Evidence of Protein Kinase C Involvement in Phorbol Diester ...

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azine was a gift from Dr. Craig Gerard, Department of Medicine,. Bowman ...... fonyl)piperazine, and Dr. Linda McPhail for assistance in the devel- opment of the ...
THE JOURNALOF BIOLOGICAL CHEMISTRY 0 1987 by The American Society of Biological Chemists, Inc.

Vol. 262, No. 11, Issue of April 15, pp. 1987 5385-5393, Printed in U.S.A.

Evidence of Protein Kinase C Involvement in Phorbol Diester-stimulated Arachidonic Acid Release and Prostaglandin Synthesis* (Received for publication, April 17, 1986)

Judy Parker$, LarryW. Daniel, and Moseley Waite From the DeDartment of Biochemistry. Bowman Grav School of Medicine of Wake Forest Uniuersity, WinstonSalem, NorthCarolina 27103 “ I

Many stimulators of prostaglandin production are The prostaglandins,hydroxyeicosatetraenoic acids, thromthought to activate the Ca2+-and phospholipid-depend- boxanes, andleukotrienes(the eicosanoids) areimportant ent protein kinase first described by Nishizuka and his local modifiers of biological function and are synthesized from colleagues (Takai, Y., Kishimoto, A., Iwasa, Y., Ka- arachidonic acid by cellular enzymes. However, before PGH’ wahara, y.,Mori, T., and Nishizuka, Y. (1979)J. Biol. synthase (cyclooxygenase) or lipoxygenase converts arachiChem. 254, 3692-3695. In this paper we report evidonicacid into the variouseicosanoids, it is thought that dence that the activation of protein kinase C caused byarachidonic acid must be released from cellular lipids where 12-0-tetradecanoylphorbol-13-acetate(TPA) is involved in the increased prostaglandin production in- it is esterified predominantly into phospholipids (1).Activaduced by 12-0-tetradecanoylphorbol-13-acetatein tion of arachidonic acid release is currently considered the Madin-Darby canine kidney (MDCK) cells. We have principal mechanism for stimulation of eicosanoid synthesis. shown that TPA activates protein kinase C in MDCK Epidermal growth factor (2), hormones (3, 4),andtumor increase thesynthesis of cells with similar dose response curve as observed for promoters (5-7) arethoughtto TPA induction of arachidonic acid release in MDCK eicosanoids by activation of arachidonic acid release. Howcells. Activation of protein kinase C was associated ever, production of eicosanoids is also controlled by the activwith increased phosphorylation of proteins of 40,000 ity of PGH synthase and lipoxygenase which catalyze the first and 48,000 daltons. We used two compounds (1-0- step in the synthesis of prostaglandins/thromboxanes and octadecyl-2-O-methyl-rac-glycero-3-phosphocholineHETEs/leukotrienes, respectively. The activity of PGH syn(ET-18-OMe) and 1-(5-isoquinolinesulfonyl)pipera- thase is increased by epidermal growth factor (8), TPA (9, zine) known to inhibit protein kinase C by different IO), and bradykinin (11).Previous studies in our laboratory if activation of protein have shown that prolonged stimulation of PGHsynthase mechanisms to further examine kinase C was involved in the increased synthesis of activity by TPA in MDCK cells was blocked by inhibitors of prostaglandins in TPA-treated MDCK cells. We found that both compounds inhibited protein kinase C par- protein synthesis (9), whereas the stimulation of arachidonic tially purified from MDCK cells and thatET-18-OMe acid release by TPA was independent of protein synthesis two mechanisms of increasinhibited the phosphorylation of proteins by protein (12). Thus, TPA appears to have of prostaglandins: 1) increased release of araing synthesis kinase C in the intact cells. Addition of either compound during or afterTPA treatment decreased both chidonic acid and 2) enhanced conversion of arachidonic acid to prostaglandins. Only the latter TPA effect is sensitive to release of arachidonic acid from phospholipids and prostaglandinsynthesis.Release of r3H]arachidonic cycloheximide, which indicates that TPA either stimulates the synthesis of PGH synthase or of an activator of this acid from phosphatidylethanolamine in TPA-treated cells was blocked by ET-18-OMe or 1-(5-isoquinoline- enzyme (9). sulfony1)piperazineaddition.However,arachidonic Many stimuli that causeproduction of eicosanoidsalso acid release stimulated by A23187 is not blocked by activate protein kinase C including tumor promoters, growth Et-18-OMe. When assayed in vitro, treatment of cells factors, hormones, and neurotransmitters (5-7, 13). Protein with Et-18-OMe did notprevent the enhanced conver- kinase C is a ubiquitous enzyme involved in the transduction sion of arachidonic acid into prostaglandins induced of biological signals (reviewed in Ref. 13). It is possible that by pretreatmentof cells with TPA. Our resultssuggest activation of protein kinase C might mediate the increase in that the stimulation of phospholipase A2 activity by TPA occurs via activation of protein kinase Cby TPA. prostaglandin synthesis caused by this diverse group of stimuli. Indeed, protein kinase C has been suggested to phosphorylate and thus modulate theactivity of a family of proteins (lipocortins) that inhibit phospholipase AP (14-17). Here we examine the role of protein kinase C in the stimulation of * This investigation was supported by grants from the American Heart Association, North Carolina Affiliate (1985-86-A-21), the Na- release of arachidonic acid and of prostaglandin synthesis by tional Institute of Health (HL31338, CA43297, DK11799), the Amer- TPA. ican Cancer Society (CH373), Forsyth Cancer Service, and National Research Service Award T32 CA 09422 from the National Cancer Institute. $’ Postdoctoral fellow under National Research Service Award T32 CA 09422 from the National Cancer Institute. Towhom correspondenceshould be addressed: Dept. of Biochemistry, Bowman Gray School of Medicine, Wake Forest University, 300 South Hawthorne Rd., Winston-Salem, NC 27103.

