An Increase in Intracellular Free Ca2+ Associated

THEJOURNALOF BIOLOGICAL CHEMISTRY

Vol. 263, No. 34, Issue of December 5, pp. 18030-18035,

0 1988 by The American Society for Bioehemistry andMolecular Biology, Inc.

Printed in

1988

U.S.A.

An Increase in Intracellular Free Ca2+Associated with Serum-free Growth Stimulation of Swiss 3T3 Fibroblasts by Epidermal Growth Factor in the Presence of Bradykinin* (Received for publication, April 4, 1988)

Richard OlsenS, Kenneth SantoneS, Deborah Melder$, S. George Oaks@, Robert Abraham$$, and Garth PowisSlI From the Departmentsof $Pharmacology and $Immunology, Mayo Clinic & Foundation, Rochester, Minnesota55905

Bradykinin gave a biphasic increase in intracellularsuch as platelet-derived growth factor (PDGF)’ to itsreceptor freeCa2+ concentration ([Ca2+]i)inserum-deprived activates a phosphoinositide-specific phospholipase C resultSwiss 3T3 fibroblasts loaded with the photoprotein ing in the hydrolysis of membrane phosphoinositol-4,5-biaequorin. Epidermal growthfactor (EGF) alone didnot phosphate to diacylglycerol and water-soluble inositol polyincrease [Ca2+Ii,but when added after bradykinin therephosphates, including inositol 1,4,5-trisphosphate (IP3(1,4,5)) was an increase in [Ca2+Ii. TheEGF-dependentin(for a review see Ref. 5). IP3(1,4,5) combines with a receptor crease in[Ca2+Iiwas maximalat 3 min and disappeared on the endoplasmic reticulum, releasing stored intracellular with a half-life of 6 min after bradykinin. Removing Ca”, which results in an increase in intracellular free Ca2+ Ca” from the external medium did not abolish either concentration ( [Ca”Ii). Diacylglycerol activates a phosphothe bradykinin or the EGF-induced [Ca2+liresponses. lipid-dependent, Ca2+-activated proteinkinase C that phosAlthough prostaglandins Ez and FZn also gave [Ca2+Ii phorylates protein serine and threonine residues (9). Protein responses and permittedan EGF-dependent [Ca2+Iire- kinase C-dependent protein phosphorylation, together with sponse, the effect of bradykinin did not appear to be Ca2+-dependentactivation of key processes, can lead to initimediated by prostaglandins since it was not blocked by ation of DNA synthesis. indomethacin. Vasopressin and phorbol 12-myristate Alternatively, binding of a growth factor to its receptor 13-acetate both gave a [Ca2*]i response but did not stimulates receptor tyrosine protein kinase activity leading to facilitate a [CaZ+li response by EGF. Bradykinin or phosphorylation of regulatory proteins that may be a stimulus EGF alone did not increaseDNA synthesis in growth- leading to DNA synthesis (10). That epidermal growth factor arrested Swiss 3T3 fibroblasts, but EGF added to- (EGF) may stimulate cell proliferation through the receptor gether with, or after, bradykinin increased DNA syn- tyrosine protein kinase pathway is supported by the findings thesis. The effect disappeared with a half-life of 180 that EGFactivates EGF receptor tyrosine protein kinase (11, min after the addition of bradykinin. It is concluded 12) and the failure to find an increase in inositol polyphosthat stimulationof receptor protein tyrosine kinaseis phates or [Ca2+]iin Swiss 3T3 fibroblasts exposed to EGF unlikely, by itself, to explain the increase in DNA (13, 14). Other workers have, however, reported that EGF synthesis produced by EGF. The observed increase in increases inositol polyphosphates in human fibroblasts (15) [Ca2+Iicaused by EGF after bradykinin probably re- and A431 epidermal carcinoma cells (16-18), as well as in flects the interaction of intracellular second messenger Swiss 3T3 fibroblasts (18-20). Thus, the second messenger pathways leading to facilitation of DNA synthesis. pathway utilized by EGF for stimulationof cell proliferation remains unclear. Nontransformed cells require more than one growth factor Growth factors are essential for the proliferation of non- for proliferation (1). EGF causes quiescent BALB/c 3T3 transformed cells as well as, in decreased amounts, for the fibroblasts to synthesize DNA and proliferate only if the cells proliferation of transformed cells (1-4). Second messenger are firstrendered “competent” by exposure to another growth factor such as PDGF, insulin, vasopressin, or bombesin (21systems transmit thesignal from activated cell surface growth 23). Synergy between EGF and othergrowth factors, or comfactor receptors to thecell nucleus where a series of molecular binations of growth factors, has also been reported in Swiss events culminate in the initiation of DNA synthesis. A num- 3T3 mouse fibroblasts (7, 24). Although there has been conber of second messenger pathways have been implicated in siderable work on second messenger pathways for individual the control of cell proliferation by growth factors (5-8). The growth factors, the role of second messenger pathways in the different pathways may cooperate or could represent alter- interaction of growth factors necessary for cell proliferation native mechanisms for regulating cell proliferation by a is poorly understood. growth factor. In this study we have investigated the synergy between One scheme proposes that the binding of a growth factor EGF and bradykinin, and some other competence-inducing mitogens both on the initiation of cell proliferation measured * This work was supported by National Institutes of Health Grant CA42286. 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” in accordance with 18U.S.C. Section 1734 solelyto indicate this fact. ll To whom correspondence and reprint requests should be addressed Dept. of Pharmacology, Mayo Clinic & Foundation, 200 First St., S. W., Rochester, MN 55905.

