mechanism of 12-0-tetradecanoylphorbol-13-acetate enhanced...

(CANCER RESEARCH 50.4650-4655 August 1. I990|

Mechanism of 12-0-Tetradecanoylphorbol-13-acetate Enhanced Metabolism ofArachidonic Acid in Dog Urothelial Cells1

Terry V. Zenser,2 Thomas E. Kling, Zofia M. Duniec, Yun-Hua Huang Wong, and Bernard B. Davis

Geriatric Research, Education, and Clinical Center, Veterans Administration Medical Center, and Department* of Biochemistry and Internal Medicine, St. l^ouisUniversity School of Medicine, St. Louis, Missouri [T. V. 2.., Y-H. H. W., B. B. Â£>./,and Laboratory of Molecular Biophysics, National Institute of Environmental HealthScience, Research Triangle Park, North Carolina [T. E. E., Z. M. D.J

ABSTRACT

The mechanism of 12-O-tetradecanoylphorbol-13-acetate (TPA) enhanced arachidonic acid metabolism Â»asinvestigated in dog urothelialcells. Primary cultures of dog urothelial cells were grown to confluencyand evaluated in the presence or absence of overnight prelabeling with['ll|arachidonic acid. High-performance liquid chromatography analysisof media from TPA stimulated cells indicated that prostaglandin !â€¢..(l'<.!â€¢;.,)was the major eicosanoid produced. Lipoxygenase products were

not detected. Control cell media contained only arachidonic acid. Effectsof selected inhibitors on TPA and exogenous arachidonic acid mediatedincreases in radioimmunoassayable !'(,! were assessed. Prostaglandin

II synthase inhibitors (indomcthacin and aspirin) prevented both TPAand arachidonic acid increases in I'(,I ,. By contrast, inhibitors of phos-pholipases (quinacrine, \V-7, and trifluoropromazine), protein synthesis(cycloheximide), and protein kinase C (staurosporine) prevented TPAbut not arachidonic acid increases in l'( â€¢!â€¢..The latter agents also reduced

TPA mediated increases in the release of total radioactivity from cellslabeled with ['lljarachidonic acid. However, aspirin reduced the amountof 3H-prostaglandins formed with TPA. A calcium requirement was

demonstrated when increases in radioimmunoassayable PGE2 elicited byTPA and the calcium ionophore A23187 were reduced with calciumdepleted media. When epidermal growth factor in combination with eitherTPA or bradykinin was used, at least additive effects were observed withrespect to release of ['Hjarachidonic acid, 'H-prostaglandins, and radioimmunoassayable I'l.l .. These experiments suggest that separate path

ways may be involved in enhanced arachidonic acid metabolism demonstrated with different agonists. For TPA, increased arachidonic acidrelease occurs by a calcium dependent process involving phospholi-pase(s), protein synthesis, and protein kinase C.

INTRODUCTION

Hormones and growth factors are required to maintain cellgrowth, proliferation, and differentiation. They communicatetheir information across cell membranes by second messengersystems (signal transduction). Second messengers include cyclicAMP, inositol 1,4,5-triphosphate, and diacylglycerol (1-3).Eicosanoids produced by arachidonic acid metabolism can serveas second or subsequent messengers in cells (4, 5). Eicosanoidsare also involved in several steps in the carcinogenic process(i.e., initiation, promotion, differentiation, and metastasis) (6-9). The tumor promoter TPA' mimics the actions of the second

messenger diacylglycerol on protein kinase C (10). Tumorpromoting effects of TPA in the mouse skin model are mediated, at least in part, by its activation of protein kinase C (11)and involve arachidonic acid metabolism (12). The importance

Received 3/16/90.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported by the Department of Veterans Affairs and byUSPHS Grant CA-28015 from the National Cancer Institute through the National Bladder Cancer Project.

2To whom requests for reprints should be addressed, at Geriatric Center(111G-JB). Veterans Administration Medical Center, St. Louis, MO 63125.