The abbreviations used are: PGH,, prostaglandin H,; TPA, 12-0tetradecanoylphorhol-13-acetate; ET-18-OMe, l-O-octadecyl-2-0methyl-rac-glycero-3-phosphocholine;protein kinase C, Ca2+/phospholipid-dependentproteinkinase; EGTA, ethylene glycol his(@aminoethy1ether)-N,N,N’,N’-tetraacetic acid; SDS, sodium dodecyl sulfate; MDCK, Madin-Darby canine kidney; TES, 2-([2-hydroxy1,l-bis(hydroxymethyI)ethyl]amino~ethanesulfonic acid.

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Arachidonic Acid Release and Protein Kinase MATERIALS ANDMETHODS

Chemicals-Arachidonic acid was from NuChek Prep. Inc., Elysian, MN; A23187 from Behring Diagnostics, and indomethacin and prostaglandins from Sigma. All phospholipids were from Serdary Research Laboratories Inc. (London, Ontario, Canada), except ET18-OMe whichwas a gift from Dr. Wolfgang E. Berdel, Munich, Federal Republic of Germany. The 1-(5-isoquinolinesulfonyl)-piperazine was a gift from Dr. Craig Gerard, Department of Medicine, Bowman Gray School of Medicine and was prepared as described (18). DEAE-Sephacel was from Pharmacia P-L Biochemicals. Histone type IIIS, bovine serum albumin, P-mercaptoethanol and phenylmethylsulfonyl fluoride were from Sigma. Diolein was from Serdary Research Laboratories Inc. Carrier-free 32P04 (approximately 285 Ci/ mg phosphorus) was from ICN and [y-32P]ATP(8-12 Ci/mmol) was from New England Nuclear Research Products. [5,6,8,9,11,12,14,15'HH]Arachidonic acid (135 Ci/mmol) was from Amersham Corp. Silica Gel 60 plates were prepared by E. Merck, Darmstadt, Federal Republic of Germany (#5721-7) and Er~[~H]ance was from NEN Research Products. Fetal bovine serum was from Gibco Laboratories, whereas medium and antibiotics for tissue culture were from Flow Laboratories, Rockville, MD. Allother chemicals were reagent grade or better. Growth of Madin-Darby Canine Kidney Cells-MDCK cells were obtained from Flow Laboratories. The cells weregrown in plastic flasks in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (heat inactivated), penicillin 100 units/ ml, and streptomycin sulfate 100 pg/ml. For studies of the toxicity of ET-18-OMe confluent MDCK cells were removed from a flask by treatment with 0.05% trypsin, 0.02% EDTA in Hanks' balanced salt solution without calcium or magnesium. The cells were counted and 5 X lo5 cells added to all wells of a 96-well microtiter plate. After incubation at 37 "C overnight, the medium was removed and replaced with dilutions of ET-18-OMe in the normal culture medium (final ethanol concentrationwas 0.1% or less and did not inhibit cell growth). After 0-6 h of incubation at 37 "C, the medium was removed from wells, the wells rinsed with trypsin solution, and then soaked in trypsin solution for 15 min at 37 "C to detach the cells from the plastic surface. The detached cells were mixed vigorously and an aliquot diluted with trypan blue (0.1% in saline) and thencounted with a hemocytometer. All samples were assayed in triplicate. For long term assays of growth, 3 X lo4 cells wereseeded in each well and cells wereexposed to ET-18-OMe, trypsinized, and counted as above a t daily intervals. Preparation of Protein Kinase C-MDCK cells were grown in 100mm plastic dishes for 4 days. The monolayer of cells was then washed with ice-cold0.9%NaCl and the cells scraped off with a rubber policeman. After centrifugation at 600 X g for 5 min at 4 "C the cell pellet was resuspended in 20 mM Tris-HC1, pH 7.5, 2 mM EDTA, 10 mM EGTA, 50 mM @-mercaptoethanol,2 mM phenylmethylsulfonyl fluoride. The cells were then sonicated for 20 s with a stepped microprobe. Unbroken cells were removedby centrifugation as above, and then the supernatantwas centrifuged at 120,000 X g for 90 min at 4 "C. The supernatant from this step (cytosol) was then fractionated on a 1 X 8-cm DEAE-Sephacel column after addition of sucrose to give 10% final concentration. The DEAE column was equilibrated in 20 mM Tris, pH 7.5,0.2 mM EGTA, 0.2 mM EDTA, 50 mM 8mercaptoethanol, 10% sucrose. After the sample was loaded onto the column, unbound material was washed through with 40 ml of the equilibration buffer. Then protein kinase C was eluted by a gradient from 0-0.5 M NaCl in equilibration buffer. Fractions of 1 ml were collected at 25 ml/h and then0.05-ml aliquots from fractions assayed for protein kinase C as below. The fractions with the most activity were pooledand used in further experiments. The particulate fraction pelleted by centrifugation at 120,000 X g was extracted by sonication in the same buffer with 1%Triton X-100 added. The Triton X-100 extract was then centrifuged at 120,000 X g for 90 min at 4 "C. The supernatant from this step (extracted particulate) was purified by DEAE column chromatography as above except that 0.1% Triton X100 wasadded to thebuffer. Assay of Protein Kinase C-The assays were done in a totalvolume of 0.25 ml and all tubes contained 25 mM Tris, pH 7.5,lO mMMgC12, 40pgof histone, 10 p~ ATP (including 1 pCi of [y-32P]ATP),0.6 mM CaCI2, 20 pg/ml phosphatidylserine, and 2 pg/ml diolein plus 0.05 ml ofprotein kinase C. Protein kinase C activity was determined as the incorporation of 3'P from [y-32P]ATPinto histone in the presence of Ca'+, phosphatidylserine, and diolein minus the incorporation in the absence of these activators. Reactions were initiated by the addition of protein kinase C and halted after 20 min at 30 "C by

