidney mesangial cells by bradykinin, antidiuretic hormone, and their analogues. Prostaglandins Leukotrienes Med. fi: 83-93. 1983. Lieberthal, W., and Levine, L. Stimulation of prostaglandin production in rat glomerular epithelial cells by antidiuretic hormone. Kidney Internat. .2.%766-770. 1984. Cantley, Jr., L.C., and Aisen, P. The fate of cytoplasmic vanadium. J. Biol. Chem. m: 1781-1784. 1979. Thastrup, O., Cullen, P.J., Drerbak, B.K., Hanley, M.R., and Dawson, A.P. Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific
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1991 VOL. 41 NO. 1
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20. 21.
22.
23.
24.
25.
26.
27.
28. 29.
30.
inhibition of the endoplasmic reticulum Ca2+-ATPase. Proc. Natl. Acad. Sci. USA 82: 2466-2470. 1990. Popescu, L.M., and Ignat, P. Calmodulin-dependent Ca2+ pump-ATPase of human smooth muscle sarcolemma. Cell Calcium 4: 219-235. 1983. Lindquist, R.N., Lynn, Jr., J.L., and Lienhard, G.E. Possible transition-state analogs for ribonuclease. The complexes of uridine with oxovanadium (IV) ion and vanadium (V) ion. J. Am. Chem. Sot. B: 8762-8768. 1973. Lopez, V., Stevens, T,. and Lindquist, R.N. Vanadium ion inhibition of alkaline phosphatase-catalyzed phosphate ester hydrolysis. Arch. Biochem. Biophys. m: 31-38. 1976. VanEtten, R.L., Waymack, P.P., and Rehkop, D.M. Transition metal ion inhibition of enzyme-catalyzed phosphate ester displacement reactions. J. Am. Chem. Sot. %: 6782-6785. 1974. Hong, S.L. The release of arachidonic acid from cellular lipids. In: Arachidonate Metabolism in Immunologic Systems. Progress in Allergy. Vo144 (Levine, L., vol. ed.; Ishizaki, K., Kallos, P., Lachmann, P.J., Waksman, B.H., eds.), Karger, Basel, 1988. pp. 99-139. Coughlin, S.R., Moskowitz, M.A., and Levine, L. Identification of a serotonin type 2 receptor linked to prostacyclin synthesis in vascular smooth muscle cells. Biochem. Pharmacol. 3.: 692-695. 1984. Felder, C.C., Kanterman, R.Y., Ma, A.L., and Axelrod, J. Serotonin stimulates phospholipase A2 and the release of arachidonic acid in hippocampal neurons by a type 2 serotonin receptor that is independent of inositolphospholipid hydrolysis. Proc. Natl. Acad. Sci. USA fl: 2187-2191. 1990. Burch,R.M., Luini, A., and Axelrod, J. Phospholipase A2 and phospholipase C are activated by distinct GTP-binding proteins in response to at-adrenergic stimulation in FRTL5 thyroid cells. Proc. Natl. Acad. Sci. USA u: 7201-7205. 1986. Carpenter, G., and Cohen, S. Epidermal growth factor. J. Biol. Chem. m: 7709-7712. 1990. Nakano, T., Hanasaki, K., and Arita, H. Possible involvement of cytoskeleton in collagen-stimulated activation of phospholipases in human platelets. J. Biol. Chem. m: 5400-5406. 1989. Humes, J.L., Sadowski, S., Galavage, M., Goldenberg, M., Subers, E., Bonney, R.J., and Kuehl, F.A., Jr. Evidence for two sources of arachidonic acid for oxidative metabolism by mouse peritoneal macrophages. J. Biol. Chem. z: 1591-1594. 1982. Editor:
JANUARY
P.W.
