Proc. Nail. Acad. Sci. USAVol. 81, pp. 6762-6766, November 1984Cell Biology

Inhibition of epidermal growth factor-induced mitogenesis byamiloride and an analog: Evidence against a requirement forNa+/H+ exchange

(growth control/anti-EGF antibodies/DNA synthesis/RNA synthesis/protein synthesis)

JEFFREY M. BESTERMAN*, SCOTT J. TYREY*, EDWARD J. CRAGOE, JR.t, AND PEDRO CUATRECASAS**Department of Molecular Biology, The Wellcome Research Laboratories, Research Triangle Park, NC 27709; and tMerck, Sharp & Dohme ResearchLaboratories, West Point, PA 19486

Contributed by Pedro Cuatrecasas, July 16, 1984

ABSTRACT We have tested the hypothesis that the rapidstimulation of Na'/H' exchange by epidermal growth factor(EGF) is a requirement for induction of mitogenesis. BALB/c3T3 cells exposed for 4 hr at 370C to both EGF at 1 ng/ml andeither 0.2-1 mM amiloride (an inhibitor of Na+/HI exchange)or 10 IAM MK-685 (an amiloride analog and more potent in-hibitor of Na+/H+ exchange) incorporated no less [methyl-3H]thymidine during a 1-hr pulse 20 hr later than did cellsexposed for 4 hr to EGF alone. Control experiments utilizinglow external pH (to dissociate EGF from its receptor) and anti-EGF antibodies indicated that the failure of amiloride to inhib-it mitogenesis when copresent with EGF during the first 4 hrwas not due to incomplete removal of EGF and complete re-moval of amiloride at t4. Cells incubated with 200 ,uM amilor-ide for 24 hr showed nearly complete inhibition of stimulationby EGF. In comparison, cells incubated with 10 ,uM MK-685for 24 hr showed only a slight inhibition of stimulation byEGF. Incubations with amiloride or MK-685 for shorter peri-ods of time indicated that only amiloride inhibited mitogenesisand that this inhibition happened between 4 (t4) and 10 (tie) hrafter EGF addition, during which time increases in RNA andprotein syntheses (required for mitogenesis) occurred. Amilor-ide inhibited both RNA and protein syntheses in intact cellsduring this prereplicative period, while MK-685 was withouteffect. We conclude that (i) inhibition of EGF-induced mito-genesis by amiloride is due not to inhibition of EGF-stimulatedNa+/H' exchange but rather to inhibition of necessary eventsoccurring during the hours immediately prior to the onset ofDNA synthesis, these events probably being RNA and proteinsynthesis and (ii) in cell culture medium buffered withCO2/HCO-, complete inhibition of EGF-stimulated Na+/H+exchange does not'inhibit EGF-induced mitogenesis and, thus,stimulation ofNa+/H+ exchange is not necessary for inductionof mitogenesis by EGF.

Recent studies on the mechanism of growth factor-inducedmitogenesis have emphasized the potentially important roleof early ionic events. Serum as well as individual growth fac-tors, such as epidermal growth factor (EGF), rapidly stimu-late Na+/H+ exchange in cultured cells (1-5). The diureticamiloride has been shown to block growth factor (e.g., EGF)stimulation of Na+/H+ exchange (1-5). Cells cultured in thepresence of amiloride for 12-24 hr after addition of mitogenshow inhibition of DNA synthesis (1, 2, 5). Therefore, wetested the hypothesis that the immediate activation ofNa+/H+ exchange by a growth factor such as EGF is a re-quirement for induction of mitogenesis (1-3). We report thatthe rapid stimulation of amiloride-sensitive Na+/H+ ex-

change by EGF appears unnecessary for induction of mito-genesis in BALB/c 3T3 cells in culture.

