ELSEVIER Biochimica et Biophysica Acta 1221 (1994) 206-210

BIt, Biochi ~mic~a et Biophysica t~ta

Bombesin stimulates transplasma-membrane electron transport by Swiss 3T3 cells

I.L. Sun a F.L. Crane ,,a H. L6w b

a Department of Biological Sciences, Lilly Hall of Life Sciences, Purdue University, West Lafayette, IN 47907-1392, USA b Department of Endocrinology, Karolinska Institute, S104 O1 Stockholm 60, Sweden

(Received 17 May 1993) (Revised manuscript 7 September 1993)

Abstract

Bombesin, a mitogenic neuropeptide, stimulates transplasmalemma reduction of diferric transferrin or ferricyanide by Swiss 3T3 cells. The stimulation of diferric transferrin reduction occurs in the range of bombesin concentrations that stimulate proliferation of Swiss 3T3 cells. Diferric transferrin reduction by the 3T3 cells is accompanied by increased proton release from the cells and bombesin increases the diferric transferrin-stimulated proton release twofold. Insulin increases the diferric transferrin reductase response and increases growth stimulation with bombesin. The effect of bombesin on the transmembrane electron transport is a new aspect of its effect on the plasma membrane in addition to increase in phosphatidylinositol turnover and protein kinase c activation. The electron transport can provide an independent mechanism of activation of the Na+/H ÷ exchange or it can change the redox state of pyridine nucleotide in the cytoplasm.

Key words: Plasma membrane electron transport; Bombesin; Growth control; Sodium ion/proton antiport; Transferrin

1. Introduction

Bombesin and related neuropeptides have been shown to induce proliferation of quiescent Swiss 3T3 cells in serum-free media [1,2]. The early signals pro- duced by bombesin binding to its receptor on the plasma membrane are activation of phosphatidyl- inositol diesterase to produce inositol 1,4,5-tri- sphosphate and diacylglycerol, which in turn activate calcium release from internal stores and protein kinase c, respectively. In addition, bombesin binding activates the N a ÷ / H + antiport to cause cytosolic alkalinization [1]. Activation of the N a + / H + antiport has been postu- lated to be partly independent of protein kinase c activation [3]. Control of cell growth, activation of D N A synthesis and specific gene activation (c fos and c myc) has been related to multiple signal expression based on increase in cytosolic Ca 2+, decrease in cytoso- lic proton concentration and activation of protein ki-

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nase c. Activation of the antiport by bombesin may not be necessary for all growth effects [2].

Proton release from cells can also be activated by external oxidants which can act as electron acceptors for a transplasma membrane N A D H oxidase [4-8]. Diferric transferrin acts as a ligand to activate this oxidase and it also acts as an external electron acceptor [5,6,9,10]. The major part of the proton release induced by the transmembrane electron transport to diferric transferrin occurs through the N a + / H + antiport [7,8,11,12]. In addition to increased proton release from cells, the reduction of external diferric transferrin causes the oxidation of cytosolic NADH with conse- quent increase in NAD concentration [13]. The in- crease in NAD may serve as an additional message by activation or inhibition of metabolic redox reactions, e.g., nucleotide transformations such as deoxyribonu- cleotide formation or guanylate synthesis [14,15]. In this paper we will show that bombesin stimulates the transplasma membrane N A D H diferric transferrin re- ductase of Swiss 3T3 cells. The stimulation of transfer- tin reduction by bombesin is accompanied by a stimu- lation of proton release from the cells. Insulin which

I.L. Sun et al. / Biochimica et Biophysica Acta 1221 (1994) 206-210 207

increases 3T3 cell proliferation with bombesin in- creases the response of the diferric transferrin reduc- tase to bombesin and gives further increase in the bombesin activation of the proton release.

2. Materials and methods

3T3 cells were cultivated in Dulbecco-modified Ea- gle's medium (DMEM) supplemented with 10% fetal bovine serum, 50 units of benzylpenicillin and 50 ~g streptomycin per ml. Cells were removed with 0.05% trypsin in salt plus 0.02% EDTA followed by resuspen- sion in DMEM plus serum to inactivate the trypsin. Cells were then washed in the buffer to be used in experimental procedures. Viability was determined by eosin Y exclusion.