C

the addition of0.05 ml of bovine serum albumin (10 mg/ml) and 1 ml of 25% trichloroacetic acid (ice-cold). The tubes were kept on ice and thenfiltered in a Millipore vacuum box using Millipore HA filters and washed with 25% trichloroacetic acid. The radioactivity bound to the filters was determined in 5 ml of Budgel Solve by scintillation counting. The amount of enzyme used was shown to result in linear activity for at least 20 min, and theassay was linearly dependent on the amount of enzyme used. ET-18-OMe or 1-(5-isoquinolinesuIfonyl)piperazinewas added directly to the reaction mixture before the addition of protein kinase C. As a control for the small amount of ethanol from the ET-18-OMe stock solution, 0.1% ethanol was included in the samples with protein kinase C but no ET-18-OMe. In addition tothe standard assay components 0.01% Triton X-100 was included in these experiments. Analysis of the Phosphorylation of Proteins by TPA-MDCK cells wereseeded into 35-mm dishes and grownfor 3 or 4 days. The medium was changed to 1 ml of minimum Eagle's medium without phosphate and cells incubated at 37 "C for 30 min. Then 100-150 pCi of 3'P04was added to each dish and incubation continued for 60 min. The labeling medium was then removed and 1 ml of 20 mM TES, 5 mM KC1, 1 mM MgCI2, 0.15 M NaCI, 10 mM glucose, pH 7.4, added to all dishes; TPA was was then added to dishes to give 50 KIM or other final concentration. After incubation at 37 "C, the medium was removed and the dishes washed three times with the above TES buffer including 2 mM EDTA, 2 mM EGTA, and 2 mM phenylmethylsulfonyl fluoride at 4 "C. Cells were scraped off the dishes in this buffer and collected by centrifugation at 600 X g for 5 min a t 4 "C. The supernatant was discarded, the cell pellet dissolved in 0.1 ml of 2% SDS, 10% glycerol, 5% P-mercaptoethanol, 0.001% bromphenol blue, 6.25 mM Tris, pH 6.8, and thenboiled for 5 min in a water bath. ET-lS-OMe, when present, was given 10 min before the TPA. Proteins were analyzed by polyacrylamide gel electrophoresis in 12.5% resolving gel with a 5% stacking gel according to Laemmli (19). The gels were fixed in 30% methanol, 10% acetic acid, dried onto filter paper, and an autoradiograph prepared by exposing Kodak X-Omat AR or SB-5 film to the gels. Autoradiographs were scanned with a Zeineh soft laser scanning densitometer (Model SL-TRFF). Detection of Changes Induced in Arachidonic Acid Metabolism by TPA or A23187"MDCK cells were grown for 2-3 days under standard conditions in 35-mm plastic dishes. The medium in each dish was replaced with 1 ml of fresh medium containing 0.5pCiof [3H] arachidonic acid and thecells grown for another 18-20 h. The labeling medium wasthen removed and thedishes washed with medium. The cells were then exposed to TPA or A23187 in 1 ml of fresh medium. After further incubation at 37 "C, the cells and medium from each dish were separated, the lipids extracted, and the radioactivity in various lipids determined by thin layer chromatography as described (20). The only modifications made were that Silica Gel 60plates were used for all chromatography and a fluorogram prepared by spraying thethin layer chromatography plate with E ~ ~ [ ~ H ] a nand c ethen exposing Kodak SB-5 film to the plate at -70 "C to aid in localizing the products for scraping the plates. Prostaglandins synthesized by the MDCK cells include PGE,,PGF,,, PG12,PGB,, and PGD, as previously confirmed by us using high pressure liquid chromatography (20). ET-lS-OMe, l-(5-isoquinolinesulfonyl)piperazine, or indomethacin were dissolved in ethanol and then diluted in medium. Addition of these compounds was directly into the medium at the same time as the stimulus (TPA or A23187) unless otherwise indicated. Final concentration of ethanol was 0.1% or less and had no effect on arachidonic acid metabolism. Assay of Prostaglandin Synthesis in Broken Cells-Prostaglandin synthesis in sonicated MDCK cells was determined as previously described (9). Samples were assayed with and without 10 pg/ml indomethacin and the sum of all radioactivity converted into prostaglandins from added [3H]arachidonic acid in the absence of indomethacin minus that in the presence of indomethacin determined. The identity of the radioactive products was determined routinely by thin layer chromatography but has been confirmed in this laboratory by high pressure liquid chromatography.' Arachidonic acid was used at 12 p ~ asaturating , condition, and included 0.5 pCiof [3H] arachidonic acid in each 1 ml of reaction mixture. Under the conditions employed, the reaction was linear with time and concentration of protein (9). Where indicated the cells were pretreated for 6 h with TPA or TPA and ET-18-OMe. G. A. Beaudry, L. Daniel, and M. Waite, unpublished results.