MATERIALS AND METHODSCell Culture. Cells were grown in Linbro 24-well plates

(Flow Laboratories, McLean, VA) in 1 ml of Dulbecco'smodified Eagle's medium (DME medium) supplementedwith serum [7.5% newborn calf serum (NBCS) for BALB/c3T3 cells, 5% calf serum for the human foreskin fibroblastline HF4, and 10% fetal calf serum for the human adult fibro-blast line A34] in a humidified atmosphere of 95% air/5%CO2 at 37°C. Wells were seeded with 2-5 x 104 cells. Thecells grew to confluence in 3-6 days, at which time the medi-um was replaced with DME medium/0.5% serum. The cellswere maintained under these "serum starved" conditions for24-48 hr prior to the start of an experiment to ensure arrestin the Go/G1 stage of the cell cycle. Cells of the BALB/c 3T3mouse fibroblast line (clone A31, passage 64) were obtainedfrom the American Type Culture Collection. The A34 cells(passage 10) and the HF4 cells (passage 16) were kindly pro-vided by P. Comens and J. Baker (University of Kansas,Lawrence).

Experimental Design. All work was performed with quies-cent cell monolayers in DME medium/0.5% serum, pH 7.4,in a humidified atmosphere of 95% air/5% CO2 at 37°C.Changing to fresh medium just prior to the start of an experi-ment had no effect on results other than to raise the level ofbasal incorporation of [methyl-3H]thymidine. Each experi-ment was initiated at time to by addition of EGF (final con-centration 1 ng/ml, unless specified otherwise). Monolayersthat were to be exposed to amiloride (final concentration 200,uM, unless specified otherwise) for the first 4 hr of the ex-periment (10-14) were preincubated with amiloride for 1 hr(t-1-to) before addition of EGF. Reducing this preincubationperiod to 15 min did not affect the results. Unless specifiedotherwise, cells were exposed to EGF for 4 hr (to-t4). At 14,monolayers were washed thoroughly using a variety of pro-cedures (see Results) and then incubated in DME medi-um/0.5% serum for 20 hr at 37°C. At t24, monolayers werewashed and then allowed to incorporate [3H]thymidine intoDNA (acid-insoluble fraction) during a 1 hr pulse (124-t25, seebelow). All cultures received the same number of mediumchanges and washes to maintain a constant background ofbasal incorporation of [3H]thymidine into DNA.

[3H]Thymidine Incorporation. At the indicated time (usual-ly t24), monolayers were washed once with DME medi-um/0.5% serum and incubated for 1 hr at 37°C in 1 ml of thesame medium containing 1 ,Ci (37 kBq) of [3H]thymidine.Incorporation of label was halted by washing the cultures six

Abbreviations: EGF, epidermal growth factor; NBCS, newborn calfserum.

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times with ice-cold phosphate-buffered saline, pH 7.4, andincubating them at 40C for .30 min in acidic fixative [50%(vol/vol) methanol/10% (vol/vol) acetic acid, 1 ml per cul-ture]. The fixative was removed and the adherent insolublefraction was dissolved in 1% NaDodSO4 (using two 150-,tlaliquots) and added to 3 ml of Aquasol-2 (New England Nu-clear). Radioactivity was determined using an LKB liquidscintillation counter. In addition, the acid-soluble fractionwas dried under nitrogen, and the radioactivity in it wasmeasured and taken as an index of the free cellular pool ofthymidine-containing deoxynucleotides.

[3H]Uridine and [3H]Leucine Incorporation. Incorporationof [3H]uridine (10 /.Ci/ml of medium) into RNA or [3H]leu-cine (5 ACi/ml of medium) into protein was determined asdescribed above for [3H]thymidine incorporation into DNA.DME medium/0.5% serum contains -1 mM leucine. At thisconcentration of extracellular leucine, it has been shown forother fibroblasts that equilibrium is established between leu-cyl-tRNA, free intracellular leucine, and extracellular leu-cine (J. N. Hildebran and R. B. Low, personal communica-tion). Thus, incorporation of [3H]leucine into an acid-insolu-ble pool probably reflects the true rate of leucine incor-poration into protein synthesized de novo.

