Phorbol Esters and Diacylglycerols Amplify Bradykinin-stimulated ...

THEJOURNALOF BIOLOGICAL CHEMISTRY

Vol. 263, No. 10,Issue of April 5, pp. 4764-4767, 1988 Printed in U.S.A.

Phorbol Esters and Diacylglycerols Amplify Bradykinin-stimulated Prostaglandin Synthesis in Swiss3T3 Fibroblasts POSSIBLE INDEPENDENCE FROM PROTEIN KINASE C* (Received for publication, July 31, 1987)

Ronald M. BurchS, Alice Lin Ma, andJulius Axelrod From the Laboratory of Cell Biology, Section on Pharmacology,National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892-

When Swiss 3T3 fibroblasts were incubated with bradykinin, prostaglandin Ez (PGEz) synthesiswas stimulated. Phorbol esters or the diacylglycerol analog 1-oleoyl-2-acetylglycerol(OAG), by themselves, did not acutely stimulate PGEz synthesis. However, when cells were preincubated with phorbol esters or OAG, bradykinin-stimulatedPGEzsynthesis waspotentiated markedly. When phorbol esters and OAG were added together, bradykinin-stimulated PGEz synthesis was potentiated in an additive manner. When cells were preincubated for 48 h with phorbol esters, then bradykinin added, amplification of bradykinin-stimulated PGEz synthesis by phorbol ester or OAG was still apparent, even though prolonged pretreatment with phorbol esters abolished protein kinase C (Ca2+/phospholipid-dependentenzyme) activity in cell-free preparations. Further, the protein kinase C antagonist, H7, only slightly inhibited phorbol ester or OAG amplification of bradykinin-stimulated PGEz synthesis. The possibility is raised that diacylglycerol, formed in response to many receptors, may serve as a transducer of receptor-receptor interactions. Since desensitization or inhibition of protein kinase C only partially reduced the amplification of bradykinin-stimulated PGEz synthesis by phorbolesters or OAG, the possibility is raised that diacylglycerol mimetics may have actions in addition to activation of protein kinase C.

ability to substitute fordiacylglycerol to activate protein kinase C (Ca2+/phospholipid-dependentenzyme) (3). Phosphorylation catalyzed by protein kinase C might potentiate bradykinin-stimulated PGE, synthesis at several sites: the receptor, the G-protein, the phospholipase, the cyclooxygenase, or PGE isomerase. While attempting to discriminate among these alternative sites of action, we found that activation of protein kinase C may not account for the ability of phorbol esters or, especially, cell-permeable diacylglycerol analogs to amplify PGE, synthesis in response to bradykinin. The data suggest that diacylglycerol amplifies bradykinin stimulation of phospholipase A, mainly independent of protein kinase C. Recent evidence suggests that the action of phorbol ester andcell-permeant diacylglycerols may beat the level of the phospholipase A,, since protein kinase C, the commonly considered target for these molecules, and phospholipase A, exhibit sequencehomology in their putative lipid-binding regulatory sites (4). EXPERIMENTALPROCEDURES

Cell Culture-Swiss albino 3T3 fibroblasts (5) were maintained in Dulbecco’s modified Eagle’s medium containing 10% calf serum and used at 40-70% confluency as previously described (1).For experiments in which the cells were incubated for 48 h with phorbol esters, the cells usually reached confluency during the incubation. PGE, Synthesis-In Swiss 3T3 fibroblasts more than 70% of arachidonic acid metabolites released in response to bradykinin is PGE, (1).Thus, PGE, synthesis was used as an index of phospholipase activity. PGE, released into the culture media was quantitated by radioimmunoassay using antiserum from Seragen (AdvancedMagIn Swiss 3T3 fibroblasts, bradykinin stimulates prostaglan- netics, Boston, MA) (1). No experimental agents used in this study interfered with the assay at theconcentrations reported in the experdin E, (PGE2)’ synthesis(1).The rate-limiting step for stim- iments. ulation of prostaglandin synthesis is the liberation of the Protein Kinase C Actiuity-Subcellular fractionation and extracprecursor arachidonic acid by phospholipase (2). In thesecells tion of protein kinase C with Triton X-100was performed as described we have demonstrated that the phospholipase activated by (6). Protein kinase C activity was assayed as transfer of 32Pfrom [ybradykinin to release arachidonic acid is a phospholipase A*, 32P]ATPto histone type 111-S(6). Statistics-PGE, synthesis experiments were performed in 12- or and we have provided evidence that a GTP-bindingregulatory 24-well plates. Each experimental manipulation was performed in (G) protein mediates bradykinin receptor activation of phos- triplicate wells. The values obtained were averaged and counted as a pholipase A, (1). single observation. Data are presented as mean & S.E. Statistical We reported that phorbol esters can amplify bradykinin- comparisons were madeusing Student’s t test for paired observations. Materials-PMA was from Behring Diagnostics. PDBu, 4-a-phorstimulated phospholipase A,-mediated breakdown of phosphatidylcholine and PGE, synthesis (1). The effects of phor- bol, OAG,and histone type 111-Swere from Sigma. H-7 and HA-1004 were from Seikagaku America, Tampa, FL.