Ramwell
1991 VOL. 41 NO. 1
Received:
9-5-90
Accepted:
lo-S-90
19
ACTIONS
OF VANADATE
Department
ON ARACHIDONIC ACID METABOLISM IN CULTURE L. Levine
BY CELLS
of Biochemietry, Brandeis Univereity, Waltham, Hasaachueetts 02254
Abstract Sodium vanadate (11 l.tM) amplified the PGI2 production of rat liver cells (the C-9 cell line) incubated with thrombin, platelet activating factor, lysine-vasopressin, the Ca2+-ionophore A-23 187, interleukin- 1B, 12-tetradecanoylphorbol- 13-acetate, teleocidin, epidermal growth factor, palytoxin, thapsigargin and colchicine but not that stimulated by exogenous arachidonic acid. Sodium vanadate (2.2 I.~M)also amplified PGF2, production of dog kidney cells (the MDCK cell line) incubated with norepinephrine and, at 0.4 pM, PGI2 production of bovine aorta smooth muscle cells stimulated by serotonin. Sodium vanadate (55 pM) did not affect production of PGE2 and PGF2o in rat basophil leukemia cells (the RBL-1 cell line) stimulated by the Ca2+ionophore A-23187, but did inhibit synthesis of peptide-containing leukotrienes and 12hydroxyeicosatetraenoic acid. When used with cultured cells at micromolar concentrations, vanadate is known to inhibit protein tyrosine-phosphate phosphatases. These results suggest that in some cells deesterification of lipids is positively regulated, at least in part, by phosphorylation of tyrosine whereas in leukocytes, lipoxygenase activities are negatively regulated, at least in part, by phosphorylation of tyrosine. Introduction Arachidonic acid metabolism by cells in culture is stimulated by a variety of compounds including growth factors, tumor promoters and physiologically active agonists (l-3). With several of these agonists, deesterification of cellular lipids also is enhanced. Many compounds that stimulate arachidonic acid metabolism also affect phosphorylations and dephosphorylations (2,3). Sodium vanadate inhibits a variety of phosphorylation and dephosphorylation reactions, the effective concentration depending on whether the activity being measured is present in intact tissues, cell-free systems, or cells. In general, millimolar concentrations are required to inhibit activities in intact tissues. In cell free systems, 40 nM vanadate effectively inhibits the Na+,K+-ATPase activity of dog kidney (4), but in intact cells, vanadate, even at 50 PM, does not inhibit Na+/K+ pump activity (5). Vanadate (1 PM) inhibits dephosphorylation of phosphotyrosine histones but not phosphoserine or phosphothreonine histones by A-431 membrane phosphatases (6) and, at 0.5 l.tM, inhibits phosphotyrosine histone dephosphorylation by a phosphatase in the plasma membranes of astrocytoma (7). The concentrations of vanadate required to inhibit different phosphotyrosine protein phosphatases may vary (reviewed in [S]).
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1991 VOL. 41 NO. 1
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PROSTAGLANDINS
In intact cells, vanadate (37.5 pM) inhibited specifically protein-tyrosine phosphate dephosphorylation in NRK-1 cells (9). Micromolar concentrations of In human fibroblasts, 4 to 10 pM vanadate vanadate also affect cell functions. stimulated thymidine incorporation into DNA and enhanced, at 6 pM, the mitogenic activities of epidermal growth factor (EGF) or fibroblast growth factor (10). Vanadate, between 5 and 50 pM, was mitogenic for Swiss mouse 3T3 and 3T6 cells (5), and at 37.5 pM induced transformation of NRK-1, and at 19 FM, induced transformation of 10 tt/2, NIH6-Cl32 and mouse embryo fibroblasts (9). The results presented here show that vanadate, at micromolar concentrations, enhances production of PGI2, measured as 6-keto-PGFl,, by rat liver cells stimulated by several agonists but not the 6-keto-PGFto synthesized from exogenous arachidonic acid and enhances synthesis of prostaglandin F2o (PGF2o) by dog kidney cells when stimulated by norepinephrine or production of 6-keto-PGFl,, by bovine aorta smooth muscle cells when stimulated by serotonin. On the other hand, vanadate inhibits synthesis of lipoxygenase metabolites, but not cyclooxygenase products, of two leukocyte cell lines when stimulated by the Ca2+-ionophore, A-23187. Materials and Methods Mate ‘als. Thrombin, 600 NM units per mg protein, platelet