'The abbreviations used are: TPA, 12-O-tetradecanoylphorbol-13-acetate;PGEÂ¡,prostaglandin E2 (other prostaglandins are similarly designated): Â«-MEM;Â»-minimalessential medium; BSA, bovine serum albumin; HPLC. high-performance liquid chromatography; HHT. 12-hydroxy-5,8.10-heptadecatrienoic acid;TFP, trifluoropromazine; EGF, epidermal growth factor.

of signal transduction pathways in carcinogenesis is furtheremphasized by the homology of several oncogenes to variouscomponents in these pathways (13) and the demonstration thatectopie expression of receptors can cause malignant transformation ( 14). Thus, an understanding of molecular mechanismsrelated to transmembrane signaling and second messenger systems will provide additional information about the multistepcarcinogenic process.

Dog is the animal model for studying aromatic amine inducedbladder cancer (15, 16). Dog urothelial cells metabolize aromatic amine bladder carcinogens (17, 18) and undergo unscheduled DNA synthesis in response to these carcinogens (19, 20).Bladder malignancies are carcinomas derived from urothelialcells. Interest in signal transduction pathways has resulted inthe initiation of studies evaluating regulation of arachidonicacid metabolism in urothelial cells. Arachidonic acid metabolism was evaluated in dog relative to human urothelial cells.Results indicate that similar cellular regulatory processes existin each species (21-24). For example, the same agonists wereshown to increase PGE2 production in both species with sub-culturing and the absence of serum or the high concentrationof serum reducing responsiveness. Thus, urothelial cell signaltransduction pathways can be evaluated by examining regulation of arachidonic acid metabolism. In the present study, theprofile of eicosanoids produced from control and TPA stimulated cells was determined. In addition, the mechanism of TPAenhanced arachidonic acid metabolism was detailed and compared to that of other agonists. These agonists include thepolypeptide hormones bradykin and epidermal growth factorand the calcium ionophore A23187 (24). This diverse group ofagonists was chosen to maximize the information obtained.Results indicate similarities and differences in the mechanismsby which agonists influence cell signal transduction pathwaysin dog urothelial cells.

MATERIALS AND METHODS

Preparation of Cell Cultures. Urothelial cells from the urinary bladderof the dog were prepared and cultured by the modification of a methodused for human urothelial cells (21, 25) as previously described (24).using aseptic procedures, freshly detached cells were centrifuged at1500 rpm for 10 min at 4Â°C.The cells in the pellet were carefullyresuspended in a small volume of medium. Approximately 5x10*

freshly detached cells in 3 ml of medium containing I% fetal calf scrumwere added to 35-mm plastic tissue culture plates coated with 0.2%gelatin (Fisher Scientific Co., St. Louis, MO) and incubated at 37Â°Cin

5% CO2-95% humidified air with medium changed biweekly. Mediumconsisted of Ham's nutrient mixture F12 (G1BCO, Grand Island, NY)

supplemented as previously described (21).Experiments were conducted with 80-90% confluent cultures (3-4

x IO5cells/35 mm plate). Growth media were aspirated, and cells werewashed twice with 1 ml of Hanks' balanced salt solution. Serum-free

Â«-MEMwas then added with or without test agents as indicated in the"Results." Cells were incubated at 37Â°Cin 5% CO2-95% air. Test

agents used were arachidonic acid, purchased from Nu-Chek Prep, Inc.,Elysian, MN; calcium ionophore A23187 and staurosporine from Cal-

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STIMULATION OF PGE2 PRODUCTION BY TPA

biochem. La Jolla, CA; and cyclohcximide, quinacrine, W-7, trifluoropromazine, indomethacin, aspirin, bradykinin triacetate, epidermalgrowth factor, and TPA from Sigma Chemical Co., St. Louis, MO.Each agonist was used at a maximally effective concentration, previously determined (24).

Prelabeling. Urothelial cells were rinsed twice with serum-free mediaand incubated with 1 Â¿iCi[JH]arachidonic acid (100 Ci/mmol; Du Pont

NEN Research Products, Boston, MA). After 1 h, fetal calf serum wasadded to a final concentration of 1%, and cells were incubated overnight(24). Cells were then rinsed twice with Â«-MEMcontaining 5 mg/mlBSA and twice with Â«-MEMcontaining l mg/ml BSA. All subsequentconditions contained Â«-MEM with 1 mg/ml BSA. After a 30-minpreincubation in the presence or absence of inhibitors, agonists wereadded in fresh media containing the corresponding inhibitor. Mediawere analyzed for products of arachidonic acid metabolism.