bo1 esters usually are considered to be mediated by their

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $Supported by the Pharmacology Research Associate Training Program of the National Institute of General Medical Sciences. The abbreviations used are: PGE,, prostaglandin E,; PMA, phorbo1 12-myristate 13-acetate; PDBu, phorbol 12J3-dibutyrate; OAG, 1-oleoyl-2-acetylglycerol.

RESULTS

Protein Kinase C Does Not Appear to Play a Role in Stimulation of PGE, Synthesis by Bradykinin-When added to cultures of Swiss 3T3 cells, bradykinin stimulates PGEz synthesis. Stimulation is complete by 5 min after bradykinin addition (1).To determine whether protein kinase C plays a role in stimulation of PGE, synthesis by bradykinin, cultures were incubated with PMA. PMA stimulates protein kinase C

4764

L;; -

Phorbol Esters and Diacylglycerols Amplify Prostaglandin Synthesis in intact Swiss 3T3 cells within seconds ( 7 ) .When added to cultures for 5 min, the time required by bradykinin to stimulate PGE, synthesis maximally, PMA (1 nM-1 pM) had no effect on PGE, synthesis (Fig. 1). Further, when added simultaneously with bradykinin, PMA (10 nM-1 p ~ had ) no effect on bradykinin (0.1 nM-1 wM)-stimulated PGE, synthesis (Fig. 1).Similarly, OAG (10-30 p ~ )when , added simultaneously with bradykinin, had no effect on basal or bradykinin-stimulated PGE, synthesis (Fig. 1).H-7 (10-100 p ~ )a , protein kinase C inhibitor (8),when added to the cultures 15 min prior to bradykinin, did not inhibitbradykinin-stimulated PGE, synthesis (Fig. 1). The ICso for inhibition of protein kinase C by H-7 in cell-free extracts from Swiss 3T3 cells was 12 p~ (see Table 111). Preincubation of Swiss 3T3 Fibroblasts with PMAAmplifies Bradykinin-stimulatedPGE, Synthesis-When PMA was added to Swiss 3T3 cells for 15 min, then PGE, synthesis monitored over the subsequent 5 min, PGE, synthesis was stimulated (Fig. 2). When PMA was added to the cells for 15 min, then bradykinin added for 5 min, bradykinin-stimulated PGE, synthesis was amplified by PMA 3.4 & 1.4-fold (Fig. 2, and see Fig.4). Preincubation of cultures with PMA markedly potentiated PGE, synthesis in response to bradykinin whether bradykinin was added at threshold concentration or maximally effective concentrations (Fig. 3). OAG (10-30 p ~

C

BK PMAOAG BK

+

c

0 .-c

-m

f

4765

: ._ .‘E > 200 300 -0 Y

z

.-

X

m

N

z

w

[L

loo O 0

0.01

0.1 1

10

10

Bradykinin, n M

FIG.3. Preincubation with PMA for 15 min amplifies PGE, synthesis in response to threshold and maximal bradykinin concentrations. In these experiments cells were grown in 24-well plates, and each manipulation was performed in triplicate to yield a single measurement. Maximum bradykinin ( B K ) stimulation was defined as the response of 1 p~ bradykinin, which was present in )triplicate wells of each plate. Data are from three experiments.

+

PMA H7

FIG.1. Protein kinase C does not play a role in bradykinin stimulation of PGE, synthesis. All incubations were for 5 min, the time required to reach maximum PGE, synthesis in response to bradykinin. Bradykinin ( B K ) was 1 p ~ PMA , was 1 p ~ OAG , was 30 p ~ H-7 , was 100 pM. Cultures were preincubated with H-7 for 15 min prior to theaddition of bradykinin. BK + PMA indicates simultaneous addition of these two agents. Mean f S.E. of four experiments. C, control.