Diferric transferrin reduction was assayed by mea- suring the formation of ferrous bathophenanthroline disulfonate (BPS) when 0.01-0.03 g wet weight of 3T3 cells were incubated with 10 /~M diferric transferrin and 10 ~M BPS in TD tris buffer (140 mM NaCI, 2.5 mM KCI, 1 mM MgC12, 25 mM Tris-HCl, pH 7.4). Ferrous BPS formation was measured using the dual beam mode on an Aminco DW2a spectrophotometer to subtract absorbance change at 600 nm from ab- sorbance change at 535 nm. At these wavelengths the net extinction coefficient for Fe(BPS)3 is 17.6 m M / c m [10]. Except for time of treatment studies the cells were pre-incubated 3-5 min with bombesin and other agents before starting the reaction by addition of diferric transferrin.

Ferricyanide reduction was measured in TD buffer by measuring the rate of decrease in ferricyanide con- centration when 0.01 g wet weight of cells were added in TD buffer with 0.1 mM ferricyanide measuring absorbance at 420 minus 500 nm [4]. The extinction coefficient used is 1 mM/cm.

Proton release was measured in salts solution (140 mM NaCI, 0.5 mM KCI, 0.1 mM MgCI 2) with a mini- mum of buffer present (1.5 mM Tris-HCl) using a Corning pH meter with glass electrode [7,8]. The 5 ml reaction mixture was continuously bubbled with air or CO 2 free air to equilibrate or remove CO 2 [7,8] and equilibrated after addition of 0.01 g wet weight of cells (150 × 106 cells). 10/.tM diferric transferrin or 100/xM ferricyanide was then added to start the reaction. Di- ferric transferrin was obtained from Boehringer Mannheim, Indianapolis, apotransferrin from Sigma, St. Louis.

3. Results

Swiss 3T3 cells which have been incubated with bombesin for 3 min show an increased rate of diferric

20 O

• ~ 10

o o

i . . . O - - /

-sO

I I I 10 20 30

Bombesin [nmolar]

Fig. 1. Effect of bombesin on diferric transferrin reduction by Swiss 3T3 cells. Bombesin added to cells (0.03 g wet wt.) in 2.8 ml TD buffer with 10/zM diferric transferrin and 10 gM BPS as described in Materials and methods.

transferrin reduction. At 15 nM stimulation can be over threefold with half maximum stimulation at 2 nM bombesin (Fig. 1). Ferricyanide reduction by the 3T3 cells is also stimulated and the percent increase in activity is similar to the increase of diferric transferrin reduction (Table 1). Both diferric transferrin and ferri- cyanide stimulate proton release from 3T3 cells. The proton release is increased two fold by both diferric transferrin and ferricyanide after cells are incubated 3 min with bombesin (Table 2). Fig. 2 shows tracings of pH changes induced by diferric transferrin with and without bombesin.

Apotransferrin does not induce proton release from the 3T3 cells but it does inhibit diferric transferrin reduction by the 3T3 cells (Table 3).

Insulin (1/xg/ml) stimulates ferricyanide reduction by 3T3 cells and increases the stimulation with bombesin (Table 4). Insulin has also been shown to enhance the bombesin stimulation of DNA synthesis by 3T3 cells [1] (Table 5). Fetal calf serum stimulates the rate of diferric transferrin reduction similarly to bombesin. On the other hand, vasopressin inhibits the diferric transferrin reduction by the 3T3 cells (Table 6).

Table 1 Effect of bombesin on ferricyanide reduction by Swiss 3T3 cells

Bombesin concentration Ferricyanide reduction rate (nmol) (nmol/min per g wet wt.)

None 180±30 (3) 6.25 330

12.5 336 25 420 50 434

Results representative of three separate experiments except for 50 nmolar bombesin tested only once. In t h e t h r e e e x p e r i m e n t s , t h e average (+ standard deviation) rate increase with 12.5 nM bombesin was 205 ± 20% and with 25 nM was 273 + 63%.

208 LL. Sun et al. /Biochirnica et Biophysica Acta 1221 (1994) 206-210

Table 2 Effect of bombesin on oxidant stimulated proton release from Swiss 3T3 cells

Bombesin Diferric transferrin Ferricyanide (nmol) stimulated proton stimulated proton

release (neq H + / release (neq H + / rain per g wet wt.) min per g wet wt.)

None 217 258 3.1 473 6.25 273 563

12.5 326 354 25 391 660

Oxidants were at 10 /~M for diferric transferrin and 100 /zM for ferricyanide. Similar results observed in five other experiments with diferric trans- ferrin and two other experiments with ferricyanide. With diferric transferrin, the increase with bombesin averaged 173 + 33% at 12.5 nM and 210+48% at 25 nM. With ferricyanide, the bombesin increase averaged 150 :t: 34% at 12.5 nM and 228% (average of two) at 25 nM.