Immunoglobulin Purification and Protein Assay. Protease-free IgG was isolated from sera (rabbit anti-mouse EGF seraand control rabbit serum) by chromatography on DEAEAffi-Gel Blue (Bio-Rad). Sera were dialyzed against 20 mMTris HCl buffer, pH 8.0/28 mM NaCl/0.02% NaN3, appliedto a DEAE Affi-Gel Blue column pre-equilibrated in thesame buffer (without NaN3), and eluted using the same buff-er. Dialysis and column chromatography were performed at4°C. Fractions containing protein (i.e., IgG) were pooled,concentrated to 0.2-0.4 mg/ml by ultrafiltration and lyophi-lization, and stored frozen. IgG fractions were preparedfrom two rabbit anti-mouse EGF antisera and designatedIgG-1 and IgG-2. Protein was assayed by the method ofBradford (6), using bovine serum albumin as a standard.

Materials. Amiloride [3,5-diamino-6-chloro-N-(diamino-methylene)pyrazinecarboxamide hydrochloride dihydrate]and MK-685 [3-amino-5-dimethylamino-6-chloro-N-(diami-nomethylene)pyrazinecarboxamide hydrochloride] weresynthesized as described (7, 8). Mouse EGF was preparedfrom the submaxillary glands of male mice by a slight modifi-cation of the HPLC method of Burgess et al. (9). Rabbit anti-mouse EGF antisera were kindly provided by P. Comensand J. Baker (University of Kansas, Lawrence) and Y.Shechter (Weizmann Institute, Rehovot, Israel). Rabbit se-rum (type 100) was from Quadroma (Escondido, CA); DMEmedium, fetal bovine serum, NBCS, and calf serum werefrom GIBCO; [methyl-3H]thymidine (20 Ci/mmol), L-[4,5-3H]leucine (50 Ci/mmol), [5,6-3H]uridine (40 Ci/mmol), andAquasol-2 were from New England Nuclear.

RESULTSKinetics of EGF-Induced Mitogenesis in the Absence and

Presence of Amiloride. Fig. 1 illustrates the dependence ofstimulation of mitogenesis on the length of exposure to EGFin the absence and presence of amiloride (200 ,uM). Expo-sure to EGF (1 ng/ml) for 24 hr gave maximal stimulation of[3H]thymidine incorporation by serum-starved (0.5% NBCS)BALB/c 3T3 cells. [The plasma concentration of EGF inboth mouse (10) and man (11) is 1 ng/ml.] Stimulation byEGF was almost completely inhibited by continuous expo-sure to amiloride during the 24 hr before the [3H]thymidinepulse.

Inhibition of EGF-Stimulated Na+/H+ Exchange Does NotInhibit Mitogenesis. Growth factors rapidly stimulateNa+/H' exchange in cultured cells (1-5). Using the pH-stattechnique to titrate extracellular pH (12), we found that EGFstimulated Na'-dependent H' extrusion from serum-

20

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FIG. 1. Dependence of EGF-induced mitogenesis on the lengthof exposure to EGF. Monolayers of serum-starved BALB/c 3T3cells were incubated at 370C in the presence of EGF (o, ^, 1 ng/ml;*, 10 ng/ml) for various lengths of time, washed three times at pH7.4, 370C (standard procedure), and then incubated in the absence ofEGF. When amiloride was present (200 gM, 0), it was present for 24hr. Twenty-four hours after exposure to EGF began, all monolayerswere again washed and then incubated with [3H]thymidine for 1 hr(see Materials and Methods). Results are expressed as means +SEM for 3-15 independent experiments; each experiment was per-formed in triplicate.

starved BALB/c 3T3 cells; the stimulation was inhibited=75% by 200 ,uM amiloride (data not shown). To determinethe role of the initial, amiloride-sensitive ionic event in EGF-induced mitogenesis, 4 hr of exposure to EGF (1 ng/ml) waschosen as the shortest exposure to the mitogen that wouldprovide sufficient stimulation (3- to 4-fold increase in[3H]thymidine incorporation, see Fig. 1). Increasing the con-centration of EGF to 10 ng/ml did not enhance the stimula-tion. Incubation of cells with EGF for 4 hr combined withcontinuous exposure to amiloride (24 hr) inhibited stimula-tion almost completely (Fig. 1).