The abbreviations used are: PDGF, platelet-derived growth factor; IP3(1,4,5),inositol 1,4,5-trisphosphate; [Ca2+]gintracellular free Ca2+ concentration; EGF, epidermal growth factor; DMEM, Dulbecco’s modified Eagle’s medium; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; EDTA, ethylenediaminetetraacetate; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraa~eti~ acid.

18030

Intracellular ea2+and DNA Synthesis by Bradykinin and EGF

18031

been discharged instantaneously. A ratio of the resting or stimulated light emission to Lmaxwas calculated and compared to an aequorin Ca2+ concentration-lightresponse curve constructedassumingan intracellular Mg' concentration of 1 mM (31).Concentration-response curves for the increase in [Ca'+], with bradykinin and EGF were fittedusing the Enzfitter nonlinear regression computer program to obtain an estimate of ECm (Elsevier-Biosoft, Cambridge, United Kingdom). fH/Thymidine Incorporation-DNA synthesis by Swiss3T3 fibroblasts was measured by [3H]thymidineincorporation. Swiss 3T3 fibroblasts were plated in 35-mm culture dishes a t 5 X lo4 cells per dish in 2 ml of DMEM containing 10% fetal calf serum and maintained for 24 h a t 37 "C ina humidified 5% CO?, 95% air environment. The dishes were rinsed twice and the cells were serum deprived by an additional 24-h incubation in 2 ml of DMEM without fetal calf serum. Preliminary experiments showed that this was the time requiredto maximally arrestthe growth of Swiss 3T3fibroblasts. EXPERIMENTALPROCEDURES DMEM containing bradykinin and EGF, alone orin various combiChemicals and Cells-Aequorin was obtained fromDr. John Blinks, nations, was added. After 24 h incubation at 37 "C with humidified Mayo Clinic, as a5 X M solutionin 0.15 M KC1 containing50 5% COS, 95% air, [3H]thymidine, 1 pCi per dish, was added for 1 h. mM HEPES, pH 7.4. EGF was purchasedfrom Calbiochem and The medium was removed and the dishes were washed three times bradykinin from ICN Biomedicals. Indomethacin, phorbol 12-myris- with 2 ml of Dulbecco's phosphate-buffered salinebefore adding 3 ml tate 13-acetate, prostaglandin E2, and prostaglandin Fzawere obtained of 10% trichloroacetic acid a t 4 "C for 10 min. Cells were detached from Sigma. [methyl-3H]Thymidine (20 Ci/mmol)was obtained from with a plastic scraper and collected by filtration through a 25-mm Du Pont-NewEngland Nuclear.Swiss 3T3fibroblastsandEGFdiameter, 0.45-pm pore membrane filter (GAG-S, Gelman Sciences nonresponsive NR-6 Swiss 3T3 fibroblasts (28) were obtained from Inc., Ann Arbor, MI) using a vacuum manifold (Amicon, Danvers, Dr. H. R. Herschman, University of California, Los Angeles. SV40- MA). The dish was washed with an additional 2 ml of 10% trichlotransformed Swiss 3T3 fibroblasts were obtained from Dr. Robert roacetic acid and the solution also passed through the filter. The filter Scott, Mayo Clinic. The meanvolume ofnormal Swiss 3T3 fibroblasts was washed with 4 ml of 5% trichloroacetic acid, then1ml of Hz0 at in suspension was determined tobe 1440 pm3/cell using a Model ZB1 4 "C and incubated with 0.5 ml of 0.01% KOH for 30 min a t room Coulter Counter used in conjunction with a Model ClOOO Coulter temperature to dissolve DNA before adding liquid scintillant (AquaChannelyzer (Coulter Electronics) calibrated with 20.01-pm diameter sol, Du Pont-New England Nuclear). [3H]Thymidine incorporation micropolystyrene spheres. assays were conducted in quadruplicate. Aequorin Loading-Swiss 3T3 cells were maintained in Dulbecco's StatisticalAnalysis-Statistical analysis of groups of data employed modified Eagle's medium (DMEM) containing 10% fetal calf serum Student's t test (32). in 75-cm2 flasks gassed with humidified 5%CO2, 95% air at 37 "C. The cells were harvested with 0.05% trypsin and 0.5 mM EDTA and RESULTS passaged before becomingconfluent. Studieswere conducted between cell passages 38 and 50. The aequorin cell loading technique was a Aequorin Loading-The low Ca2+-centrifugation method for modification of the method of Borle et al. (29). Cells fromone 75-cm2 loading Swiss 3T3 fibroblasts with aequorin gave an intracelflask were harvested andwashed by centrifuging at 200 X g for 5 min lular aequorin