Analysis of Arachidonic Acid Metabolites in Media. Arachidonic acidmetabolites were identified by HPLC analysis. Media were acidified topH 3.5 with glacial acetic acid, ethanol was added to a final concentration of 10%, and media were applied to a disposable extraction(PrepSep-C18) column. Retained material was eluted with methanol,concentrated, and used for subsequent analysis. Metabolites were separated by reverse-phase HPLC, with a C|8 Ultrasphere ODS column(Altex Scientific. Inc., Beckman Instruments. Inc., Berkeley, CA) usingmethanol and buffered water (pH 5.05) at a flow rate of 1.1 ml/min asdescribed previously (26). The PGE2 and PGD2 fraction cluting fromthe reverse-phase HPLC column was collected, concentrated, and reap-plied to a straight-phase column (/j-Porasil, 10 /jm, from Waters Associates, Milford, MA). The prostaglandins were eluted with hex-ane:ethanol:acetic acid (994:6:1) for 25 min followed by a linear gradient to 809; solvent of hexane:ethanol:acetic acid (90:10:1) for anadditional 60 min at a flow rate of 3.0 ml/min (27).

To determine effects of inhibitors on radiolabeled nonpolar versuspolar material in media, solvent extraction was used as previouslydescribed (28). The percentage of arachidonic acid (nonpolar) andprostaglandins (polar) was determined. Protein was precipitated bycentrifugation following addition of two volumes of cold acetone. Mediawere then extracted with two volumes of petroleum ether. After removalof the organic phase, the aqueous phase was extracted an additionaltwo times with petroleum ether. All organic phases were combined andevaporated. Radioactivity in the organic and aqueous fractions wasexpressed as percentage of arachidonic acid and prostaglandins, respectively. The organic and aqueous fractions were characterized by thin-layer Chromatographie analysis using silica gel F-254 plates. Plateswere developed with chloroform:methanol:acetic acid (90:5:5). Prosta-glandin standards and arachidonic acid were applied as carriers andvisualized with iodine vapors. The radioactivity of zones correspondingto standards was determined (29). The organic fraction was found tocontain arachidonic acid, whereas the aqueous polar fraction was predominantly prostaglandins. PGE2 was the major prostaglandin. Theseresults are qualitatively consistent with those obtained with HPLCanalysis and reported in Fig. 1.

Radioimmunoassay of Prostaglandin E2. The PGE2 content of themedia was measured by double-antibody radioimmunoassay (30). Rabbit antiserum to PGE2 was obtained from Regis Chemical Co., MortonGrove, IL. Tritium-labeled PGE2 was purchased from Du Pont NENResearch Products, and goat antiserum to rabbit 7-globulins was purchased from Antibodies, Inc., Davis, CA. The medium from each platewas analyzed in duplicate, and the value of this duplicate determinationwas averaged and considered as N = 1. Data were expressed as themean of duplicate experiments or as the mean Â±SEM of ng PGE2/105

cells. All experiments were repeated at least three times.

RESULTS

Metabolite Characterization. The profile of eicosanoids produced by urothelial cells was assessed using ['HJarachidonic

acid prelabeled cells. Media from control and cells stimulatedwith TPA for 2 h were assessed by reverse-phase HPLC. Asshown in Fig. 1, arachidonic acid was the only radiolabeled

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Fig. 1. Reverse-phase HPLC profile of radiolabeled eicosanoids derived from[3H)arachidonic acid prelabeled cells. Positions at which eicosanoid standardselute are indicated. Chromatographie conditions are described in "Materials andMethods."* . media from control cells; , media from TPA treated cells.