Fig. 2 shows that when 200 ,uM amiloride was present dur-ing but not after the 4-hr exposure to EGF, inhibition ofEGF-induced mitogenesis did not occur. Cells were preincu-bated with amiloride for 1 hr, then incubated with EGF andamiloride. After 4 hr, the monolayers were washed and thenincubated in medium devoid of EGF and amiloride for an

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FIG. 2. Effect of 4-hr exposure to EGF and amiloride on[3H]thymidine incorporation. Monolayers of serum-starved (0.5%NBCS) BALB/c 3T3 cells were incubated at 370C in the absence orpresence of EGF and/or amiloride for 4 hr, washed three times atpH 7.4, 370C (standard procedure), and incubated in the absence ofboth EGF and amiloride for 20 hr. All monolayers were againwashed and then incubated with [3H]thymidine for 1 hr. Data areexpressed as the mean ± SEM for two independent experiments;each experiment was performed in triplicate.

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additional 20 hr, at which time a 1-hr pulse of [3H]thymidinewas administered. To ensure that the Na+/H+ antiporterwas completely inhibited, 1 mM amiloride was tested simi-larly. This concentration also failed to inhibit EGF stimula-tion. Control cells exposed for 4 hr to amiloride (200 ,uM or 1mM) without EGF showed basal [3H]thymidine incorpo-ration.To be certain that the failure of amiloride to inhibit subse-

quent mitogenesis when copresent with EGF for 4 hr was notdue to complete removal of amiloride and incomplete remov-al (washout) of EGF, four approaches were used to ensure ascomplete a removal of EGF as possible. (i) To minimize anyresidual nonspecific binding of EGF to cells or culture dish-es, the standard washing procedure (three washes with DMEmedium/0.5% NBCS, pH 7.4, at 37°C) was modified to in-clude 0.1% bovine serum albumin. The results were unaf-fected (data not shown). (ii) Binding ofEGF to its receptor ispH-sensitive. Haigler et al. (13) and Krupp et al. (14) haveshown that at pH s4.5 at 4°C, >95% of bound EGF rapidlydissociates. Thus, after exposure to EGF and amiloride, thecells were washed by the standard procedure and incubatedeither with DME medium/0.5% NBCS, pH 5.5, for 15 min at22°C or with 150 mM NaCl/100 mM sodium acetate, pH 4.5,for 10 min at 4°C. Incubation under acidic conditions did notalter the results (Table 1). Control experiments indicatedthat subsequent basal incorporation of [3H]thymidine wasstimulated slightly by brief incubation under acidic condi-tions. (iii) The cells were washed according to the standardprocedure and incubated in DME medium/0.5% NBCS con-taining anti-EGF IgG or control IgG at 5-10 ug/ml. EGF-induced mitogenesis was unaffected by addition of anti-EGFIgG or control IgG (Table 2). Anti-EGF IgG at this concen-tration blocked stimulation by EGF (1 ng/ml) when addedsimultaneously at to rather than subsequent to exposure toEGF. (iv) Approaches ii and iii were combined. Cells ex-posed to EGF, washed by the standard procedure, incubatedbriefly under acidic conditions, and then incubated with anti-EGF antibody showed similar incorporation of [3H]thymi-dine to that of cells exposed to EGF, washed by the standardprocedure, incubated at pH 7.4, and then incubated withcontrol IgG (Table 3). The results of the four approachessuggest that the failure of amiloride (when copresent withEGF at a concentration sufficient to completely inhibitEGF-induced Na+/H+ exchange) to inhibit mitogenesis isnot likely to be due to residual EGF.Time Frame for Amiloride Inhibition of Mitogenesis. As a

first approach to understanding the mechanism of amiloride

Table 1. Effect of incubation under acidic conditions on abilityof amiloride to inhibit EGF stimulation of [3H]thymidineincorporation in BALB/c 3T3 cells