concentration, assuming a uniform distribution and resuspended three times in 135 mM NaCl, 4 mM KC1, 11 mM M. This of aequorin, between and within cells, of 2 X glucose, and 0.5 mM potassium phosphatebuffer, pH 7.4, a t 4 "C.The mediumusedfor the last wash also contained 1 mM EGTA. The indicates that cells took up approximately 0.5% of their volloadingprocess. The washed cells were suspended in 0.5 ml of solution containing5 X lo5 ume of external medium during the M aequorin, 0.15 M KCl, and 50 mM HEPES, pH 7.4, and incubated loaded cells lost approximately50% of their aequorin content with gentle shaking for 10 min at 4 "Cbefore being centrifuged at 200 during the first4 h of incubation and then lost aequorin more X g for 30 s. The aequorin solution could be reused for loading cells 22 h. Eighteen hours after aequorin slowly with a half-life of up to 30 times. The cells were then suspended in DMEM containing 10% fetal calf serum and plated at 1 X lo6 cells per dish in 35-mm loading the intracellular concentration of aequorin was 6 X lo-@M. An advantage of an 18-h attachment period in medium culture dishes (Falcon, Becton Dickinson Labware) containing 2 ml of medium per dish. The cells were allowed to attach to the surface containing fetal calf serum was that irreversiblydamaged of the culture dish and they were incubated for 18 h a t 37 "C in a cells did not attach to the surface of the culture dish. Also, humidified 5% Con, 95% air environment.After this time the medium the remaining cells had time to recover from the stress of was replaced with 2 ml of DMEM without fetal calf serum, and the trypsinization and the loading procedure. Aequorin was not dishes were incubated for 2 h a t 37 "C and gassed with humidified cytotoxic and the loaded cells continued to grow normally. 5% CO,, 95% air. Measurement of [Ca2+l,-An estimate of [Caz+liwas made by light The resting light emission from aequorin-loaded cells was a t emission from the serum-free aequorin-loaded cells. Culture dishes 18 h much more stable than at earlier times when a pattern containing the cells were placed in a temperature-controlled holder of irregular spiking was seen. The reason for the spiking is a t 37 "C over a radiofrequency interference-shielded photomultiplier damagedcells or to cells tube (9635QA, Thorn EM1 Gencom, Fairfield, NJ) in a light-tight not known but may bedueto box. The surface of the medium in the culture dish was continually detaching from the surface of the culture dish.Physically few attached cells caused an increase in flushed with humidified 5% CO2, 95% air. Serum, EGF, bradykinin, displacing even a and other agents dissolved in 0.2-1 ml of warm DMEM were added light emission. to the dish through separate temperature-controlled, light-tight inlet Altered [Ca2+/iby Serum-Adding 10% fetal calf serum to lines. Some cells were exposed to Ca2+-free DMEM containing 0.5 Swiss 3T3 fibroblasts incubated in DMEM without fetalcalf mM EGTA for 5 min, ortoDMEMcontaining M indomethacin for 5 min before adding EGF or bradykinin in the samemedium. At serum for 2 hcaused an increasein the apparent [Ca2+]; produce a[Ca2+]. , Increase. ' the end of each experiment the cells were lysed with 1 ml of 1% (Table I). Medium alone did not The response was transient, lasting 1 min, and [Ca2+IiinTriton X-100 containing 5 mMCaC12 and the total light signal was integrated. It took approximately 15 min for all of the cells to be creased from an average resting value of 0.2 to 1.1PM. Varying lysed. Light emitted from the cells was converted to an estimate of the period of incubation with serum-free medium between 2 [Ca"]; by the calibration methoddescribed by Allen and Blinks (30). In this method the integral of the light ( L )signal obtained during the and 48 h before adding fetal calf serum had noeffect on the Triton X-100 exposure was multiplied by the peak-to-integral ratio size or duration of the [Ca2+Iiresponse (results not shown). Altered [Ca"]; by Bradykinin andEGF-A typical increase for aequorin (2.6 s-' a t 37 "C) to obtain a calculated peak intensity (Laax)that would have been seen if all the aequorin in the cell had in apparent [Ca2+]iproducedby bradykinin inSwiss 3T3