compound detected in control media. Following TPA stimulation, additional peaks of radioactivity were observed. The peakwhich coeluted with the PGE2 and PGD2 standards containedthe most radioactivity (25% of recovered radioactivity). Twosmall peaks were observed that coeluted with 6-keto-PGF,,,(6%) and 12-hydroxy-5,8,10-heptadecatrienoic acid (2.5%) butwere not further characterized. Leukotrienes (i.e., LTB4) andhydroxyeicosatetraenoic acids (i.e., 15-HETE and 5-HETE)were not detected, indicating the lack of lipoxygenase activity.The peak of radioactivity which Ã©lÃ»teswith the PGE2 and PGD2standards in Fig. 1 (20-22 min) was further analyzed bystraight-phase HPLC. A single peak was observed that coelutedwith PGE2 standard and confirmed the identity of the majoreicosanoid product as PGE2. Thus, in subsequent experiments,PGE2 was determined by radioimmunoassay. The amount ofradioactivity present in media from cells treated with TPA wassignificantly higher than that from control cells, suggesting thatTPA may act in part by stimulating phospholipase activity.

Effects of Inhibitors. To further characterize the response toTPA, specific inhibitors were used (Fig. 2). TPA increasedPGE2 formation from endogenous arachidonic acid from 0.27Â±0.09 in control (data not shown) to 3.52 Â±0.44 ng/105 cells

as measured by radioimmunoassay. PGE2 synthesis was significantly decreased in the presence of inhibitors of prostaglandinH synthase (indomethacin), phospholipases (quinacrine, W7,and trifluoropromazine), protein synthesis (cycloheximide), orprotein kinase C (staurosporine). Indomethacin and quinacrinewere the most effective and trifluoropromazine the least effective inhibitors. The concentrations of inhibitors used were maximal, as determined in previous experiments. A lower concentration of cycloheximide (10 MM)than used in Fig. 2 inhibitsprotein synthesis in human urothelial cells by 90-95% (22).

Effects of these inhibitors on PGE2 production from exogenous arachidonic acid were investigated (Fig. 2). The use ofexogenous arachidonic acid bypasses the requirement for phospholipase activity (endogenous arachidonic acid) and focusesspecifically on the ability of the prostaglandin H synthaseenzyme to produce PGE2. The addition of arachidonic acid tocells increased PGE2 from 0.27 Â±0.09 in control (data notshown) to 6.58 Â±0.87 ng/105 cells. Indomethacin and aspirin

(data not shown) significantly reduced arachidonic acid increases in PGE2. Inhibitors of phospholipases, protein synthe-

4651



STIMULATION OF PGE; PRODUCTION BY TPA

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prostaglandin H synthase. Thus, prostaglandins synthesizedfollowing aspirin treatment would require synthesis of newenzyme. ['HJArachidonic acid prelabeled cells were pretreated

with l mM aspirin for 30 min and then washed twice to removeaspirin before incubation with TPA (Fig. 4). The amount ofradiolabeled prostaglandins was completely abolished. In aparallel experiment, TPA increases in radioimmunoassayablePGE2 were correspondingly inhibited by preincubation of cellswith aspirin. However, the amount of radiolabeled arachidonicacid released was not significantly altered by aspirin pretreatment. Similar effects were observed following bradykinin addition to aspirin treated cells (data not shown). Thus, these resultssuggest that the agonists TPA and bradykinin are not inducingthe synthesis of prostaglandin H synthase.

Calcium Requirement. The calcium requirement for agoniststimulated PGE2 production was evaluated (Fig. 5). Cells werepreincubated in media that contained or was devoid of calciumfollowed by treatment with agonists for 60 min. In the absenceof media calcium PGE2 synthesis was reduced by 90% withTPA and A23187. These results suggest the involvement ofphospholipase(s) which require calcium for TPA and A23187increases in PGE2.

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Fig. 3. Effects of different inhibitors on TPA mediated release of endogenousarachidonic acid, [3H]Arachidonic acid prelabeled cells were preincubated withthe same inhibitors used in Fig. 2. Media radioactivity was assessed 2 h afteraddition of 100 nM TPA. The amount of radioactivity in media from control cellswas 19,600 Â±940 cpm.

sis, and protein kinase C were ineffective. These results suggestthat effects of the latter inhibitors on TPA increases in PGE2synthesis must be due to their regulation of endogenous arachidonic acid levels.