EGF Amiloride pH of post-wash(t-o4) (t1-4) incubation* cpm- - 7.4 790 ± 92- + 7.4 1052 ± 62+ - 7.4 2788 ± 190+ + 7.4 3250 ± 347- - 5.5 962 ± 8- + 5.5 857 ± 33+ - 5.5 4338 ± 408+ + 5.5 4094 ± 590- - 4.5 1895 ± 111+ + 4.5 4742 ± 895

Times of exposure are shown in parentheses. Concentrations:EGF, 1 ng/ml; amiloride, 400 ,uM. Data are expressed as the mean± SEM (n = 3) of the radioactivity incorporated into the acid-insolu-ble fraction during a 1-hr pulse (124-t25)-*Post-wash incubations began at 14, and were at 22°C for 15 min (pH5.5) or at 4°C for 10 min (pH 4.5).

Table 2. Effect of anti-EGF antibody on EGF stimulation of[3H]thymidine incorporation in BALB/c 3T3 cellsEGF Control IgG Anti-EGF IgG-1 cpm(t0t4) (t4-tN4) (t4-tN4)- - - 1,284 ± 27- + - 1,376 ± 14- - + 1,210 ± 141+ - - 2,200 ± 190+ + - 2,966 ± 59+ - + 2,374 ± 244

(1r-4) (10-4) (o-04)+ + - 3,464± 251+ - + 1,485 ± 60

0(tUt) (to-tN24) (t0-t24)+ - - 25,231 ± 2,523+ + - 21,635 ± 2,420+ - + 2,549 ± 138Times of exposure are shown in parentheses. Concentrations:

EGF, 1 ng/ml; IgGs, 5-10 Ag/ml. Incorporation occurred duringt24-t25. Results of one representative experiment are shown; similarresults were obtained in eight other experiments.

inhibition of EGF-induced mitogenesis, we attempted to de-fine a period of time during which amiloride must be presentfor inhibition to occur. With cells exposed to EGF for 10 hr(to-t1o), incubation with amiloride from to to t1o was as effec-tive in inhibiting EGF-induced [3H]thymidine incorporationas incubation from to to t24 (68% and 72% inhibition, respec-tively). Exposure to amiloride from t1o to t24 was ineffective.Control cells exposed to amiloride alone for 10 hr showedonly a slight reduction in basal [3H]thymidine incorporation.In addition, amiloride had no effect on [3H]thymidine trans-port as determined by measuring the total radioactivity in theacid-soluble cellular pool of thymidine-containing deoxynu-cleotides (results not shown). These findings, in conjunctionwith those shown in Fig. 2, suggested that a critical amilor-ide-sensitive event(s) occurs between t4 and t1o. Consistentwith this hypothesis, Table 4 shows that for cells exposed toEGF from to to t4, the presence of amiloride during t4-t1oinhibited mitogenesis by 65%, although the presence of amil-oride during to-t4 had no effect.An Amiloride Analog Inhibits Na+/H+ Exchange but Not

Mitogenesis. To define more precisely the nature of the amil-oride-sensitive event(s), we utilized the amiloride analogMK-685 (dimethylamiloride), a more potent inhibitor of theNa+/H+ antiporter (ref. 15; see Materials and Methods for adescription of the structure of MK-685). Table 4 indicates

Table 3. Effect of incubation under acidic conditions and of anti-EGF antibody on EGF stimulation of [3H]thymidine incorporationin BALB/c 3T3 cells

pH of Control Anti-EGFEGF post-wash IgG IgG-2(t0-4) incubation* (14-124) (14-t24) cpm

- 7.4 + - 790 ± 92- 7.4 - + 541 ± 23+ 7.4 + - 2788 ±190+ 7.4 - + 2699±114_ 5.5 + - 962± 8_ 5.5 - + 798 ± 71+ 5.5 + - 3970 ± 305+ 5.5 - + 2858 ± 31

Times of exposure are shown in parentheses. Concentrations:EGF, 1 ng/ml; IgGs, 5-10 ,g/ml. Radioactivity incorporated (124-t25) is expressed as the mean + SEM (n = 3).*Post-wash incubations were at 22°C for 15 min.