by DNA synthesis and on changes in [Ca2+];in serum-free Swiss 3T3 fibroblasts. Bradykinin was studied because it has been reported toincrease [Ca2+];in a number of cells (25-27), although its role as a competence-inducingfactorhasnot been reported. Wefound that EGFby itself neither stimulated DNA synthesis nor produced an increase in [Ca2+Ii.Bradykinin gave a biphasic increase in [Ca2+];but by itself did not stimulate DNA synthesis. EGF added after bradykinin induced an increase in [Ca2+Ii and stimulated DNA synthesis. The results of the study support a relationship between the bradykinin-facilitated increase in [Ca2+]i by EGF and the synergistic stimulation of DNA synthesisby this combination of mitogens.

Intracellular Ca2+and DNA Synthesis Bradykinin by and

18032

TABLEI Changes in[Ca"]; in Swiss3T3 fibroblasts produced by growth factors Aequorin-loaded Swiss 3T3 fibroblasts were plated at 1 X IO6 cells/ dish and incubated for 18 h in DMEM with 10% fetal calf serum and then for 2 h in DMEM without serum. The cells were exposed to either 10% fetal calf serum, IO-' M bradykinin, 5.4 X lo-' M EGF, or M vasopressin in the combinations shown, and [Ca2*];was measured. The second agent was added 4 to 5 min after the first agent. Values are the mean ? S.D. of n different preparations. Resting

Growth factor(s)

IC~*+I~ "

Fetal calf serum Bradykinin EGF EGF after bradykinin EGF after bradykinin/ wash' Vasopressin EGF after vasopressin

Peak [ca2+li

5 0.17 f 0.05 25 0.21 k 0.05 5 0.15 f 0.00 11 0.22 f 0.06 3 0.32 It 0.03

I

0

5

CcM min 1.08 f 0.06" 1.0 f 0.1 1.51 k 0.21" 4.0 f 1.0 0.15 f 0.00 NAb 1.95 f 0.74" 1.9 ? 0.7 0.32 f 0.03NA

A

5

10

4 0.29 ? 0.14 1.59 f 0.47" 4.0 ? 0.7 4 0.30 & 0.00 0.33 f 0.06" NA

E4

Bf 5

!

0

FIG. 2. Effect ofEGF on [Ca2+]iin serum-free Swiss 3T3 fibroblasts in the absence and presence of bradykinin. A , EGF, 5.4 X IO-' M, was added atthe arrows E , before and after M bradykinin which was added at the arrow B. B , bradykinin, lo-' M, was added to another cell preparation at the arrow B before 5.4 X lo-' M EGF, which was added at the arrow E. The right-hand scale is 20 nA photomultiplier current.

2.0r

O:

Bf

J

10

Time (mid

Duration

" p < 0.01 compared to resting value. NA, not applicable. e The cells were washed immediately after exposure to bradykinin before adding EGF.

0

EGF

10

"? 16

6

4

6 8 Time (mid

IO

12

1:

FIG. 3. Increase in [Ca2+Iiin serum-free Swiss 3T3 fibroblasts produced by EGF, 6.4 X lo-' M, added at various times after bradykinin, 10" M. I