activating factor (PAF), A-y3187 12-0-tetradecanoylphorbol-13-acetate (TPA) sodium vanadate (NajVOd), colch&ine and arachidonic acid were purchased from Sigma Chemical Co. (St. Louis, MO). Teleocidin and palytoxin were gifts from Dr. H. Fujiki, National Cancer Center, Tokyo, Japan. Lysine-vasopressin (Lys-ADH) was purchased from Vega Biotechnologies, Inc. (Tuscan, AR). Thapsigargin was purchased from LCServices Co., Woburn, MA; epidermal growth factor (EGF) was purchased from Collaborative Research, Lexington, MA; and recombinant interleukin- lp (IL-1j3) was a gift from Dr. Dagmar Ringe, Department of Biochemistry, Brandeis University, Waltham, MA. 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 (11). Cells were seeded at 0.4 x 106 tells/35-mm tissue culture dishes (P60; Falcon Plastics Co., Oxnard, CA) in 1 or 2 ml of the serumsupplemented medium. After 1 day of growth, the medium was removed and the cells were washed twice with MEM and then treated as described under design of cell culture experiments with rat liver cells. Bovine aorta smooth muscle cells (SMC) were prepared from a primary culture of freshly obtained calf aorta (12). Cultures were maintained in serum-supplemented MEM. For assays of the effects of agonists on prostaglandin production, cells were plated at a density of 105 tells/35-mm dish and incubated overnight at 37” in serum supplemented MEM. The medium was removed, and the cells were washed twice and
8
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1991 VOL. 41 NO. 1
PROSTAGLANDINS
were then incubated at 37°C in an atmosphere of 95% 02-5% CO2 in MEM with penicillin and streptomycin containing various concentrations of agonists. Rat basophil leukemia (RBL-1) cells and 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 (13). 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 incubated in 1 ml of MEM or MEM containing vanadate in the presence and absence of A-23 187. Exponentially growing dog kidney (MDCK) cells were seeded at 2 x l@ cells per 35-mm Falcon tissue culture dish in 1 or 2 ml of MEM containing 2 mM L-glutamate and 10% (v/v) fetal bovine serum and incubated for 20 hr. In the experiments described, the cells were washed twice and incubated with 1 ml of the MEM or MEM containing vanadate in the presence and absence of norepinephrine (14).
es D in f 11 Is, The experimental design was as follows: The cells, in the P 35-mm dishes, were preincubated for 60 min with either non-serum supplemented MEM or non-serum supplemented MEM containing Na3V04, after which they were washed twice with 1 ml of MEM and incubated with 1 ml of various agonists diluted in MEM or, for the NagVO4 preincubated cells, with the agonists diluted in MEM containing Na3V04. Experiments were performed with cells prepared for that day. The number of cells as well as unknown factors probably account for some small differences in absolute levels of 6keto-PGF1, sometimes found in different experiments. At various times, the culture fluids were collected and stored at -20°C until assayed by radioimmunoassay (RIA) for cyclooxygenase or lipoxygenase products. RTA. Culture fluids were assayed for 6-keto-PGFla (C-9 and SMC cells), PGF2o (MDCK cells), peptide-containing leukotrienes, 12-hydroxyeicosatetraenoic acids, PGF2, and PGE:! (RBL-1 and WEHI- cells) with previously characterized antisera. The serologic specificities of these immune systems have been described (15). 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 with different rat liver cell populations, i.e. cells preincubated with MEM compared to cells preincubated with MEM containing vanadate, statistical significance was evaluated by the unpaired Student’s t-Test. A P value of ~0.05 was considered significant.
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1991 VOL. 41 NO. 1
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PROSTAGLANDINS
Results Vanadate (11 l&i) does not affect the viability of rat liver cells (the C-9 cell line) after incubation for 20 hr as measured by cell number and trypan blue exclusion. However, vanadate (11 PM) amplified 6-keto-PGFl, production stimulated by PAF (1.9 nM), teleocidin (23 nM) and Lys-ADH (10 nM) after incubation with the agonists for 20 hrs (see experimental design) (Table 1). Table 1. Effect of Vanadate (11pM) on the Levels of 6-keto-PGFl, in Culture Fluids of Rat Liver Cells (the C-9 Cell Line) Stimulated by PAF, Teleccidin and Lys-ADH* Vanadate (11 l.W Agonist None PAF Teleocidin Lys-ADH