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Table 4. Ability of amiloride or MK-685 to inhibit EGFstimulation of [3H]thymidine incorporation inBALB/c 3T3 cells

EGF Addition(t0t4) to-4 t4-t1o cpm

- - - 1278 ± 76- Amiloride - 1117 ± 82- - Amiloride 1070 ± 145- MK-685 - 1140 ± 101- - MK-685 1395 ± 105+ - - 4461 ± 278+ Amiloride - 4066 ± 469+ - Amiloride 2107 ± 270+ MK-685 - 4571 ± 169+ - MK-685 4295 ± 467

EGF was present at 1 ng/ml, amiloride at 200 AM, and MK-685 at10 AM. At t4, anti-EGF IgG was added at -4 Ag/ml to all groups.Radioactivity incorporated (t24-t2l) is expressed as the mean ± SEM(n = 3).

that 10 AtM MK-685, a concentration sufficient to completelyinhibit Na+/H+ exchange (15, 16), did not affect basal orEGF-induced mitogenesis when present at any time betweento and t1o. In addition, cells incubated with 10 MM MK-685for 24 hr showed only a slight inhibition of stimulation byEGF (data not shown). Thus, stimulation of Na+/H+ ex-change by EGF is not a necessary step in induction of mito-genesis by EGF and, moreover, inhibition of EGF-inducedmitogenesis by amiloride is not due to inhibition of Na+/H+exchange.

Amiloride Interferes with RNA and Protein Syntheses, butMK-685 Does Not. The onset ofDNA synthesis (S phase), asmeasured by [3H]thymidine incorporation, is usually ob-served -12 hr after exposure of cultured cells to mitogens(unpublished observations for BALB/c 3T3 fibroblasts stim-ulated by EGF; refs. 1, 17, 18 for other cultured cell types).During the hours preceding the onset of S phase, RNA syn-thesis and protein synthesis, both necessary prereplicativeevents, increase (18, 19). To explore the possibility that amil-oride inhibited mitogenesis by inhibiting RNA and proteinsyntheses during the prereplicative period, RNA synthesiswas measured by [3H]uridine incorporation and protein syn-thesis was measured by [3H]leucine incorporation. Table 5shows that, after exposure of cells to EGF for 4 hr, bothRNA and protein syntheses increased during the prereplica-tive period. The presence of amiloride inhibited both RNAand protein syntheses by -50%. Both basal and EGF-stimu-lated RNA and protein syntheses were inhibited by amilor-ide. Note that 10 ,M MK-685 was without effect. Inhibition

Table 5. Effect of amiloride and MK-685 on RNA and proteinsynthesis in EGF-stimulated BALB/c 3T3 cells

EGF Addition Incorporation (t4-t.o), cpm(t0t4) (t4-t40) [3H]Uridine [3H]Leucine

- - 3873 ± 300 4241 ± 270- Amiloride 1548 ± 174 2448 ± 230- MK-685 3837 ± 131 4972 ± 258+ - 6688 ± 468 6447 ± 140+ Amiloride 2408 ± 208 3633 ± 234+ MK-685 5875 ± 293 6805 ± 613

Cells were incubated with or without EGF (1 ng/ml) for 4 hr. Themedium was removed and the cells were washed (standard proce-dure) and then incubated for 6 hr in medium containing amiloride(400 MM) or MK-685 (10 gM) as indicated and either [3H]uridine or[3H]leucine. Incorporation into the acid-insoluble fraction is ex-pressed as the mean ± SEM for triplicate determinations. Two addi-tional experiments yielded similar results.

of radiolabeled leucine incorporation into acid-insoluble ma-terial by amiloride was not due to inhibition of leucine trans-port, as the acid-soluble cellular pool of radiolabeled leucineat both t6 and t1o was unaffected by amiloride (and MK-685)(data not shown). However, the acid-soluble pool of radiola-beled uridine-containing nucleotides was decreased 25% byamiloride treatment but was unaffected by MK-685 (data notshown). This indicates that partial inhibition of uridine up-take may contribute to the decrease in [3H]uridine incorpo-ration into RNA.