Release and Protein Kinase C

Arachidonic Acid

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on the48-kDa protein was similar (not shown). Occassionally phosphorylation of a protein of 29,000 daltons was also stimEffect of TPA on Distributionof Protein KinaseC in MDCK ulated by TPA but this was not always seen perhaps due to Cells-Protein kinase C activity was notdetectedinthe overlap with a nearby heavily phosphorylated protein. cytosol or in a Triton X-100 extract from the particulate Protein KinaseC from MDCK Cells Is Inhibited by ET-18fraction of MDCK cells. However, after the crude samples OMe and 1-(5-Isoquinolinesulfonyl)piperazine-The partially were partially purified by DEAE ion exchange column chromatography, protein kinaseC activity could be detected (Fig. purified protein kinase C from MDCK cells was inhibited by 1).In control cells most of the protein kinase C activity was addition of either ET-18-OMe or l-(5-isoquinolinesulfonyl)found in the cytosol, whereas in TPA-treated cells protein piperazine (Fig. 4). Half-maximal inhibition was observed at kinase C activity was found in the Triton X-100 extract of about 10 PM of each compound. In other in vitro assays ETthe particulate fraction(Fig. 1).The protein kinaseC activity 18-OMe inhibitedprotein kinase C50% a t 12 p M (28),whereas from MDCK cells eluted from DEAE columns with the con- l-(5-isoquinolinesulfonyl)piperazinerequired 20 pM for 50% centration of NaCl (80-100 mM) previously reported to elute inhibition (18). Addition of high concentrations of ET-18OMe failed to completely inhibit theenzyme, perhaps because the enzyme from bovine brain (21) and was dependent on ET-18-OMe is amphipathic and a nonhomogenous mixture ea2+ and phosphatidylserine. Thus previously as reported for other cells (22-24) addition of TPA to MDCK cells altered forms at high concentrations. Addition of ET-18-OMe during the TPA exposure inhibited the distribution of protein kinase C between cytosolic and the phosphorylation of all three proteins (48-, 40-, and 29particulate fractions. dose Protein Phosphorylation Is Stimulated by TPA in MDCK kDa)stimulated by TPA (Fig. 5).Furthermore,the dependence of ET-18-OMe for inhibition of TPA-stimulated Cells-Addition of TPA stimulated the phosphorylation of two prot.eins of 40,000 and 48,000 daltons in the MDCKcells protein phosphorylation was similar to that required for the (Fig. 2). In order to quantitate the extentof TPA-stimulated inhibition of proteinkinase C(Fig. 5). Thus, ET-18-OMe cells which phosphorylation of these two proteins, autoradiograms were inhibited protein phosphorylation in the intact resulted from TPA addition. scanned witha densitometer (seeexample in Fig. 5). The Protein Kinase C Inhibitors Reduce Arachidonic Acid Redensity of the film image from the 40,000-dalton protein was then compared to that of a protein whose phosphorylation lease andProstaglandinSynthesis in TPA-treatedMDCK was not stimulated by TPA (Ref.). TPA stimulation of the Cells-MDCK cells release [3H]arachidonicacid and prostaphosphorylation of the 40-kDa protein was dependent on the glandins from prelabeled cells into the medium upon stimudose of TPA (Fig. 2) and on time (Fig. 3). The effect of TPA lation by TPA (6). Addition of ET-18-OMe decreased both the TPA-stimulatedrelease of arachidonic acid and synthesis of prostaglandins (Fig. 6). Addition of 1-(5-isoquinolinesulfony1)piperazine had a similar effect (not shown). The inhi0.25 bition of arachidonic acid release and prostaglandin synthesis by either protein kinase C inhibitor continued for 24 h, the 0.20 3 longest period tested. Addition of increasing concentrations of either ET-18-OMe or l-(5-isoquinolinesulfonyl)piperazine 0. 15 f caused a dose-dependent reduction in the changes in arachi0.10 i 3 donic acid metabolism induced by TPA (Fig. 7). A maximum z of 50-60% of the release of arachidonic acid or the production 0.05 of prostaglandins induced by TPA was inhibited by 20-30 p~ of eitherinhibitor,noadditionalinhibition occured with 0. 00 higher concentrations. In most experiments we used 100 nM TPA to get maximum release of arachidonic acid (6), although ET-18-OMe also inhibited arachidonic acid release and prostaglandin production with1 or 10 nM TPA. Furthermore, ET18-OMe could be added after TPA and still inhibited arachidonic acid release (not shown) and prostaglandin synthesis (Fig. 8), which was maintained for hours. Regardless of the time of ET-18-OMe addition there was always some arachidonic acid release and prostaglandin synthesis stimulatedby TPA which was not inhibited by ET-18-OMe. ET-18-OMe Inhibitionof TPA-induced Prostaglandin Synthesis Is Not the Result of Reduction of Prostaglandin H 5 1035 30 15 25 20 40 Synthase Activity-Since both arachidonic acid release and F r a c t 1 on prostaglandin synthesis were reduced by ET-18-OMe or 1-(5FIG. 1. TPA induces redistribution of protein kinase C in isoquino1inesulfonyl)piperazinewe examined theeffect of ETMDCK cells. MDCK cells were grown for 4-5 days and then given 18-OMe on the synthesis of prostaglandins in cell homogefresh DMEM-10% fetal calf serum with or without 100 nM TPA. nates. TPA-treated homogenates had elevated capacity for After 30 min at 37 “C, the cells were collected and “cytosol” and a prostaglandin synthesis (Table I), reflecting the increase in Triton X-100 extract of the particulate fraction prepared as under “Materials and Methods.” These samples were then chromatographed de novo synthesis of PGH synthase or an activator of this on a DEAE-Sephacel column and a 0-0.5 M NaCl gradient applied to enzyme (9,lO). Therewas some variation in the prostaglandin elute the protein kinase C. Fractions eluted from the column were synthesis of differentpreparations from TPA-treatedand assayed for protein kinase C in the presence and absence of Ca2+,PS, control cells, probably because PGH synthase activity is afand diolein and thepmol 32Ptransferred histone that was dependent on these activators is plotted. A , control cells; B, TPA-treated cells. fected by unknown variables such as growth factors in the 0-0, cytosol; m”,. Triton X-100 extract of particulate fraction; serum (2) and the age of the cell culture. However, TPA consistently increased prostaglandin synthesis 3-5-fold in all A-A, NaC1. RESULTS

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Arachidonic Acid Release and Protein KinaseC

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FIG. 2. TPA stimulates phosphorylation of MDCK proteins. Left, MDCK cells were labeled with 32P04 and then stimulated by different concentrations of TPA for 60 min as described under “Materials and Methods.” The proteins were separated by SDS-polyacrylamide gel electrophoresis and the autoradiogram from the gel is shown. Right,32P04-labeledMDCK cells were stimulated for 30 min with different concentrations of TPA. After electrophoresis the autoradiogram of the gel was scanned. The density in the 40,000-dalton protein relative to the density of the reference band (REFin Fig. 5) is plotted. Several experiments were combined in this figure.