Concentration
1.9 nM 23 nM 1onM
P value Unpaired r-Test t
ng/ml ** 0.08 0.16 0.19 0.12
* f * f
0.004 0.018 0.010 0.012
(5) (5) (5) (5)
0.06 0.29 0.25 0.23
f f f +
0.005 0.020 0.012 0.012
(5) (5) (5) (5)
0.072 0.001 0.006
* The C-9 cells were preincubated in MEM or vanadate for 60 min, at which time they were washed with MEM and incubated in MEM or vanadate or the agonist diluted in MEM or the agonist diluted in MEM containing vanadate for 20 hr. ** Mean + SEM. ( ) represents number of dishes. t Statistical significance between 6-keto-PGF1, produced in the presence and absence of vanadate. The data in Table 1 represent one experiment in which 5 dishes were analyzed. The data obtained in five separate treatments with cells (triplicate dishes) treated with PAF (1.9 nM), thrombin (0.1 unit/ml), Lys-ADH (10 nM), the Ca2+-ionophore A23187 (0.1 PM), TPA (17 nM), IL-lj3 (1.1 PM), teleocidin (23 nM), palytoxin (20 PM), EGF (17 nM), thapsigargin (23 nM), colchicine (250 nM) and arachidonic acid (10 PM) for 20 hr in the presence and absence of vanadate (11 l&l) are shown in Table 2. Only when the cells were treated with exogenous arachidonic acid was amplification m seen. The combination of vanadate and the agonists also did not affect the viability of the cells as measured by cell number and trypan blue exclusion after the 20 hr of incubation. The effects of vanadate on the time of appearance of 6-keto-PGFlo stimulated by PAF (1.9 nM), arachidonic acid (10 PM), the CaZ+-ionophore A-23187 (0.1 PM) and thrombin (0.1 units/ml); palytoxin (20 PM) and teleocidin (23 nM) are shown in Fig. 1 and 2. Again, the amplification of 6-keto-PGFlo synthesis after preincubation of the
10
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1991 VOL. 41 NO. 1
PROSTAGLANDINS
Table 2. Effect of Vanadate (11 pM) on the Production of 6-keto-PGFto by Rat Liver Cells (the C-9 Cell Line) in the Presence of Various Agonists *
Na3V04 Agonist
6-keto-PGFlo ng/ml t
Concentration
0.07 + 0.006 (15)
None
P Value Unpaired r-Test $
0.10 * 0.010 (15)
0.071
Arachidonic acid Thrombin
1OpM 0.1 units/ml
1.45 zb 0.123 (6)
1.49 + 0.010 (6)
0.12 f 0.018 (5)
0.23 f 0.025 (5)
0.872 0.007
PAF
1.9 nM
0.17 + 0.010 (5)
0.42 + 0.072 (5)
0.010
LysADH
1onM 0.1 pM
0.18 + 0.017 (10)
0.50 f 0.036 (10)
A-23187
0.23 f 0.041 (5)
0.42 f 0.066 (5)
0.044
IL-@
1.1 pM
0.06 zb 0.005 (3)
0.14 + 0.02 (3)
TPA
17nM
0.12 + 0.016 (5)
0.22 zb 0.022 (5)
Teleocidin
23 nM
0.18 & 0.015 (4)
0.26 zb 0.013 (4)
EGF Palytoxin
17nM 20 pM
0.10 f. 0.003 (5) 0.12 f 0.013 (3)
0.36 f 0.083 (3)
Thapsigargin
23 nM
0.37 + 0.026 (5)
3.30 f 0.205 (5)
Colchicine
250 nM
0.14 zb 0.008 (4)
0.28 + 0.030 (4)
0.14 + 0.008 (5)
0.006 0.007
* Cells were preincubated in MEM or vanadate (11 pM) for 60 min. They then were washed 2 times with MEM and incubated for 20 hr in the presence of the agonists diluted in MEM or agonists diluted in MEM containing Na3V04 (11 PM). t Mean +_ SEM ( ) = Number of experiments. Each experiment was done with triplicate dishes. The values within the triplicate dishes agreed within 20%. $ Statistical significance between 6-keto-PGF1, of vanadate.
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1991 VOL. 41 NO. 1
produced in the presence and absence
11
PRMTAGLANDINS
PAF b 0.4
0.3
0.2
0.1
C E 3l C II?
0
5
B
0
15
Arachidonic Acid
B
d
10
0.3
2.0
3
t
10
Teleocidin
; A
1.5
2
0.2
I=1.0 t
0.1
o.3L 0
3
10
0
13
I
I
10
20
Hours
Fig. 1, Effect of time on 6-keto-PGFf, production by rat liver cells stimulated by PAF, arachidonic acid, palytoxin or teleocidin in the presence and absence of vanadate (11 PM). The cells were preincubated with MEM or vanadate for 60 min, and then incubated with: PAF, 1.9 nM; PAF in MEM (D ), PAF in vanadate ( b). Palytoxin, 20 PM; palytoxin in h4EM ( q ), palytoxin in vanadate ( 0 ). Arachidonic acid, 10 pM; arachidonic acid in MEM (A), arachidonic acid in vanadate (A). Teleocidin, 23 nM; teleocidin in MJ3M ( O), teleocidin in vanadate ( 0). Cells preincubated with MEM and treated with MEM ( 0) and cells preincubated with vanadate and treated with vanadate (W).