Release of Endogenous I^HjArachidonic Acid. To measure

release of endogenous arachidonic acid, cells were prelabeledwith ['Hjarachidonicacid. Effects of inhibitors were determined

by assessing radioactivity of media (Fig. 3). TPA stimulationresulted in an increased release of 109,000 Â±3,000 cpm overcontrol values. This corresponds to a 5.5-fold increase in radioactivity elicited by TPA. Thus, TPA clearly increased the releaseof endogenous arachidonic acid. All inhibitors tested reducedTPA mediated release of radioactivity. Cycloheximide was themost (96% inhibition) and TFP the least effect (70%) inhibitor.

Synthesis of Prostaglandin H Synthase. TPA has been shownto enhance de novo synthesis of the enzyme prostaglandin Hsynthase in several cells including an epithelial cell line (31-34). To detect increased synthesis of prostaglandin H synthase(35), cells were preincubated with aspirin, an irreversible inhibitor of prostaglandin H synthase (36), to inhibit any preexisting

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and radioimmunoassayable PGE; elicited by TPA. Cells were pretreated in thepresence and absence of 1 mM aspirin (ASA) for 30 min and then TPA for 2 h.Both unlabeled and |3H]arachidonic acid prelabeled cells were used. With the

former, radioimmunoassayable (RIA) PGE2 was assessed in media. Media fromprelabeled cells were monitored for ('Hjarachidonic acid (AA) and 'H-prostaglan-dins (PCs). See details in "Materials and Methods."

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Fig. 5. Calcium requirement for agonist stimulated PGEÂ¡synthesis was assessed. Cells were preincubated for 30 min in the presence or absence of calciumdepleted media containing 0.05 mM EDTA. Agonists were added for 1 h. andmedia PGEj content was assessed. The absolute response was obtained bysubtracting control from agonist stimulated values. Control values in the presenceand absence of calcium were 5.8 Â±0.7 and 1.9 Â±0.5 ng/10* cells, respectively.

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Effect of Other Agonists. The effect of other agonists onarachidonic acid metabolism was evaluated (Fig. 6). EGF wasevaluated alone and in combination with TPA and bradykinin.Each agonist was used at maximum effective concentrations(24). Radioactive and nonradioactive experiments were done inparallel. EGF produced modest increase in each parameter.Increases in radioimmunoassayable PGE2 were significantlygreater in the presence than absence of EGF. In addition, theamount of 3H-prostaglandins observed was greater in the pres

ence than absence of EGF. However, increases were only significant with bradykinin plus EGF compared to bradykininalone. Values observed with release of [3H]arachidonic acid

were increased in the presence compared to the absence ofEGF, although none achieved significance.

DISCUSSION

This is the first investigation to determine the profile ofeicosanoids produced by dog urothelial cells. Cellular prosta-glandin H synthase was demonstrated by the detection of pros-taglandins. PGE2 was the most abundant eicosanoid produced.6-Keto-PGFiQ and HHT were also detected in smaller amountsand only tentatively identified. 6-Keto-PGF,â€ž is a decomposition product of PGI2 metabolism (37). HHT is a product ofboth the thromboxane and prostaglandin pathways. However,because thromboxane was not detected, HHT was probablyderived from the prostaglandin pathway (38). These results aresimilar to those of a previous study assessing arachidonic acidmetabolism of human urothelial cells (21). In human cells,PGE2 is the major eicosanoid synthesized, with significantamounts of 6-keto-PGFiâ€ž and HHT also being detected.HPLC analysis of dog urothelial cell media failed to detectlipoxygenase products including leukotrienes. In contrast, human cells produced small (6% of the total eicosanoids detected)but significant amounts of lipoxygenase products. The latterwere only tentatively identified. The current study does not ruleout the possible presence of lipoxygenases in dog urothelialcells. Other unsaturated lipids may prove to be better substratesfor these enzymes than arachidonic acid (39, 40). Thromboxaneand leukotrienes were not detected with either dog or humanurothelial cells.