FIG.4. Dose-response for augmentation of bradykininstimulated PGE, synthesis by PMA and OAG. PMA or OAG was added to triplicate wells 15 min prior to the addition of bradykinin, 1 p ~ After . the addition of bradykinin, PGE, synthesis was monitored for 5 min. On the ordinate, “100%”indicates the response to bradykinin alone. Data are mean f S.E. of five experiments. OAG was taken only to 30 p~ since higher concentrations interfered with the PGE, radioimmunoassay.

also amplified bradykinin-stimulated PGE, synthesis (Figs. 4 and 5). When PMA (1p ~ )a, concentration which maximally po) tentiates bradykinin’s effect (Fig. 4), and OAG (30 p ~ were ) added to thecultures together, then bradykinin (1p ~ added 15 min later, bradykinin-stimulated PGE, synthesis was potentiated in an additive manner. In the presence of PMA alone, bradykinin-stimulated PGE, synthesis was amplified 2.6 & 0.8-fold. In the presence ofOAG alone, bradykininstimulated PGE, synthesis was stimulated 3.6 & 1.1-fold. When PMA and OAG were added simultaneously, bradyki” C BK PMA PMA nin-stimulated PGE, synthesis was amplified 5.4 & 1.8-fold J ( n = 3, p 0.02). BK The effect of length of preincubation with PMA or OAG FIG.2. Amplification of bradykinin-stimulated PGE, synthesis by pretreatment with PMA. Cells were preincubated with on amplification of bradykinin-stimulated PGE, synthesis is PMA, 10 nM, for 15 min prior to addition of bradykinin ( B K ) ,1 p ~ . shown in Fig. 5. Amplification was readily apparent when Wells were then rinsed once with media. PGE, synthesis was meas- bradykinin was added 5 min after PMA, and it continued to ured for 5 min. Mean f S.E. of six experiments. C, control. increase until PMA was added 30 min prior to bradykinin.

Phorbol Esters and Diacylglycerols Amplify Prostaglandin Synthesis

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TABLEI1 Desensitization of protein kinase C by prolonged exposure to phorbol esters blocks stimulation of basal PGEZ synthesis but not amplification of bradykinin-stimulated PGE, synthesis Mean f S.E. of four experiments. Cultures were preincubated with PMA or OAG for 15 min or 48 h then bradykinin was added for 5 min. PGE2

Length of exposure

48 h

15 min pglweu

Time, hours

FIG. 5. Time course for pretreatment with PMA to amplify bradykinin-stimulated PGEz synthesis. Zero percent amplification represents PGE, synthesis in response to 1 p M bradykinin. In these experiments cultures were pretreated with 1 pM PMA for the times indicated, then PGE, synthesis was measured in response to 10 nM PMA or 10 PM OAG, added along with 1 PM bradykinin for 5 min. In the otherexperimentscultures were pretreated with 4aphorbol for the indicated times, then thewells wererinsed and 10 nM 4wphorbol was added along with 1 PM bradykinin for 5 min. Mean f S.E. for four experiments. TABLEI Desensitization of protein kinase C by prolonged exposure to phorbol esters Mean f S.E. of triplicate determinations in one of three similar experiments. Incorporation of 32Pfrom [-y-3ZP]ATPinto histone was assayed in the presence or absence of phosphatidylserine (400 pg/ ml), CaC12 (1 mM), and the indicated phorbol ester (100 nM) for 2 min. The cultures had been preincubated with phorbol ester for the times indicated. Each point was obtained from four IO-cm dates. Length of exposure

0

Control PMA PDBu

144 f 22 846 f 112 1012 f 136

48

l hh

h

24 cpm "P into histone

1 2 8 f 28 7 1 6 f 88 8 7 5 2 102

212 f 28 423+-56 302 + 4 0

3 2 6 f 26 366f46 340f58

Phorbol esters desensitize protein kinase C when they are added to cells for prolonged periods of time (3). Inthe present study, protein kinase C activity was found to be completely desensitized by 48 h after the addition of PDBu (Table I).As shown in Fig. 5, after preincubation of Swiss 3T3 cells with PMA for 24-48 h, PMA or OAG still potentiated bradykininstimulated PGE, synthesis, despite complete desensitization of protein kinase C . However, the enhanced basal PGE, synthesis observed after 15 min treatment with PMA alone, was completely blockedby 48-h preincubation with PMA (Table 11). Similar effects were observed when the cultures were preincubated with PDBu (0.4 p ~ for ) 48 h (data not shown). Preincubation of cultures with 1 p~ PDBu for 48 h did not block amplification of bradykinin-stimulated PGE, synthesis by OAG (Fig. 5). The phorbol analog 4a-phorbol, which does notactivateprotein kinase C , did not affect bradykinin-stimulated PGE, synthesis (Fig. 5). H-7 Only Partially Blocked Amplification of BradykininStimulated PGE, Synthesis-Since prolonged preincubation of the cultures with PMA or PDBu desensitized protein kinase C but did not abolish amplification of bradykinin-stimulated PGE, synthesis, the possibility was raised that themechanism for amplification is independent of protein kinase C . To test the role of protein kinase C further, the effects of the protein kinase C inhibitor H-7 were tested. H-7 only partially blocked the potentiation of bradykinin-stimulated PGE, synthesis by PMA, PDBu, or OAG (Table 111). In contrast, H-7 nearly completely inhibited PMA or PDBu enhancement of protein kinase C activity (Table I).HA-1004, an analog of H-7 which