12
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1991 VOL. 41 NO. 1
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Thrombin
0
_
r 10
20
Hours
Fig. 2, Effect of time on 6-keto-PGFt, production by rat liver cells stimulated by A23 187 or thrombin in the presence and absence of vanadate (11 @VI). The cells were preincubated with MEM or vanadate for 60 min, and then incubated with: A-23187, 0.1 l.tM; A-23187 in MEM (V), A-23187 in vanadate (‘I). Thrombin, 0.1 unit/ml; thrombin in MEM (a), thrombin in vanadate ( 4). Cells preincubated with MEM and treated with MEM ( 0) and cells preincubated with vanadate and treated with vanadate (H). cells with vanadate was found with all agonists except when exogenous arachidonic acid was used as a substrate for the cyclooxygenase. The MDCK cells are about 5 times more sensitive with respect to toxic effects of NajVOq than the C-9 cells. Vanadate (2.2 ltM> amplified PGF2o production by MDCK cells incubated with norepinephrine and, at 0.4 FM, amplified 6-keto-PGFlo production by SMC cells stimulated by serotonin (Table 3). In these.experiments toxicity as measured by cell number and trypan blue exclusion was not determined; however, the treatments did not obviously affect the morphology or the number of cells as judged microscopically. RBL-1 and WEHI- cells, when stimulated by the Ca2+-ionophore, A-23187, metabolize arachidonic acid via cyclooxygenase and lipoxygenase pathways (13). When stimulated by the Ca2+-ionophore, A-23187, the production of both
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1991 VOL. 41 NO. 1
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PROSTAGLANDINS
Table 3. Effect of Vanadate on the Levels of PGF2o in Culture Fluids of MDCK or 6-keto-PGF1, in Culture Fluids of SMC Incubated with Norepinephrine and Serotonin, Respectively * In absence of
In presence of
Ligand
Cell
J.&&**
MDCK
Noreninenhrine
Noreoinenhrine PGF2a
1.45 + 0.097 (11) Norepinephrine (59 pM) Vanadate (2.2 ttM) t
1.56 f 0.108 (3) 7.50 _+0.350 (3)
1.33 i 0.030 (3) Serotonin
SMC
Serotonin 6-keto-PGF1o
0.42 f 0.031 (8) Serotonin (5.7 PM) Vanadate (0.4 t&I) t
1.63 f 0.190 (8) 5.10 f 0.472 (4)
0.44 + 0.008 (4)
* The cells were incubated in presence and absence of norepinephrine or serotonin for 20 hrs. ** Mean + SEM. ( ) = number of dishes. t Higher doses of vanadate (11 l.tM, MDCK; 2.2 l,tM, SMC) were toxic as judged microscopically.
Table 4. Effect of Vanadate on Arachidonic Acid Metabolism of RRL-1 Cells Stimulated by the Ca2+-Ionophore A-23 187
Arachidonic acid metabolite
A-23 187 (0.4 w) and Vanadate (55 ClM) *
A-23187 (0.4 PM)
Q&Lo PGE2 PGFza LTC4 12-HETE
0.90 1.02 12.98 13.43
* f f f
0.150 0.037 0.654 0.511
(8) (8) (8) (8)
1.14 0.91 2.09 3.15
f f f f
0.085 0.020 0.199 0.349
(4) (4) (4) (4)
* Vanadate at levels < 55 pM were not tested. t In MEM alone, ~0.2 ng of the cyclooxygenase and lipoxygenase products were present. Vanadate (55 JIM) did not affect these basal levels. Mean f SEM. ( ) = number of dishes.