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Minutes FIG. 3. Time course of TPA stimulation of phosphorylation of the40,000-dalton protein. MDCK cells were labeled as in Fig. 2, stimulated with 50 nM TPA, and cells harvested a t the times shown. After electrophoresis the autoradiogram was scanned and the ratio of the density of the 40,000-dalton band relative to thereference (REF)band is plotted. 0-0, TPA-treated; W-W, control.

experiments. The addition of ET-18-OMe during TPA treatment of the cells failed to block the increase in prostaglandin synthesis (Table I). No diminution of the TPA enhancement of conversion of arachidonic acid to prostaglandins occurred even with 26 PM ET-18-OMe. Thus, ET-18-OMe fails to block the second effect of TPA, that is, the stimulation of the conversion of arachidonic acid into prostaglandins. As previously published (9), however, we do not known if this stimulation of prostaglandin synthesis by TPA is the result of enhanced synthesisof PGH synthase of ora co-factor required for activation of the PGHsynthase. We confirmed that ET-18-OMe blocked only the release of arachidonic acid but not its metabolism to prostaglandins since addition of ET-18-OMe directly into the in vitro assay

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I NH I B I TORS (pM> FIG. 4. Inhibition of protein kinase C from MDCK cells in vitro by ET-18-OMe and 1-(5-isoquinolinesulfonyl)-piperazine. Protein kinase C was prepared from MDCK cells by DEAESephacel column chromatography as described under “Materials and Methods.” ET-18-OMe or l-(5-isoquinolinesulfonyl)piperazinewere added into the standardassay described under “Materials and Methods.’’ Residual protein kinase C activity in thesesamples is presented as percent of that without addition of these compounds. 0-0, ET18-OMe;0-0, l-(5-isoquinolinesulfonyl)piperazine.

did not inhibit prostaglandin synthesis by preparations from either control or TPA-treatedcells (not shown). Furthermore, in the presence of indomethacin, free arachidonic acid accumulated in the TPA-treatedMDCK cells since the indomethacin blocked the conversion of arachidonic acid to prostaglandins (Fig. 9A). However, in cells exposed to TPA andET-18OMe, arachidonic acid did not accumulate in the presence of indomethacin (Fig. 9B). This indicates that conversion of arachidonic acid to prostaglandins is not the limiting step in arachidonic acid metabolism when ET-18-OMe is present and the reduction of prostaglandin synthesis caused by ET-18OMe occurs before the action of PGH synthase. Effect of ET-18-OMe and 1-(5-Isoquinolinesulfonyl/-piper-

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FIG. 5. ET-18-OMe inhibition of TPA-induced phosphorylation of MDCK cell proteins. Left, autoraand then diogram of the SDS-polyacrylamide gel electrophoresis separation of MDCK proteins labeled with 32P04 stimulated for 60 min with TPA or TPA plus ET-18-OMe as described under “Materials and Methods.” Left arrows indicate migration of standard proteins of molecular weights shown. Right arrows indicate the 48-, 40-, and 29-kDa bands which are stimulated by TPA. Densitometer scans are from a portionof the autoradiogram indicated by the bracket. Letter designations correspond to those of the inset. Arrows mark 48-, 40, and 29-kDa bands which are stimulatedby TPA exposure. REF arrow marks a band that does not vary with TPA exposure. Lane a, 0-time; lune b, 0.005% dimethyl sulfoxide; lune c, 50 nM TPA plus 40 FM ET-18-OMe; lane d, 50 nM TPA. Right, MDCK as above and thenstimulated with 50 nM TPA for 30 min in the presence of different cells were labeled with 32P04 concentrations of ET-18-OMe. Autoradiograms of SDS-polyacrylamide gels were scanned and the density in the control; A, ET-18-OMe 50 p~ 40,000- or 48,000-dalton protein plotted as a ratio to the REF band. 0, TPA; without TPA; K, kDa.

.,

azine on Deacylation of MDCK Phospholipids-We next determined the effect of ET-18-OMe and 1-(5-isoquinolinesulfony1)piperazineon the deacylation of arachidonic acid from the cellular lipids of MDCK cells. The source of the arachidonic acid released by TPA was studied using cells prelabeled with [3H]arachidonic acid. At the end of the labeling period, the [3H]arachidonic acid was distributed among the cellular lipids: phosphatidylcholine, 18%; phosphatidylethanolamine, 57%; phosphatidylinositol, and phosphatidylserine, 17%; arachidonic acid, 0.6%; triglycerides, 1-3%; other 3-6%.As previously observed (6), the bulk of the [3H]arachidonic acid released with TPA was from phosphatidylethanolamine with lesser amounts derived from phosphatidylinositol and phosphatidylcholine (Fig. 10). In TPA-treated cells, ET-18-OMe inhibited the loss of [3H]arachidonic acid from phosphatidylethanolamine without effecting the loss from the otherphospholipids. The same result was obtained with 1-(5-isoquino1inesulfonyl)piperazine (not shown). Thus, when release of arachidonic acid was reduced by addition of the protein kinase C inhibitors there was a retention of [3H]arachidonic acid in phosphatidylethanolamine. Effect of ET-18-OMe on A23187-induced Arachidonic Acid Release-We also examined the effect of ET-18-OMe on the

deacylation of arachidonic acid from phospholipids caused by a second stimulus. The calcium ionophore A23187-stimulated arachidonic acid release and prostaglandinsynthesis in MDCK cells; however, ET-18-OMe did not alter the release of arachidonic acid stimulated by A23187 (Fig. 11).In fact, addition of ET-18-OMe increased the release of arachidonic acid induced by A23187, although ET-18-OMe itself did not stimulate arachidonic acid release. This may be because ET18-OMe also inhibits reacylation of arachidonic acid into phospholipid^.^ The treatment of MDCK cells with A23187 also caused the release of the majority of the [3H]arachidonic acid from phosphatidylethanolamine (not shown). These results demonstrate that ET-18-OMe is not a direct inhibitor of phospholipase activity and that the mechanisms of TPA and A23187 stimulation of arachidonic acid release are distinct. Toxicity of ET-18-OMe to MDCK Cells-In order to ensure that the effect of ET-18-OMe was unrelated to a change in cell number, we exposed confluent cultures of MDCK cells to ET-18-OMe, thus duplicating the conditions used in the other experiments. The number of intact cells in cultures treated with ET-18-OMe for up to 6 h was no different than control J. Parker, L. W. Daniel, and M. Waite, unpublished results.