Fe2Tf 101abl

c o n t r o l ~ 7 J Fe2Tf IOIlM

25nM / -

b°m 7 30 n

pH decrease

moles H +

1 rain

Fig. 2. Activation of proton release from Swiss 3T3 cells by diferric transferrin with and without 25 nM bombesin. Proton release mea- sured as described in Materials and methods with 0.02 g wet weight of cells equilibrated in salts solution before addition of 10 ~M diferric transferrin at the arrow.

Table 3 Effect of apotransferrin on the bombesin stimulated diferric transfer- rin reduction by Swiss 3T3 cells

Additions Diferric transferrin reduction (nmol/ min per g wet wt.)

None 5.5 + 1.2 (3) 25 nM bombesin 14.8 + 0.8 (3) 25 nM bombesin + 1.7/~ M apotransferrin 10.3 25 nM bombesin + 3.4/~M apotransferrin 6.5 25 nM bombesin + 6.8 ~M apotransferrin 4.7 6.8/xM apotransferrin 1.4

Assay with 10 p,M diferric transferrin added to start the assay after 3 min incubation of cells with bombesin and apotransferrin.

Table 4 Effect of insulin on bombesin stimulation of ferricyanide reduction by Swiss 3T3 cells

Addition Ferricyanide reduction (nmol/min per g wet wt.)

Experiment 1 Experiment 2

None 174 + 31 (3) 217 (2) Insulin 1/~g/ml 288 324 Bombesin 3.1 nM 314 371 Bombesin 6 nM 464 Bombesin 3 nM+ insulin 598 462 Bombesin 6 nM + insulin 787

In a third experiment, insulin (1 ~g/ml) increased the activity with 12.5 nM bombesin by 260% and with 25 nM bombesin by 250%.

Table 5 Effect of insulin on stimulation of Swiss 3T3 cell DNA synthesis (thymidine incorporation) by bombesin

Addition [3H] thymidine incorporation (% of 10% FCS)

None 3 Insulin 1 ~g /ml 15 Bombesin 10 nM 15 Bombesin 10 nM+ insulin 1/zg/ml 80

Similar combined effects of bombesin and insulin have been re- ported by others [1,27].

Peptides with no effect include PDGF, somatostatin and beef serum albumin (not shown).

In contrast to growth of transformed cells, prolifera- tion of 3T3 cells is not increased by diferric transferrin alone in serum-free media. Bombesin alone, without transferrin, induces some growth, but both diferric

Table 6 Comparison of effects of bombesin vasopressin and fetal calf serum on the rate of diferric transferrin reduction by Swiss 313 cells

Addition Ferrous BPS formation (nmol/min per g wet wt.)

Experiment 1 Experiment 2

None 3.9, 4.5 12.5 nM bombesin 6.4 25 nM bombesin 8.6 50 nM bombesin 12.2 0.5% FCS 11.6 1.0% FCS 14.7 None 100 nM vasopressin 200 nM vasopressin 300 nM vasopressin

7.6 :t: 1.6 (3) 6.7 1.0 0

48 h Swiss 3T3 cells grown in 10% serum assayed in TD buffer, 10 /xM BPS, 10 IxM Fe2Tf, 0.015 g wet wt. cells, by measure of absorbance at A535_600, after 3 min incubation with bombesin or serum before transferrin addition. Similar bombesin stimulation observed in five other experiments with an average activity increase of 222 + 55 % at 12.5 nM and 353 + 113% at 25 nM. In three experiments, 1.0% FCS gave an average stimula- tion of 330+ 65%.

LL. Sun et al. / Biochimica et Biophysica Acta 1221 (1994) 206-210 209

Table 7 Effect of bombesin and transferrin on 3T3 cell growth

Cells × lO-5/flask

Mo addition 6.3 Bombesin 12.5 nM 10.1 Bombesin 25 nM 9.6

Bombesin 12.5 ~ M + ferric Tf3.4/.LM 13.8 Bombesin 25 nM+ ferric Tf 3.4/x M 24.6

Ferric transferrin 3.4/~M 7.4

Cell culture as described in Materials and methods without serum for 48 h. In another experiment, cell count after 48 h was 2.8 cells" 105/flask with no addition, 4.0 with 6.25 nM bombesin and 6.6 with 6.25 mM bombesin plus 3.4/zM ferric transferrin.

transferrin and bombesin are required to stimulate maximum growth (Table 7).