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PROSTAGLANDINS
cyclooxygenase and lipoxygenase products is increased (13). In the presence of vanadate (55 PM), only the levels of lipoxygenase products were affected - their production by RBL-1 cells was inhibited (Table 4). Neither the morphology nor the number of the leukocytes was affected by these treatments. Arachidonic acid metabolism by WEHI- cells stimulated by the Ca2+- ionophore A-23 187 was affected by vanadate (55 PM) similarly, i.e. the levels of the peptide-containing leukotrienes were inhibited while those of cyclooxygenase products, PGE2 and TxB2, were not (data not shown). Discussion Sodium vanadate amplified 6-keto-PGFlo production by rat liver cells that had been incubated with several agonists, PGF2o production by MDCK cells that had been incubated with norepinephrine, and 6-keto-PGFld synthesis by SMC that had been stimulated by serotonin. The production of PGE2, PGF2d and 6-keto-PGFlo by rat mesangeal cells (16) and 6-keto-PGFld by glomerular epithelial cells (17) stimulated by Lys-ADH also was amplified by vanadate (11 @) (unpublished data). Vanadate, however, inhibited production of peptide-containing leukotrienes and 12hydroxyeicosatetraenoic acid by RBL- 1 cells, and peptide-containing leukotrienes by WEHI- cells stimulated by the Ca2+-’ionophore A-23187 - the levels of cyclooxygenase products of the two leukocyte cell lines stimulated by A-23187 were not affected. At none of these levels of vanadate used with these cells was a toxic response observed, at least as measured by the number of the cells and trypan blue exclusion or by microscopic examination after a 20-hour incubation. The amplification of PGF2, (MDCK) and 6-keto-PGFld (SMC and C-9) production by vanadate probably reflects regulation at a step distal to specific receptor binding as it was effective (at least with C9 cells) with several agonists that stimulate arachidonic acid metabolism by non-receptor mediated mechanisms, e.g. colchicine, A-23187 and thapsigargin (thapsigargin mobilizes intracellular Caz+ [ 191). With rat liver cells the regulation by vanadate is proximal to cyclooxygenase activity - no amplification was found when exogenous arachidonic acid was used as substrate. This is not surprising since several of the agonists used in this study (Table 2) stimulate arachidonic acid metabolism by increasing deesterification of phospholipids (l-3). Vanadate has been reported to affect several enzymes. Vanadate decreases Ca2+ATPase (20), ribonuclease (21), alkaline phosphatase (22) and acid phosphatase (23) activities - the effective concentrations used for some of these inhibitions are 2 to 3 orders of magnitude higher than those used in the present study. In studies with enzymes in a cell free system, concentrations of vanadate even less than those used in the present study were effective, especially inhibition of Na+,K+-ATPase (4). In intact cells, however, even 50 pM vanadate did not inhibit the Na+/K+-ATPase (5). At micromolar levels comparable to those used in this study, vanadate has been shown to be a specific inhibitor of protein-tyrosine-phosphate phosphatase -not an inhibitor of protein-serine (threonine)-phosphate phosphatase. The content of phosphotyrosine in
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1991 VOL. 41 NO. 1
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normal rat kidney cells (NRK-1) treated with vanadate (37.5 PM) for 3 days and incubated with 5 mCi phosphate for 24 hr was increased as much as 36 fold, but levels of protein-serine(threonine)-phosphate did not change (9). Vanadate is found in the cytoplasm of human red blood cells as the vanadyl ion associated with hemoglobin (18) - the vanadyl ion is much less effective than vanadate at inhibiting Na+/K+-ATPase (18). If vanadate is acting by inhibiting protein-tyrosine phosphatase, the relative effectiveness of the vanadyl ion would be of interest. If vanadate is reduced to the vanadyl ion in the cytoplasmic compartments of C-9, SMC, kidney cells or leukocytes, as it is in human erythrocytes, and if vanadyl is much less effective at inhibiting phosphatases, then vanadate may be acting on non-cytoplasmic protein-tyrosine phosphatases. The most likely pathway being regulated in the rat liver, dog kidney and smooth muscle cells is deesterification of cellular lipids. Several enzyme reactions have been implicated in deesterification of cellular lipids, including coupled phospholipase Cdiacylglycerol lipase reactions and phospholipase A2 activities (24). Vanadate amplifies arachidonic acid metabolism after incubation of cells with thrombin, PAF, Lys-ADH, A-23 187, IL- 1p, TPA, EGF, palytoxin, thapsigargin, colchicine, norepinephrine and serotonin. The mechanisms of stimulated arachidonic acid metabolism by such diverse agonists are likely to be very different. For example, serotonin acts by way of a type 2 receptor (2526) to