20

r m (W

2A). A second application of EGF then gave a much smaller

increase in [Ca2+];. Washing the cells free of bradykinin abolished the [Ca2+];response to EGF (Table I). The largest increase in [Ca"]; by EGF occurred when EGF was added 3 min after bradykinin; thereafter the response declined with a half-life of about6 min (Fig. 3). Concentration-response curves gave values for the increase in [Ca2+Iiby bradyfibroblasts deprived of serum for 2 h is shown in Fig. 1. There kinin of 1.2 x lo-' M, for the permissive effect of bradykinin was a rapid increase in [Ca2+]iwithin 10 s of adding the on the increase in [Ca2+]iby 5.4 X lo-' M EGF of 1.6 X lo-' bradykinin, lasting about 1 min, followed by a second smaller M, and for the increase in [Ca"]; by EGF in the presence of M bradykinin of 1.2 X lo-' M (Fig. 4). It is interesting rise in [Ca2+];which lasted 2-3 min. The average peak [Ca2+Iiresponse of a number of cell preparations to bradykinin thatatEGF concentrations greater than M thereapis shown in Table I. There was a rapid decline in the size of peared to be inhibition of the [Ca2+];response (Fig. 4C). the [Ca2+];response to repeated doses of bradykinin, although Neither the [Ca2+];response to bradykinin nor the bradykivasopressin could still produce a [Caz+];response at a time nin-facilitated EGF [Ca2+]iresponse was altered by exposing when the [Ca2+];response to bradykinin had completely dis- the cells to medium containing no Ca2+ and0.5 mM EGTA or appeared (Fig. 1). The [Ca2+];response produced by vasopres- by lo-' M indomethacin, an inhibitor of cyclooxygenase (33) sin added after bradykinin was only slightly smaller than the (Fig. 5). Varying the time of incubation with serum-free response produced by vasopressin alone (compare Fig. 1with medium between 2 and 48 h before exposing the cells to the data in Table I). The results suggest that desensitization growth factors had no effect on the size or pattern of the to the bradykinin-induced increase in [Ca2+];was primarily [Caz+];responses to either bradykinin or EGF in the presence of bradykinin (results not shown). homologous in nature. [Ca'+]i Responses with Other Mitogens-Although vasoEGF at concentrations up to M failed to produce a [Ca2+];response in any preparation of serum-free Swiss 3T3 pressin alone caused a [Ca2+Iiincrease, it did not facilitate a fibroblasts studied. However, EGF added a few minutes after [Ca2+Iiincrease with EGF added after vasopressin (Table I). M) by themselves bradykinin induced a marked increase in [Caz+];which lasted PDGF (3.3 X lo-' M) and bombesin about 2 min (Fig. 2 and TableI). When bradykinin was added caused a [Ca'+]; increase, but only bombesin facilitated a after EGF, the second phase but not the first phase of the [Ca2+];increase with EGF (results notshown). Prostaglandins bradykinin-induced [Ca2+];response was increased. This may Ez and FPa both caused an increase in [Ca2+];and facilitated represent superimposition of the EGF-induced [Ca2+];re- an increase in [Ca"]; by EGF (Fig. 6). The [Ca2+];response sponse on the second phase of the bradykinin response (Fig. to EGFin the presence of prostaglandin EPwas delayed about FIG. 1. Increase in [Ca2+]i producedby bradykinin and vasopressin in serum-free Swiss 3T3 fibroblasts. Bradykinin, lo-' M, was added at the arrows B, vasopressin, lo-' M, was added at the arrow V. The right-hand scale is 20 nA photomultiplier current.

18033

Intracellular Ca2+and DNA Synthesis by Bradykinin and EGF

B

C A 1 2 0 "A

Ru

Time (mid

Concontr8tIon (M)

FIG. 6. Effect of prostaglandins on resting [Ca2+]iand the FIG. 4. Concentration-responserelationships of theinfibroblasts. crease in [Ca2+Iiin serum-free Swiss 3T3 fibroblasts to dif- [Ca2+]iresponse to EGF in serum-free Swiss 3T3 M;and B, prostaglandin E, (PGE,), ferent concentrations of: A (O), bradykinin ( B K ) :B (m), bra- A , prostaglandin F,, (PGF,,) M, added before EGF, 5.4 X IO-' M, at the arrows E. The rightdykinin inducing a change in [Ca2+]iby 5.4 X lo-' M EGF; hand scale is 20 nA photomultiplier current. and C (A),EGF added 5 min after 10" M bradykinin.