DISCUSSIONEGF has been shown to rapidly stimulate Na+/H' exchangein cultured cells (2, 5). The diuretic amiloride can inhibitEGF stimulation of Na+/H' exchange (2, 5) without affect-ing EGF binding and subsequent internalization (2). Also,cells cultured in the presence of amiloride for up to 24 hrshow decreased [3H]thymidine incorporation but not de-creased viability (2, 18). These observations have led to thehypothesis that the rapid stimulation of Na+/H' exchangemay be a necessary step in induction of mitogenesis by EGF(1-3). The data presented here do not support that hypothe-sis. When cells were exposed to EGF and to amiloride or theamiloride analog MK-685 simultaneously for only 4 hr, amil-oride and its analog completely failed to inhibit EGF-stimu-lated [3H]thymidine incorporation, even at concentrationsknown to inhibit EGF-stimulated Na+/H' exchange com-pletely.

In experiments designed to define a critical period of timeduring which amiloride must be present in order to inhibitEGF-induced mitogenesis, we found that t4-tio was such atime frame. Koch and Leffert (18), studying peptide hor-mone stimulation of hepatocyte proliferation in culture, ob-served a time-pattern for amiloride sensitivity very similar tothat which we report for 3T3 cells. During continuous expo-sure to mitogens, if amiloride was present only during thefirst 4 hr, no inhibition of hepatocyte proliferation was ob-served. If amiloride was present for the first 12 hr or longer,complete inhibition was observed. Thus, the time interval t4-t12 appeared to be critical. Likewise, Stiernberg et al. (20)found that an amiloride-sensitive event appeared necessaryfor thrombin stimulation ofDNA synthesis in mouse embryofibroblasts, and that this amiloride-sensitive event occurred7-9 hr after thrombin addition. Stiernberg et al. (20) postulat-ed that this amiloride-sensitive prereplicative event involvedan amiloride-sensitive Na+ influx that peaked 8 hr afterthrombin addition. Although we have not investigated thepossibility that EGF stimulates a second amiloride-sensitiveNa+ influx in 3T3 fibroblasts during the prereplicative peri-od, our finding that the continuous presence of MK-685 doesnot inhibit mitogenesis argues against such a required event.During the prereplicative period we observed that amilor-

ide inhibited both RNA and protein synthesis but that 10 ,uMMK-685 did not inhibit either process (Table 5). Consistentwith these observations, amiloride inhibited EGF-stimulatedmitogenesis but 10 AM MK-685 did not (Table 4). The inhibi-tion of mitogenesis by amiloride clearly cannot be attributedto inhibition of Na+/H+ exchange. The literature indicatesthat amiloride can affect a myriad of cell functions. In addi-tion to inhibiting incorporation of uridine into RNA andincorporation of leucine into protein in intact cells (as report-ed here and in ref. 18), amiloride can inhibit (i) cell-free pro-tein synthesis (16, 21), (ii) Na+-dependent amino acid trans-port (18), (iii) protein kinase activity in cell-free preparations(15, 22) and in intact cells (15), and (iv) the Na+/H+ ATPase(23). Half-maximal inhibition for most of these effects occursin the same concentration range as that for inhibition ofNa+/H' exchange: 0.1-1 mM amiloride. Thus, we wish toemphasize the need for extreme caution in coupling mitogen-

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stimulated (amiloride-sensitive) Na+/H' exchange and theinitiation of DNA synthesis as cause and effect.Furthermore, recent evidence indicates that stimulation of

Na+/H' exchange alone, even if necessary, is not sufficientfor induction of mitogenesis: (i) EGF stimulates cytoplasmicalkalinization in A431 cells via stimulation of Na+/H' ex-change, without stimulation of mitogenesis (24), and (ii) Lys-bradykinin stimulates amiloride-sensitive Na' uptake in WI-38 cells, without stimulation of DNA synthesis (25).