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Arachidonic Acid Release and Protein KinaseC

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FIG. 6. ET-18-OMe inhibits TPA-induced release of arachidonic acid and prostaglandin synthesis. MDCK cells prelabeled with [3H]arachidonicacid were exposed to TPA with or without ET-18-OMe. Radioactive lipids accumulating in the mediumwere analyzed as described under “Materialsand Methods.” A , arachidonic acid; B, prostaglandins (sum of PGE,, PGGKF,,, PGF2,, PGB,, and PGD,). A typical experiment is shown with the results represented as percent of the total radioactivity in the cells at the start of the experiment (1 X lo6 dpm). 0-0, 100 nM TPA; H”., 100 nM TPA plus 20 PM ET-18-OMe.

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5

10 15 20 25 Hours FIG. 8. ET-18-OMe added after TPA inhibits prostaglandin synthesis. MDCK cells were labeled with [3H]arachidonicacid as in Fig. 6 and stimulated by addition of 1 nM TPA at zero time. ET-18OME (40 PM) was added at zero time (0-El), 2 h (A-A), 4h (0-O), 8 h (A-A), or 12 h (H-H). Cells treated only with TPA are also shown (0-0) Radioactivity . in prostaglandins was quantitated as for Fig. 6 and is given as percent of total radioactivity at the start of the experiment. Arrows indicate the time of addition of ET-18OMe.

TABLE I ET-18-OMefails to prevent the TPA-indued stimulation the of conversion of arachidonic acid to prostaglandins Prostaglandin synthesis was assayed in sonicated preparations of MDCK cells with and without pretreatment with TPA or TPA plus ET-18-OMe as described under “Materials and Methods.” Treatment 0 1 0 2 0 3 0 4 0 5 0 0 ET- 18-OMe CM)

25 50 100 lsoquinolinesulfonarnide ( rM )

Prostaglandin synthesis pmol min” mg protein” 186 f 153 (8)b 700 f 410‘ (7)

DMEM, 10% fetal calf serum FIG. 7. Protein kinase C inhibitors ET-18-OMe and 1-(510 nM TPA isoquinolinesulfony1)piperazine inhibit arachidonic acid and 10 nM TPA 17 I . ~ MET-18-OMe 805 f 446‘ (3) prostaglandin releasein TPA-treatedMDCK. MDCK cells were Values are the mean f standard deviation. prepared as in Fig. 6, except the concentration of each inhibitor was Number of separate preparations assayed. varied and all samples extracted at 2 h. Radioactivity is expressed as Different from DMEM, 10% fetal calf serum by Student’s t test, the percent of the sum of arachidonic acid and prostaglandins with TPA alone (control). Prostaglandins (0-0), arachidonic acid p < 0.006. (0-El), and sum of arachidonic acid + prostaglandins (M-m).

+

cultures. No change in the number of intact cells was observed at 6 h even with 40 PM ET-18-OMe. In other experiments ET-18-OMe was added to subconfluent cells and the growth of the cells observed for several days. Theseexperiments showed that ET-18-OMe decreased the ability of the cells to proliferate; however, this effect occurs much later than the effects we observed on protein phosphorylation and arachidonic acid release. Since the inhibition of arachidonic acid release caused by ET-18-OMe in TPA-treated cells is observed even before 1 h, the effect of ET-18-OMe on arachidonic acid release is not due to cell death andmust represent a change in cellular metabolism. DISCUSSION

We have shown that TPA has the same effect on protein kinase C in MDCK cells as it hasin many other cell types. In

the MDCK cells TPA caused the redistribution of protein kinase C to theparticulate fraction as previously observed for other cells (21-23). Furthermore, TPA stimulated the phosphorylation of 40- and 48-kDa proteins in the MDCK cells. In platelets a40-kDa protein has been shown to be a substrate for protein kinase C (26, 27), and TPA-stimulated phosphorylation of 48-kDa protein has been reported in neutrophils (28). However, the MDCK cells respond to TPAmore slowly than platelets and neutrophils in the phosphorylation of the 40- and 48-kDa proteins. Since the dose response for the stimulation of protein phosphorylation by TPA is the same for MDCK cells as for other (26, 29-31), we feel the slower response of MDCK cells is probably due to higher levels of protein kinase C inhibitors in the MDCK cells which activation by TPA can only slowly overcome. This would be consistent with our inability to detect protein kinase C in crude cell fractions. Furthermore, protein kinase C activity is low

5391

Arachidonic AcidRelease and Protein Kinase C

:: i

I

I

I

I

I

A

40

20 0

0

0.01 0.1

1

0

10

0.01 0.1

1

10

Indomethacin pg I ml

FIG. 9. Indomethacin causes arachidonic acid to accumulate in TPA-treated but not in TPA- and ET-18-OMe-treated MDCK cells. MDCK cells were labeled with [3H]arachidonicacid as

B

in Fig. 3. The cells were stimulated for 3 h at 37 “C with various combinations of TPA, ET-18-OMe, and indomethacin, and then the lipids in the medium were analyzed as described under “Materials and Methods.” The percent of total radioactivity in prostaglandins or arachidonic acid was determined and then all other values were normalized to thatof the sum of prostaglandins and arachidonic acid from TPA-treated samples set to 100% (8.2% of total radioactivity). A , TPA 100 nM; B, TPA + ET-18-OMe 40 p~ cells. 0-0, prostaglandins; 0-0, arachidonic acid; and A-A, arachidonic acid and Prostaglandins.