4. Discussion

Bombesin has been shown to activate three transplasma membrane signaling systems in 3T3 cells and the activation of these signals has been related to bombesin stimulation of DNA synthesis [1] and specific oncogene expression [16]. The signals activated through hydrolysis of phosphatidylinositol bis phosphate are increased cytosolic calcium and protein kinase c activa- tion. In addition, the Na+/H + antiport is activated to increase cytosolic pH. The studies reported here show in addition that bombesin can activate diferric transfer- rin reduction by activation of the transplasma mem- brane electron transport system. Since diferric trans- ferrin or ferricyanide alone can activate the Na+/H + antiport mediated proton release from the 3T3 cells, the activation of the Na+/H + antiport by bombesin in 3T3 cells [3] may be based on increased electron trans- port through the plasma membrane. This is especially true for cell growth studies where the culture media contains ferric transferrin or other ferric compounds which can act as electron acceptors for the plasma membrane electron transport. In studies with cells transferred from growth media into physiological salt solutions where no electron acceptor is added, the activation of the antiport may be through other signals, or oxygen can act as the electron acceptor [17,18] for the plasma membrane enzyme. It is clear that diferric transferrin and ferricyanide can activate proton release from the 3T3 cells in the absence of bombesin and that this oxidant-dependent proton release is greatly en- hanced by bombesin.

Proliferation of the 3T3 cells is not induced by diferric transferrin and only partially by bombesin in- dependently. There is good growth of these ceils when both diferric transferrin and bombesin are present. This suggests that the initiation of growth of these cells

requires maximum activation of the Na+/H + antiport to increase cytosolic pH. The further stimulation of growth in the presence of low concentrations of bombesin by insulin may also be based on activation of increased proton release [1]. Low concentrations of insulin alone (1 ~g /ml ) give only a small increase in redox activity and growth. In addition, the bombesin can activate the other signals previously listed [1,19,20] which may be additional requirements for growth acti- vation. The diferric transferrin has been shown to increase oxidation of cytosolic NADH to increase NAD concentration in the cytosol [13,21] and the relation of NAD levels to cell growth has not been fully explored. The NAD increase could constitute a signal uniquely under control of the dehydrogenase and the activation of electron transport activity by bombesin could in- crease this signal which would affect the prooxidant state in the cell which has been related to growth control [22,23].

The basis for bombesin activation of transplasma membrane electron transport is not known. Based on inhibition of diferric transferrin reduction by mono- clonal antibodies to the transferrin receptor, the reduc- tion of diferric transferrin at the plasma membrane requires the presence of the transferrin receptor. On the other hand, ferricyanide reduction occurs at a site which is not sensitive to these antibodies [24]. Since bombesin stimulates both diferric transferrin and ferri- cyanide reduction, its action can be directly on the electron transport components through a bombesin receptor. Since phorbol myristate acetate (PMA) which activates protein kinase c does not activate diferric transferrin reduction in 3T3 cells (data not shown), the bombesin effect on electron transport is not through protein kinase c activation [1,2]. The activity is not time-dependent since cells exposed to bombesin for 48 h still show a threefold increase in diferric transferrin reductase activity over controls treated without bombesin for the same time (data not shown). If bombesin sites are lost after 40 h exposure of 3T3 cells, the activation of the redox system is not dependent on the continued presence of bombesin [25]. The com- bined action of bombesin and insulin to stimulate ferricyanide reduction by 3T3 cells is paralleled by the combined effect of these factors on 3T3 cell prolifera- tion. The effect on ferricyanide reduction indicates that increased transferrin receptor expression is not necessary for the action of these factors but transferrin binding to its receptor does increase the response and can provide a natural avenue of oxidase activation to stimulate growth. Inhibition of diferric transferrin re- duction by vasopressin is a surprise, since vasopressin stimulates growth of 3T3 cells [1,2]. This indicates that transmembrane electron transport alone cannot stimu- late growth. On the other hand, there is evidence that oxygen can compete with ferric transferrin for elec-

210 LL. Sun et al. /Biochimica et Biophysica Acta 1221 (1994) 206-210

trons in 3T3 cells [26], so the measurement of transfer- rin reduction does not give a complete understanding of plasma membrane redox activity.

The mechanism by which the ligand-activated plasma membrane electron transport influences growth control remains to be established. In addition to change in N A D / N A D H ratio and Na+ / H + antiport activation electron transport to ferricyanide activates Ca 2+ up- take [28] and protein tyrosine kinase phosphorylation [29] as well as c myc and c fos oncogene expression [30] in other cells.

5. Acknowledgments

Supported by NIH career award K06 21839 to F.L.C and a Swedish Medical Research Grant to H.L.

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