Basal PMA, 1 p M OAG, 30 p M Bradykinin, 1p~ Bradykinin PMA Bradykinin + OAG

+

132 f 12 164 f 28" 288 f 118 30" 475 f 36" 1012 f 42" 1070 1485 f 84"

106 rf: 17 97 rf: 12b rf: 8b 524 f 40" rf: 62" 1248 f 54"

" p < 0.05 or less compared to basal.

Not significantly different from basal.

TABLEI11

H-7inhibits phorbol ester stimulation of protein kinase C activity but only partially inhibitsphorbol ester or OAG potentiation of PGE, synthesis Mean f S.E. from three experiments for PGE, synthesis and from triplicate determinations from one of three similar experiments for protein kinase C activity. PGE, pglwell

Control Bradykinin, 1 p~ PMA, 10 nM + bradykinin H-7, 100 p~ + PMA + bradykinin Control Bradykinin PDBu, 10 nM + bradykinin H-7 PDBu + bradykinin Control Bradykinin OAG, 10 p M + bradykinin H-7 + OAG + bradykinin

+

126 rf: 14 344 rf: 28 1296 f 74 846 f 52 143 f 38 544 f 32 2076 f 88 1416 f 152 206 f 25 500 f 28 1719 f 112 932 f 45 cpm 3zP into

histone

Control PMA, 100 nM + H-7,lO p M H-7,100 p M PDBu, 100 nM + H-7, 10 p M + H-7, 100 /LM

285 f 28 1265 f 212 816 rf: 42 312 +. 36 1428 f 175 904 f 118 274 f 36

+

does not block protein kinase C at the concentrations used, had no effect on PMA amplification of bradykinin-stimulated PGE, synthesis (data not shown). DISCUSSION

The present results demonstrate that preincubation of Swiss 3T3 cells with phorbol esters or diacylglycerol analogs potentiate the ability of bradykinin to stimulate PGE, synthesis. Protein kinase C did not appear to play a role in the stimulation of PGE, synthesis by bradykinin alone. In addition, desensitization experiments, and experiments using the protein kinase C inhibitor H-7,suggested that protein kinase C is not responsible for most of the amplification of bradykinin-stimulated PGE, synthesis elicited by preincubation of the cells with phorbol esters or OAG. The mechanisms responsible for amplification by phorbol esters and OAG may be distinct, since PMA and OAG added simultaneously amplify subsequent bradykinin-stimulated PGE, synthesis additively.

Phorbol Esters and

Diacylglycerols Amplify Prostaglandin Synthesis

Phorbol esters and OAG have been found to potentiate arachidonic acid release and metabolism in response to the calcium ionophore A23187 (9, lo), epinephrine (ll),and endotoxin and zymosan (12) in several cell types. Among those studies, the protein kinase C inhibitor H-7blocked endotoxin, zymosan, and epinephrine-stimulated arachidonic acid metabolism, consistent with a role for protein kinase C in the mediation of receptor activation of arachidonic acid metabolism in response to those agonists. In one study (9), the augmentation of A23187-stimulated arachidonic acid metabolism by PMA was found to require much larger concentrations of PMA than did stimulation of protein kinase C activity. In the present study, PMA, PDBu, nor OAG stimulated PGE, synthesis when added to the cultures for 5 min, the time required for bradykinin to stimulate PGE, synthesis maximally (2). However, preincubation with phorbol esters or OAG did stimulate PGE, synthesis. Stimulation of basal PGE synthesis by phorbol esters appeared to involve protein kinase C, since desensitization of protein kinase C by exposure of the cultures to PMA or PDBu for 48 h, or addition of the protein kinase C inhibitor H-7, blocked the stimulation of PGE, synthesis. However, neither desensitization nor inhibition of protein kinase C blocked phorbol ester or OAG potentiation of bradykinin-stimulated PGE, synthesis. These results aresimilar to those recently reported for the effects of phorbol esters and OAG on the fMLP-elicited respiratory burst in neutrophils (13). In that study, H-7 inhibited the effects of phorbol esters or OAG by themselves to elicit a respiratory burst, but was without effect on phorbol ester or OAG amplification of fMLP-induced respiratory burst. The mechanism by which phorbol ester andOAG enhanced bradykinin-stimulated PGE, synthesis is at present unclear. The dose-response relationship for PMA to stimulate PGE synthesis was similar to those reported to stimulate protein kinase C. OAG, in the present study, wasmore potent in stimulating PGE, synthesis than has been reported for its enhancement of protein kinase C activity (13). However, rigorous dose-response curves would have to be generated for both processes under our conditions to confirm that suggestion. Perhaps bothphorbol esters and OAG stimulated a protein kinase C which is resistant to desensitization and H-7. If so, the activity of such an enzyme would have to account for only a minute portionof total cellular protein kinase C, since both desensitization and H-7 blocked detectable protein kinase C activity in cell-free preparations from Swiss 3T3 cells. Alternatively, phorbol esters and OAG may have altered the phospholipid bilayer structure of cellular membranes, making the substrate more available for the phospholipase which releases