liberate arachidonic acid from phospholipids by phospholipase A2 activity (26) while norepinephrine stimulates liberation of arachidonic acid by way of a (rl receptor and G-protein interaction to stimulate both phospholipase C and phospholipase A2 activity (27). EGF binds to its receptor to activate phospholipase C-y (28), although whether this activated phospholipase C-y liberates arachidonic acid via coupled diacylglycerol lipase activity is not known. Colchicine’s stimulation of 6-ketoPGFt, production probably reflects perturbation of the cytoskeleton (29) and activation of phospholipase AZ. Most likely both pathways of deesterification are being regulated by tyrosine phosphorylation, but not necessarily by the same proteins. Such regulation may not be attributable directly to tyrosine phosphorylation but indirectly to other kinases activated in a cascade that phosphorylates serine or threonine. Within the limits of our analyses, the initial rates of 6-keto-PGFt, production by the rat liver cells after incubation with vanadate are not affected. The increased stimulation of arachidonic acid metabolism by vanadate appears to be occurring at a relatively late stage of deesterification. Therefore, the possibility that vanadate is affecting other metabolic reactions in addition to inhibition of protein-tyrosine phosphatase can not be ruled out. The lipoxygenase, not the cyclooxygenase, pathway of the RBL-1 (Table 4) and WEHI- cells is inhibited by vanadate (55 l.tM) after stimulation by the Ca2+-ionophore A-23187. Presumably, phosphorylation of an enzyme is negatively regulating production of peptide-containing leukotrienes and hydroxyeicosatetraenoic acids. Most likely it is the lipoxygenase molecules that are inhibited by phosphorylation, but regulation of enzymes distal to 5-lipoxygenase activity, e.g. the 5-hydroperoxyeicosatetraenoic acid dehydrase or the glutathione-S-transferase are also possible.
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Although the effects of vanadate at levels less than 55 @I on RBL-1 cells were not measured, the effects of a higher level (275 pM) were. The A-23187-stimulated levels of LTC4 and 1ZHETE were inhibited even more effectively at 275 @4 than at 55 PM but those of PGEz and PGF2o still were not altered. Vanadate, even at 275 @l, caused no obvious morphological changes in the cells. The reasons for the lack of effect of vanadate on the cyclooxygenase products of the RBL-1 cells at levels higher than those found to be effective in other cells are not known. Possibly different pathways of deesterification exist in the leukocytes (30) or if the same pathways do exist, their threshold of regulatory control is higher. In either case, an enzyme in the lipoxygenase pathway is sensitive to negative regulation. In rat liver cells, the increases of PGI;! produced over basal levels after stimulation by physiological agonists in the presence of vanadate ranged from 2-fold with EGF to 7-fold with vasopressin; in SMC, 12-fold with serotonin; and in the dog kidney cells, Sfold with norepinephrine. The physiologic consequence of these amplifications (or lipoxygenase inhibitions) is not known. In addition, at the non-toxic levels used in this study, it is possible that not all protein-tyrosine phosphatase activity is being inhibited - the amplification (or inhibition) could be much higher if protein-tyrosine phosphatase activities were eliminated. Sodium vanadate, however, is toxic to cells in culture - the toxicity varies among cells (55 ~,LMfor C-9; 11 pM for MDCK, 2.2 l.tM for SMC, and greater than 55 @4 for the leukocytes). Earlier studies (9) showed that 50 p.M vanadate affected survival of sparse cultures of NRK-1 cells. NM-6Cl-32 cells and 10 Tic! were even more sensitive to the toxic effects of vanadate. This toxicity to vanadate may be caused by sustaining an activity that is regulated by phosphorylated tyrosines. Each cell’s sensitivity would reflect that sustained activity.
Acknowledgements This work was supported by Grant RDP- 12K from the American Cancer Society. I wish to thank Nancy Worth for technical assistance and Inez Zimmerman for preparation of the manuscript.
References 1, Curtis-Prior, P.B., ed. Prostaglandins: Biology and Chemistry of Prostaglandins and Related Eicosanoids. Churchill Livingstone, Edinburgh, 1988. 2. Levine, L. Tumor promoters, growth factors and arachidonic acid metabolism. In: Prostaglandins in Cancer Research (Garaci, E., Paoletti, R., and Santoro, M.G., eds.), Springer-Verlag, Berlin, 1987, pp. 62-73. 3. Levine, L. Tumor promoters and prostaglandin production. In: Eicosanoids, Lipid Peroxidation and Cancer (Nigam, S.K., McBrien, D.C.H., and Slater, T.F., eds.) Springer-Verlag, Berlin, 1988, pp. 11-21.
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Received:
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Accepted:
lo-S-90
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