I

0

20

0

I

5

B

A

EGTA

5

0

0

5

40 60 80 100

0

Time

2

1

3

4

5

EGF (nM)

Bradykinin (nM)

FIG. 5. Effect of removing external Ca2+and of indometh-

FIG. 7. ['HIThymidine incorporationin 24-h serum-deacin on the [Ca2+]iresponse of serum-free Swiss 3T3 fibro- prived Swiss 3T3 fibroblasts exposed to bradykinin and EGF. blasts to bradykinin and EGF. h f t panel, control cells; middle A , concentration-response curve to bradykinin without (0)and with panel, cells exposed to medium containing no Ca2+and 0.5 mM EGTA (e)EGF, 3.3 X IO-' M. B, concentration-response curve to EGF for 5 min; and right panel, cells exposed to IOT5M indomethacin for without (A) and with (A)bradykinin, 5 X M. EGF was added 1 5 min. Bradykinin, M, was added at the arrows B and EGF, 5.4 min after bradykinin. Values are mean of four determinations, bars X lo-' M, at the arrows E. The right-hand scale is 20 nA photomulare standard error. tiplier current.

1 min. Phorbol12-myristate13-acetate M) produced a small transientincreasein [Ca2+]i but did notpermit a [Ca2+Iiresponse to EGF (results not shown). EGF-nonresponsive and SV4O-transformed Swiss 3T3 Cells-In an EGF-nonresponsive Swiss 3T3 fibroblast line that, presumably,lacks functional EGF receptors (28) the [Ca2+Ii response to bradykinin was decreased compared to normal Swiss 3T3 fibroblasts, whereas the [Ca2+Iiresponse to EGF after bradykinin was almost completely absent (results not shown). The [Ca2+]i responses of SV40-transformed Swiss 3T3 fibroblasts were studied because the cells reportedly had a decreased growth factor requirement for proliferation (33). The cells showed [Ca2+Ii responses to bradykinin and EGFof a magnitude similar to that in non-transformed Swiss 3T3 fibroblasts (results not shown). DNA Synthesis-Bradykinin and EGF alonedid not stimulated [3H]thymidine incorporation in serum-free Swiss 3T3 fibroblasts, but in the presence of both growth factors there was a large increase in [3H]thymidine incorporation (Fig. 7). The lowest growth factor concentration producing maximal DNA synthesis was lo-@M for bradykinin and lo-' M for

n

m

0 7

W

I

a 0

lo^"""" """""""""!

u

00

40

80

120

160

200

240

TIME (min) FIG. 8. Stimulation of ['Hlthymidine incorporation in 24-h serum-deprived Swiss 3T3 fibroblasts by EGF, 3.3 X lo-' M, added at various times after bradykinin, 5 X lo-' M. Values are mean of four determinations, bars are standard error. The dotted line is [3H]thymidine incorporation without growth factors.

EGF. Maximal [3H]thymidine incorporation was seen when EGF was added 10 min after bradykinin; thereafter theeffect disappeared with a half-life of about 180 min (Fig. 8). EGF

18034

Intracellular Ca2+and DNA Synthesis by Bradykinin and EGF

added 10 min before bradykinin did not stimulate [3H]thymidine incorporation (results not shown). DISCUSSION