P

0

1

2

3

4

5

Hours

FIG. 11. ET-18-OMe fails to inhibit A23187-induced arachidonic acid and prostaglandin release. MDCK cells labeled with [3H]arachidonic acid as for Fig. 6 were transferred into fresh DMEM, 10% fetal calf serum (0-O), 10 PM A23187 (0-O), ET-18OMe 40 pM (0-O), or A23187 + ET-18-OMe (A-A). A t times shown, medium was removed from the cells and the lipids of the medium extracted into ch1oroform:methanol. Results are presented as percentof total radioactivity in cells at startof the experiment. A , arachidonic acid; B, prostaglandins.

C inhibitors to further test theinvolvement of protein kinase C in the release of arachidonic acid and synthesis of prosta0 4 8 12 16 20 24 glandins stimulated by TPA. We found that addition of ETHours 18-OMe or l-(5-isoquinolinesulfonyl)piperazine decreased FIG. 10. Effect of ET-18-OMe on deacylation of [‘Hlarachidonic acid from phospholipids in TPA-treated MDCK cells. both the release of arachidonic acid and the synthesis of The extracted lipids of the cells from Fig. 6 were separated by thin prostaglandins in TPA-treated MDCK cells. The concentralayer chromatography and radioactivity in various phospholipids tions of these compounds which inhibited arachidonic acid quantitated by scraping appropriate areas of the plate and counting release and prostaglandinsynthesis were similar to those the silica in scintillation fluid. Results are presented as percent of required to inhibit partially purified protein kinase C from total radioactivity in the cells at the start of the experiment. 0-0 and 0-0, phosphatidylethanolamine; 0-0 and W”,. phosphati- MDCK cells and toinhibit phosphorylation of proteins stimdylcholine; and 0- -0 and 0- -0, phosphatidylinositol + phospha- ulated by TPA in intact cells. Since the protein kinase C tidylserine. 100 nM TPA (circles);100 nM TPA + 20 pM ET-18-OMe inhibitors we used have distinctstructures and unrelated (squares). modes of inhibition of protein kinase C ( 2 5 , 32); it is unlikely that they share other effects on MDCK cells. Furthermore, in the MDCK cells which agrees with previous reports that although the isoquinolinesulfonamides inhibit other protein kidney has less protein kinase Crelative to platelets (21). The kinases (32), ET-18-OMe is unable toinhibitCAMP- and dose response for stimulation of protein phosphorylation by cGMP-dependent kinases (25). ET-18-OMe did not inhibit TPA in MDCK cells is similar to that for the release of conversion of arachidonic acid to prostaglandins inthe intact arachidonic acid stimulated by TPA in these cells (6). Fur- cells and was unable to prevent the TPA-induced stimulation thermore, both l-oleoyl-2-acetyl-rac-glycerol and 12-diocta- of the conversion of arachidonic acid into prostaglandins. The noyl-rac-glycerol, two other activators of protein kinase C, latter result suggests that TPA increases the conversion of stimulated arachidonicacid release and prostaglandin synthe- arachidonic acid to prostaglandins by a mechanism which is sis equivalent to that induced by TPA.3 Since all three acti- independent of the ability of TPA to activate protein kinase vators of protein kinase C stimulate prostaglandin synthesis, C. However, since we found that protein kinase C was not it appears that protein kinase C is involved in one or more completely inhibited by inhibitors, it is possible that residual low level of protein kinase C activity suffices for the TPA steps of prostaglandin synthesis. We have used two compounds known to be protein kinase enhancement of the conversion of arachidonic acid into prosI

I

I

I

I

I

5392

Arachidonic Acid

Release and

taglandins. We think the best interpretationof our results is that inhibition of protein kinase C in MDCK reduces the release of arachidonic acid caused by TPA withoutdecreasing the ability of the cells to synthesizeprostaglandinsfrom arachidonic acid. Previous work from this laboratory suggested that a phospholipase A2 which primarily cleaves phosphatidylethanolamine is activated by TPA in MDCK cells (33). The results reported here suggest that the activityof a phospholipase A,, which is responsible for the release of arachidonic acid from phosphatidylethanolamineinMDCK cells, is regulated by protein kinase C. There are some reports of regulation of a phospholipase A, by a mechanism which involves phosphorylation in other cells. Addition of CAMP, ATP, and M$+ stimulated phospholipase A, activity in a preparation from bovine brainsynaptic vesicles (34).Wightman et al. (35) observed a similaractivation of phospholipase A, by ATP and of murine macrophages and showed Mg2' or Ca2+ in sonicates direct activation of phospholipase A, by CAMP-dependent kinase from bovine heart. Furthermore,phospholipase A, has previously been suggestedto be regulatedby phosphorylationdephosphorylation of a regulatory protein (14, 36, 37). Although we do not know the function of the 40- and 48-kDa proteins phosphorylated by protein kinase C in MDCK cells, the 40-kDa protein of human plateletsknown to be a substrate for protein kinaseC(26, 27) has been reportedto bea phospholipase A, inhibitor when in a dephosphorylated state (17). Weobserved thatarachidonic acidrelease was still susceptible to inhibition by addition of ET-18-OMe many hours after the TPA was applied. This suggests that the phospholipase A, activity might be continually regulated by protein kinase C. It is possible that in MDCK cells, protein kinase C directly phosphorylates and thus activates a phospholipase A, or alternativelyintermediatesteps mightbe involved. At this point it is notclear why a portion of the release of arachidonic acid stimulated by TPA and all of the release stimulated by A23187 in MDCK cells was resistant to inhibition by ET-18-OMe. A23187 causes an influx of Ca2+ into the cell and leads to an increase in the activityof phospholipase A, (38), probably because phospholipase A, activity is dependent on calcium (39). The activation of phospholipase A, by an increase in intracellular Ca2+appears to be distinct from the activation induced by TPA through protein kinase C. This is supportedby the fact thatin combination TPA and A23187 induce more release of arachidonic acid from MDCK cells than either stimuli alone (12). Our results are consistent with the presence of two mechanisms to activate release of arachidonic acidin MDCK cells. Itis difficult topredict whether a single phospholipase A, is activated via multiple pathways or if separate phospholipases are involved in each pathway. Indeed, it is possible that the action of the protein kinase C on phospholipase A, activity is mediated through a change in the concentration of Ca2+ required to activate the phospholipase. If this were the case it would explain why the Ca2+ionophore A23187 overcomes the inhibition of the protein kinase C by ET-18-OMe. The Ca'+-independent arachidonic acid release stimulated by vasopressin (40), angiotensin I1 (40), and thrombin (41)might also be mediated by activation of phospholipase A2 by proteinkinase C since these stimuli are thought to activate protein kinaseC (13, 27, 42). Thus, there appear be to two mechanisms (which may or may not be part of the same pathway) for the activation of phospholipase A, in kidney cells: one probably controlled by protein kinase C and the other a result of an increase in the