The increase in prostaglandin production observed with TPAand other agonists has been attributed to de novo synthesis ofthe enzyme prostaglandin H synthase (31-34, 41-43). In those

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radioimmunoassayable (RIA ) PGE2 was assessed in media. Media from prelabeledcells were monitored for [3H]arachidonic acid (AA) and 3H-prostaglandins (PCs).

TPA (0.1 ^M), EGF (0.1 /jg/ml), and bradykinin (BK) (0.8 ^M) were presentwhere indicated. See details in "Materials and Methods." The absolute response

was obtained by subtracting control from agonist stimulated values.

studies, cycloheximide prevented the increase in prostaglandinH synthase and the subsequent increase in prostaglandin production. Aspirin is an irreversible inhibitor of prostaglandin Hsynthase (36). Thus, new enzyme synthesis is required beforeeicosanoids can be produced. In cells pretreated with aspirin,agonist (i.e., TPA) increases in prostaglandin production dueto synthesis of new enzyme are not prevented (31, 42, 43). Inthe current study, cycloheximide prevented increases in PGE2elicited by TPA. However, aspirin pretreatment prevented TPAincreases in PGE2 and radiolabeled prostaglandins but not therelease of arachidonic acid. Thus, TPA does not appear to beincreasing synthesis of new prostaglandin H synthase enzymeduring the 2-h incubation period. TPA mediated increases inurothelial cell prostaglandin H synthase synthesis may occurlater.

Increased availability of arachidonic acid substrate is anothermechanism by which agonists regulate increases in prostaglandin production. TPA dramatically increased the release of radioactivity from [3H]arachidonic acid prelabeled urothelial cells.

These results are consistent with this material being initiallyarachidonic acid. That is, arachidonic acid is the only compoundidentified in media from TPA stimulated cells pretreated withaspirin (Fig. 4). Thus, TPA elicits an increased release ofendogenous arachidonic acid which is metabolized by prostaglandin H synthase to prostaglandins, primarily PGE2. Cycloheximide treatment reduced the TPA mediated release of radioactivity and synthesis of PGE2 which is consistent with reducedavailability of arachidonic acid. Inhibitors of phospholipases(quinacrine, W-7, and TFP) (44) and protein kinase C (stauros-porine) (45) also inhibited both release of radioactivity andPGE2 production mediated by TPA. The lack of effect of theseinhibitors on increased PGE2 production mediated by exogenous arachidonic acid demonstrates the specificity of theireffects (Fig. 2). Thus, TPA regulated increases in eicosanoidsappear to require protein synthesis and involve phospholi-pase(s) and protein kinase C dependent pathways.

In this study, treatment of cells with cycloheximide preventedthe increase in prostaglandin production and suggests thatprotein synthesis is required for TPA stimulation. The dependency of arachidonic acid release on protein synthesis and geneexpression has been previously observed. Clark et al. (46) haveshown that cycloheximide and actinomycin D prevented theleukotriene D4 stimulated release of arachidonic acid and eicosanoid production by smooth muscle and endothelial cells. Thestimulatory effects of leukotriene D4 were attributed to synthesis of a phospholipase A2 stimulatory peptide (47). In humanand dog urothelial cells, TPA increases in PGE2 synthesis aretime delayed and dependent upon protein synthesis (22, 24).The present study is the first to demonstrate that cycloheximideactually blocks urothelial cell release of radioactivity mediatedby TPA. In dog urothelial cells, TPA increased PGE2 synthesisis also prevented by actinomycin D (24). Although our resultsindicate dependency of arachidonic acid release on proteinsynthesis and gene expression, it is not clear whether phospholipase A2 and/or phospholipase C pathways are involved.

Protein kinase C is the major cellular receptor for TPA (10).The tumor promoting effects of TPA in the mouse skin modelare mediated, at least in part, through protein kinase C (11).Protein kinase C has been suggested to phosphorylate andmodulate the activity of a family of proteins (lipocortins) thatinhibit phospholipase A2 (48). Phosphorylation mediated activation of phospholipase A2 is thought to be responsible forTPA mediated increased release of arachidonic acid and prostaglandin synthesis in MDCK cells (49). Staurosporine inhibi-

4653




tion of TPA mediated urothelial cell release of radioactivityand synthesis of PGE2 suggests that protein kinase C is involvedin regulation of arachidonic acid metabolism in this modelsystem.