Valid measurement of changes in [Ca2+];with an intracellular Ca2+-sensitiveprobe requires that the insertion of the probe does not damage the cell, resulting in pathological changes in [Ca2+];which obscures physiological responses, while the presence of the probe should notalter normal [Ca"]; responses (34). The hyperpermeabilization loading procedure we used gave an estimated intracellular aequorin concentration of 6 X lo-' M, 18 h after cell loading. This is greater than the intracellular aequorin concentration of 1-5 X lo-' M reported by Borle et al. (29) in hyperpermeabilized kidney cells. The increased loading of aequorin achieved by us was probably due to our use of a higher concentration of aequorin during cell loading. Aequorin at these intracellular concentrations should not buffer [Ca2+];.Blinks et al. (35) have calculated that buffering of [Ca2+];by intracellular aequorin inan otherwise unbuffered system should not be significant below about 1 X M aequorin. The cells, after an initial growth delay due to passage, continued to grow normally, demonstrating the absence of cytotoxicity by the aequorin. Exposure of serum-free Swiss 3T3 fibroblasts to bradykinin resulted in a transient, biphasic increase in [Ca"]i. Studies in which fluorescent Ca'+-sensitive cell-permeant probes were used to measure [Ca2+]ihave shown only a single [Ca2+Iipeak with bradykinin (17,26,27,36) or, in some studies, a sustained [Ca2+Ji plateau after the initial response (25). The absence of a clearly defined second [Ca'+]; peak after bradykinin in these studies maybedue tothe well known abilities of Ca2+sensitive fluorescent probes to buffer changes in [CaZ+li (34). The increase in [Ca2+];produced by bradykinin in Swiss 3T3 fibroblasts probably occurs through bradykinin-stimulated IP3(1,4,5)formation and subsequentrelease of Ca" from intracellular stores, as reported for other cell types (26, 27, 37, 38). Support for this mechanism comes from the finding that removing extracellular Ca2+ did not affect the rapid [Ca"]; response to bradykinin. Other investigators have found that removing external Ca2+has no effect on the peak [Ca'+]; response to bradykinin (25, 26), although Reiser and Hamprecht (27) reported that omitting external Ca'+ completely blocked the bradykinin-induced [Ca2+];increase in a neural cell line. We also found that thesecond slower [Ca2+];response to bradykinin was not affected by removing external Ca2+. Some studies have reported that removing external Ca2+ shortens or abolishes the plateau phase of the [Ca'+]; response to bradykinin (17, 37, 38), although the exact relationship of this plateau phase to our observed second [Ca2+]iincrease remains unclear. We observed rapid desensitization to the bradykinin-induced increase in [Ca2+Ii.This has been seen in other cell types with bradykinin (26, 27, 37). Vasopressin, which increases [Ca2+Iiby an IP3(1,4,5)-mediated release of Ca2+from the endoplasmic reticulum in Swiss 3T3 cells (391, still gave an increase in [Ca*+Iiin bradykinin-desensitized Swiss 3T3 fibroblasts. This suggests that the effect probably represents bradykinin receptor desensitization, rather than depletion of internal Ca2+stores. In our studies we found no increase in [Ca'+];by EGF alone, M, with serum-free Swiss even at concentrations up to 3T3 fibroblasts. This is in agreement with reports by McNeil et al. (13) and Tsuda et al. (14), but differs from reports by Hesketh et al. (19), Morris et al. (20), andPandiella et al. (18), in which an increase in [Ca2+];by EGF was seen in Swiss 3T3 fibroblasts. We found that pretreatment of the cells with

bradykinin was required for EGF to produce an increase in [Ca'+];. It is possible that, in the studiesreporting an increase in [Ca2+]iwith EGF alone, an endogenous or serum-derived growth factor remained that facilitated the EGFresponse. In the A431 epidermal carcinoma cell, EGF hasbeen reported to increase [Ca2+];by inducing an influx of extracellular Ca2+ across the cell membrane, possibly through avoltage-dependent Ca2+channel (16, 40). We found that removing external Ca2+did not block the bradykinin-facilitated [Ca2+Iiresponse to EGF inSwiss 3T3 fibroblasts, suggesting that inthese cells the Ca2+may be released from intracellular stores. Bradykinin can stimulate arachidonic acid and prostaglandin formation (41-44). Arachidonic acid is itself an intracellular second messenger (46,47). We found that prostaglandins E' and FZUboth increased [Ca2+Iiand facilitated an increase in [Ca2+Iiby EGF in Swiss 3T3 cells. However, neither arachidonic acid nor prostaglandins appear to be involved in the facilitation of the EGF-induced [Ca2+];increase by bradykinin since it was not blocked by indomethacin at a concentration that blocked the bradykinin-induced release of arachidonic acid and prostaglandin formation in Swiss 3T3 fibroblasts (47).' Furthermore, although vasopressin was found to increase arachidonic acid formation in the Swiss 3T3 fibroblasts,' it did not facilitate an EGF-dependent [Ca2+],response. Vasopressin has been reported to increase IP3(1,4,5) and DG formation in Swiss 3T3 fibroblasts (39), so that activation of this pathway alone would not appear to be responsible for facilitation of the EGF-induced [Ca2+];increase by bradykinin. The mechanism may involve changes in the levels of other inositol polyphosphates, the functions of which are only now being investigated (5, 48). One possibility is that binding of EGF to itsreceptor, which is known to lead to increased receptor phosphatidylinositol kinase activity as well as increased protein tyrosine kinase activity (49), resultsin increased formation of IP3(1,4,5)by increasing the pool of phosphoinositol 4,Ei-biphosphate available to the bradykinin-activated phospholipase C.By this mechanism EGF indirectly stimulates the release of intracellular Ca2+ only when phospholipase C activity is increased by bradykinin. There is a precedent for the facilitation of the EGFinduced [Ca2+Iiresponse by bradykinin. Insulin-like growth factor I1 does not by itself increase [Ca2+Iiin BALB/c 3T3 cells, but in PDGF-treated cells it facilitates an increase in [Ca2+];by EGF (50). An interesting featureof the bradykinin- andEGF-induced [Ca2+];responses is that they were seen after only 2 h serum deprivation, before all the cells were arrested in the GOcell cycle phase, and they did not increase with longer times of serum deprivation up to 48 h. This suggests that all cells, or at least the fraction of cells that are going to respond, give a [Ca2+];response independent of the stage of the cell cycle. A key question is whether the facilitation of the EGFinduced [Ca2+];response by bradykinin is important for the control of cell proliferation. Neitherbradykinin nor EGF alone increased DNA synthesis in growth-arrested Swiss 3T3 fibroblasts, although the combination of bradykinin and EGF produced a marked stimulation of DNA synthesis. This is similar to the synergy reported between EGF and other mitogens in causing cell proliferation in BALB/c 3T3 fibroblasts (21-23) and in Swiss 3T3 fibroblasts (7, 24). Mitogens such as PDGF and phorbol ester cause a paradoxical decrease in EGF receptor binding through phosphorylation of the EGF receptor by an increase in [Ca"]; and activation of protein kinase C (51). This has been suggested to be related to the stimulation of cell proliferation by EGF (52). However, EGF