It has been suggested that growth factor (e.g., EGF)-in-duced mitogenesis requires persistent occupation of cell sur-face receptors (17, 26). This hypothesis was based on experi-ments in which the addition of anti-EGF antibodies to EGF-stimulated human fibroblasts appeared to markedly inhibitmitogenesis when added during the first 8 hr of exposure toEGF (17, 26). In our efforts to make certain that the washingprocedure used at t4 completely removed all of the EGF, wefound that addition of anti-EGF antibody, subsequent to nor-mal washing after 4 hr of EGF stimulation, failed to inhibitEGF-induced mitogenesis of BALB/c 3T3 fibroblasts.These experiments were repeated with human foreskin fi-broblasts (HF4 cells) and with human adult fibroblasts (A34cells), using IgG fractions prepared from two different rabbitanti-mouse EGF sera. Under no condition did we find repro-ducible inhibition of mitogenesis by antibody addition after 6hr of EGF-stimulation (data not shown), although some inhi-bition was observed on occasion. At the concentration used(10ug/ml), antibody completely inhibited EGF-inducedmitogenesis if added simultaneously with EGF at to. Also,inhibition by subsequent addition of antibody was not ob-served over a 3000-fold concentration range (0.003-10,ug/ml) with either of the two antibodies (data not shown).Singh et al. (27) have addressed the question of antibody

reversal for the induction of mitogenic competence by plate-let-derived growth factor. They found that, if quiescentBALB/c 3T3 cells were exposed to this factor for .30 min,subsequent addition of anti-factor antibody did not preventthe mitogenic response.During the preparation of this manuscript, L'Allemain et

al. (16) reported that, in HCOj-free medium, stimulations ofNa+/H+ exchange activity and DNA synthesis are linked.This conclusion was based on finding that a series of amilor-ide analogs (of which MK-685 was one) inhibited growth fac-tor-stimulated DNA synthesis in CCL39 cells (at 25 or 50mM extracellular Na+) with the same rank order as for inhi-bition of Na+/H+ exchange, and that the two most potentamiloride analogs prevented growth factor-induced DNAsynthesis at 140 mM extracellular Na+. However, it is evenmore important that L'Allemain et al. (16) also found thatwhen the medium was buffered with CO2/HCO- (as is thecase in the work we report here and in vivo) complete inhibi-tion of Na+/H+ exchange activity by amiloride analogs(e.g., MK-685) failed to inhibit stimulation ofDNA synthesisby growth factors. This finding is in agreement with ourown.The inability of amiloride or amiloride analogs to inhibit

growth-factor induced mitogenesis in the presence ofCO2/HCO- cannot be attributed to a mechanism in whichHCO- prevents amiloride inhibition of Na+/H+ exchange.Schuldiner and Rozengurt (28) have reported that growthfactor-induced cellular alkalinization (resulting from acceler-ated Na+/H+ exchange) was inhibited by amiloride in thepresence of CO2/HCO .

In conclusion, our findings suggest that (i) inhibition of

EGF-induced mitogenesis by amiloride is not due to inhibi-tion of EGF-stimulated Na+/H' exchange, but to inhibitionof necessary events occurring during the hours immediatelybefore the onset of DNA synthesis, these events probablybeing RNA and protein syntheses; (ii) in medium bufferedwith CO2/HCO-, complete inhibition of EGF-stimulatedNa+/H' exchange does not inhibit EGF-induced mitogene-sis; and (iii) in contrast to earlier reports (17, 26), but inagreement with recent findings (P. Comens and J. Baker,personal communication), addition of anti-EGF antibodysubsequent to exposure to EGF for 4 or 6 hr does not inhibitEGF stimulation of mitogenesis.

We thank Lee Rose for preparation of the manuscript and Dr.Harry LeVine III for critical comments.

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Proc. NatL Acad Sci. USA 81 (1984)

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