Calcium can function as a second messenger and/or be anecessary cofactor for subsequent steps in signal transductionpathways. Previous studies including those with human urothelial cells have demonstrated a calcium requirement for TPAresponses (22). Phospholipase A2 (50), phospholipase C (51,52), and protein kinase C ( 10) require calcium. Calcium requirements of the latter enzymes could explain the reduced synthesisPGE2 observed in calcium depleted media with TPA andA23187.

Effects of inhibitors have been attributed, in the precedingdiscussion, to specific processes. However, other effects of theseagents should be considered. Trifluoperazine and W-7 are classified as calmodulin antagonists. Calmodulin antagonists including W-7 have antiproliferative and antitumor effects (53).Since calmodulin does not activate purified phospholipase A2(44), the effects of trifluoperazine and W-7 on arachidonic acidrelease may relate to the calcium dependency of this enzyme orinvolve phospholipase C. The proposed effects of trifluoperazine and W-7 on phospholipase(s) are consistent with theinhibition observed with quinacrine. Quinacrine is often usedto assess the involvement of phospholipase pathways (44). Highconcentrations of nonsteroidal antiinflammatory agents mayinhibit phospholipase A2 (44). However, nonsteroidal antiinflammatory agents did not reduce agonist mediated release ofarachidonic acid. Instead, they prevented increases in PGE2production by way of their inhibitory effects on the fatty acidcyclooxygenase activity of prostaglandin H synthase. Althoughthe inhibitors used have a variety of effects on different processes, the use of multiple inhibitors with different chemicalstructures that have overlapping effects on phospholipases suggests a role for these enzymes in TPA mediated PGE2 synthesis.

Cell viability was used as a basis for selecting maximallyeffective concentrations of test agents. Cell count and arachidonic acid responsiveness were used as indexes of cell viability.When conducting dose response studies, concentrations of testagents which decreased cell count or arachidonic acid stimulation were not chosen. Indomethacin was the one exception.Indomethacin is an inhibitor of the fatty acid cyclooxygenaseactivity of prostaglandin H synthase. These studies also demonstrated that effects of test agents were dose responsive. Thus,the dose response effects of test agents and the lack of effect oftest agents on cell viability provide additional support for theproposed actions of these agents.

Nonequilibrium metabolic labeling with ['Hjarachidonic acid

can result in measurements of radioactivity released which arenot representative of the actual mass of arachidonic acid released. In the current study, the measurement of radioimmuno-assayable PGE2 closely correlated with radioactivity released.Thus, our results are not a consequence of nonequilibriummetabolic labeling.

Urine has components which stimulate cell proliferation andare tumor enhancing. EGF and EGF-related growth factorsrepresent a majority of this activity in urine (54). Previousstudies demonstrated that this polypeptide hormone increasedPGE2 synthesis in dog urothelial cells (24). When EGF wasused in combination with either TPA or bradykinin, at leastadditive effects were observed with respect to release of |'H]-rachidonic acid, 'H-prostaglandins, and radioimmunoassayable

PGE2. Since maximal concentrations of each agonist were used,additive results suggest that EGF is acting by mechanisms

distinct from TPA or bradykinin. EGF stimulated release ofarachidonic acid and production of eicosanoids are necessaryfor the proliferative response it elicits in quiescent BALB/c3T3 fibroblasts (55). Eicosanoids represent important intracel-lular mediators of hormonal responses. This report demonstrates that evaluation of mechanisms involved in TPA enhanced arachidonic acid metabolism will provide an understanding of signal transduction and second messenger pathwaysin urothelial cells.

ACKNOWLEDGMENTS

The authors appreciate the expert technical assistance of PatriciaEaston, Cindee Rettke, and Alma Schisla Â¡nconducting the experiments.

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1990;50:4650-4655. Cancer Res Terry V. Zenser, Thomas E. Eling, Zofia M. Duniec, et al. Metabolism of Arachidonic Acid in Dog Urothelial Cells

-Tetradecanoylphorbol-13-acetate EnhancedOMechanism of 12-

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