* R. Abraham, unpublished observations.

Intracellular Ca2+and DNA Synthesis by Bradykinin and EGF receptor phosphorylation by protein kinase C cannot explain the bradykinin-facilitated [Ca2+];response to EGF we observed because phorbol12-myristate 13-acetate andvasopressin were unable to mimic this effect of bradykinin. The effects of EGF and bradykinin in increasing [Ca2+Iiand DNA synthesisboth occurred at nanomolar concentrations of the growth factor, yet the two effects showed different time dependencies. The effect of bradykinin in facilitating an increase in [Ca2+]iby EGF disappeared with a half-life of 6 min, whereas the facilitation of the increased DNA synthesis with EGF lasted much longer, disappearing with a half-life of around 180 min. A second increase in [Ca2+];by EGF after bradykinin by itself is unlikely to account for the stimulation of DNA synthesis since vasopressin, which causes an increase in [Ca2+]; eitherbefore or after bradykinin, could not replace EGF in stimulating DNA synthesis. It is likely that the increase in [Ca2+Iiby EGF after bradykinin is only part of a complex series of changes in intracellular second messengers that lead to initiation of cell proliferation after exposure to the growth factors. An EGF-nonresponsive cell line that may not have functional EGFreceptors (28) did not give a bradykinin-facilitated EGF [Ca2+Ii response. The [Ca"]; response to bradykinin was also decreased in these cells, so that a lack of EGF receptors may not be the only mechanism responsible for the EGF insensitivity. SV4O-transformedSwiss 3T3 fibroblasts showed bradykinin- andEGF-induced [Ca2+Iiresponses similar to the [Ca2+],responses in non-transformed Swiss 3T3 cells. SV40transformed cells have normal levels of functional EGF receptors (53), butSV40-transformed Swiss 3T3 fibroblasts do not require EGF for continued proliferation (54). It seems most likely that SV40-transformed Swiss 3T3 fibroblasts have bypassed a requirement for a [Ca2+Iiincrease and biochemical signals delivered by EGF in order to maintain the constitutive state of proliferation. In conclusion, evidence for an association between the bradykinin facilitation of an EGF-induced [Ca2+];response and stimulation of DNA synthesis by bradykinin and EGF has been presented. The evidence is still circumstantial and more work is needed to determine how the increase in [Ca2+]; is related to other actions of EGF such as receptor autophosphorylation, protein phosphorylation, and changes in inositol polyphosphate synthesis. Acknowledgments-The helpful discussions of Dr. John Blinks and the excellent secretarial assistance of Wanda Rhodes are gratefully acknowledged. REFERENCES 1. Goustin, A. C., Leof, E. B., Shipley, G. D., and Moses, H. L. (1986) Cancer Res. 46,1015-1029 2. Scherr, C. J. (1987) Mol. Biol. Med. 4 , 1-10 3. Wahl, M. I., and Carpenter, G. (1987) Recent Adu. Biochem. Bid. Cancer 5 , 130-139 4. Weinstein, I. B. (1987) J. Cell. Biochem. 33,213-224 5. Berridge, M. J. (1987) Bbrhim. Biophys. Acta 907,33-45 6. Rasmussen, H. (1986) N. Engl. J. Med. 147,1094-1101,1164-1170 7. Rozengurt, E. (1986) Science 2 3 4 , 161-166 8. Taylor, C. W. (1986) Trends Pharmacol. Sci. 7,467-471 9. Takai, Y., Kikkawa, U., Kaihuchi, K., and Nishizuka, Y. (1984) Adu. Cyclic

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