Protein Kinase

C

concentration of intracellular Ca2+. Acknowledgments-We thank Dr. Wolfgang E. Berdel for the gift of the ET-WOMe, Dr. Craig Gerard for the 1-(5-isoquinolinesulfonyl)piperazine, and Dr. Linda McPhail for assistance in the development of the protein kinase C assay. REFERENCES 1. Imine, R. F. (1982) Biochem. J. 204, 3-16 2. Levine, L., and Hassid, A. (1977) Biochem. Biophys. Res. Comnun. 76,1181-1187 3. Zusman, R. M., and Keiser, H. R. (1977) J. Biol. Chem. 2 5 2 , 2069-2071 4. Blackwell, G. J., Duncombe, W. G., Flower, R. J., Parsons, M. F., and Vane, J. R. (1977) Br. J. Pharmacol. 59, 353-366 5. Levine, L., and Hassid, A. (1977) Biochem. Biophys. Res. Commun. 79,477-484 6. Daniel, L.W., King, L., and Waite, M. (1981) J. Biol. Chem. 256,12830-12835 7. Ohuchi, K., Watanabe, M., Yoshizawa, K., Tsurufuji, S., Fujiki, H., Suganuma, M., Sugimura,T., and Levine, L. (1985) Biochem. Biophys. Acta834,42-47 8. Bailey, J. M., Muza, B., Hla, T., and Salata, K. (1985) J. Lipid Res. 26,54-61 9. Beaudry, G.A., Waite, M., and Daniel, L. W. (1985) Arch. Biochem. Biophys. 239,242-247 10. Ohuchi, K., and Levine, L. (1978) J. Biol. Chem. 253,4783-4790 11. Morrison, A. R., Moritz, H., and Needleman, P. (1978) J. Biol. Chem. 253,8210-8212 12. Daniel, L. W., Beaudry, G . A,, King, L., and Waite, M. (1984) Biochem. Biophys. Acta7 9 2 , 33-38 13. Nishizuka, Y.(1984) Science 225, 1365-1370 14. Hirata, F. (1981) J . Biol. Chem. 2 5 6 , 7730-7733 15. Rothhut, B., Russo-Marie, F., Wood, J., DiRosa, M., and Flower, R. J. (1983) Biochem. Biophys. Res. Commun. 117,878-884 16. Hirata, F., Matsuda, K., Notsu, Y., Hattori, T., anddel Carmine, R. (1984) Proc. Natl. Acad. Sci. CJ. S. A. 8 1 , 4717-4721 17. Touqui, L., Rothhut, B., Shaw, A. M., Fradin, A., Vargaftig, B. B., and Russo-Marie, F. (1986) Nature 3 2 1 , 177-180 18. Gerard, C., McPhail, L. C., Marfat, A,, Stimler-Gerard, N. P., Bass, D. A,, and McCall, C. E. (1986) J . Clin. Znuest. 77,6165 19. Laemmli, U. K. (1970) Nature 2 2 7 , 680-685 20. Beaudry, G. A., King, L., Daniel, L. W., and Waite, M. (1982) J. Biol. Chem. 257, 10973-10977 21. Kikkawa, U., Minakuchi R., Takai, Y., and Nishizuka, Y. (1983) Methods Enzymol. 99, 288-298 22. Kraft, A. S., and Anderson, W. B. (1983) Nature 301,621-623 23. Kraft, A. S., Anderson, W. B., Cooper, H. L., and Sando, J. J. (1982) J . Biol. Chem. 257, 13193-13196 24. Uratsuki, Y., Nakanishi, H., Takeyama, Y., Kishimoto, A., and Nishizuka, Y. (1985) Biochem.Biophys. Res. Commun. 1 3 0 , 654-661 25. Helfman, D. M., Barnes, K. C., Kinkade, J. M., Vogler, W. R., Shoji, M., and Kuo, J. F. (1983) Cancer Res. 43,2955-2961 26. Yamanishi, J., Takai, Y., Kaibuchi, K., Sano, K., Castagna, M., and Nishizuka, Y. (1983) Biochem.Biophys. Res. Commun. 112,778-786 27. Sano, K., Takai, Y., Yamanishi, J., and Nishizuka, Y. (1983) J. Biol. Chem. 258,2010-2013 28. Andrews, P. C., and Babior, B. M. (1983) Blood 61,333-340 29. Miyake, R., Tanaka, Y., Tsuda, T., Kaibuchi, K., Kikkawa, U., and Nishizuka, Y. (1984) Biochem.Biophys. Res. Commun. 1 2 1,649-656 30. Trevillyan, J. M., Kulkarni, R. K., and Byus, C. V. (1984) J . Biol. Chem. 259,897-902 31. Werth, D. K., and Pastan, I. (1984) J . Biol. Chem. 2 5 9 , 52645270 32. Hidaka, H., Inagaki, M., Kawamoto, S., and Sasaki, Y. (1984) Biochemistry 23,5036-5041 33. Beaudry, G. A,, Daniel, L. W., King, L., and Waite, M. (1983) Biochim. Biophys. Acta 750,274-281 34. Moskowitz, H., Puszkin, S., and Schook, W. (1983) J . Neurochern. 41,1576-1586 35. Wightman, P. D., Dahlgren, M. E., and Bonney, R. J. (1982) J . Biol. Chem. 257,6650-6652

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