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arachidonate for PGE, synthesis. Such a mechanism has been proposed to account for the action of diacylglycerois onplatelet arachidonate metabolism (14, 15). Since phospholipase A, is a calcium-dependent enzyme, another potential mechanism for potentiation of bradykinin-stimulated PGE, synthesis by phorbol esters or OAG might be a rise in intracellular free calcium, or a lowering of the calcium requirement for the phospholipase. Phorbol esters and OAG do increase free intracellular calcium in Swiss 3T3 cells (16). Finally, it has recently been suggested that protein kinase C and phospholipase A, possess similar regulatory sequences (4).If this suggestion proves true, then phorbol esters and OAG may havestimulated PGE, synthesis by direct activation of phospholipase A,. Such activity suggests a novel mechanism for receptor-receptor interaction: diacylglycerol “priming” of phospholipase A, activation in response to activation of other receptors. The present results suggest that diacylglycerols maytransduce receptor-receptor interactions within cells by mechanisms independent of their activation of protein kinase C. Several receptor types stimulate formation of diacylglycerol. The diacylglycerol produced may potentiate the effects of subsequent activation of other receptors. REFERENCES 1. Burch, R. M., and Axelrod, J. (1987) Proc. Natl. Acad. Sci. U. S. A. 84,6374-6378 2. Kunze, H., and Vogt, W. (1971) Ann. N. Y.Acad. Sci. 180, 123125 3. Niedel, J. E., and Blackshear, P. J. (1986) in Receptor Biochemistry and Methodology (Putney, J. W., Jr., ed) Vol. 7, pp. 4788, Alan R. Liss, New York 4. Maraganore, J. M. (1987) Trends Phurmucol. Sci. 12, 176-177 5. Todaro, G. J., and Green, H. (1963) J. Cell Biol. 17,299-313 6. Rodriguez-Pena, A., and Rozengurt, E. (1984) Biochem. Biophys. Res. Commun. 120, 1053-1059 7.Zachary, I., Sinnett-Smith, J. W., andRozengurt, E. (1986) J. Cell Biol. 102,2211-2222 8. Hidaka, H., Inagaki, M., Kawamoto, A., and Sasaki, Y. (1984) Biochemistry 23,5036-5041 9. Halenda, S. P., Zavoico, G. B., and Feinstein, M. B. (1985) J . Biol. Chem. 260,12484-12491 10. Billah, M. M., and Siegel, M. I. (1987) Biochem. Biophys. Res. Commun. 144,683-691 11. Jeremy, J. Y., and Dandona, P. (1987) Eur. J . Pharmacol. 136, 311-316 12. Burch, R. M. (1987) Eur. J. Phurmucol., 142, 431-435 13. Bass, D. A., Gerard, C., Olbrantz, P., Wilson, J., McCall, C. E., and McPhail, L. C. (1987) J. Biol. Chem. 2 6 2 , 6643-6649 14.Dttwson, R. M. C., Hemington, N. L., andIrvine, R. F. (1983) Biochem Biophys. Res. Commun. 1 1 7 , 196-201 15. Dawson, R. M. C., Irvine, R. F., Bray, J., and Quinn, P. J. (1984) Biochem. Biophys. Res. Commun. 125,836-842 16. Mendoza, S. A., Schneider, J. A., Lopez-Rivas, A., Sinnett-Smith, J. W., and Rozengurt, E.11985) J. Cell Bwl. 102,2223-2233