Studies on a neuronal-like transport system for serotonin in two cell lines

Pharmacological Research Communications, Vol. 14, No. 9, 1982

851

STUDIES ON A NEURONAL-LIKE TRANSPORT SYSTEM FOR SEROTONIN IN TWO CELL LINES

M. Vaghi, L. Curatolo, S. Algeri, N. Brunello °, T. Mennini, L. Morasca, A.L. Salmona, L. Mella ~, M. Locati ~, and E. Dolfini Istituto di Ricerche Farmacologiche 'MARIO NEGRI' Via Eritrea, 62 - 20157 MILAN, Italy ~Ospedale L. Sacco, I Clinica Medica, Universit~ di Milano, Via G.B. Grassi, 74 - 20157 MILAN, Italy

Received in fina/ form 2 7 July 7982

SUMMARY The active transport of serotonin (5-HT) was studied in two cultured cell lines; FET ~a finite fibroblast-lika cell line derived from the osteomuscular structures of C~H embryo mice) and EUE (a continuous cell line derived from a human emb~yonal epithelium). These transport mechanisms are characterized by a high affinity carrier mediated system (km approx 1.3 uM and I.O uM ~espectively; Vmax approx 8 pmol/20 m i n x iQ6 cells and 2 pmol/20 mln x iO cells respectively) and temperature dependence. Fluoxetine (Lilly 110140) and Chlorimipramine preferentially block FET but not EUE serotonin uptake. Our results suggest that a serotonin-liEe transport system is present in unspecialized cultured cells, but it d~ffers kinetically from the neuronal serotonin transport mechanism. INTRODUCTION Uptake, storage and metabolism of neurotransmitters are important biochemical parameters in specialized nerve cells (Narotzky and Bondareff, 1974; platelets

Suddith et al., 1978) and in cultured cells and

(Wielosz et al., 1976).

Recently some Authors reported that

isolated cultured aortic endothelial cells from pigs possess a saturable O

Present address: Lab. Preclinical Pharmacology, William A. White Bldg. Saint Elizabeths Hospital, washington D.C. 20032 U.S.A.

0031-6989/82/090851-09/$03.00/0

© 1982 The Italian Pharmacological Society

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852

high affinity serotonin uptake system (Pearson et el., 1977). While in nervous tissueE the uptake system represents the major mechanism of neurotransmitter deactivation at receptor sites, in other cell types this carrier medlated transport system could represent an "ancestral"membrane activity still remaining on the cell surface, or a functionally active receptor site.

To test this possibility we measured the uptake of 5HT in

one epithelial-like continuous cell line and in one fibroblast-%ike finite line, which should represent two different states of cells not expected to be involved with neurotransmisslon. MATERIALS AND METHODS Cells and culture techniques FET: a fibroblast-like cell line was derived from the osteomuscular structures of embryo C3H mice as previously described (Curatolo et al., 1980).

Growth medium was Eagle's Minimum Essential Medium (MEM) on Hank's

Balanced Salt Solution (BSS) plus Hepes 20 m_M, 20% fetal bovine serum (Gibco), glutamine (5.84 mg/ml) and 50 ug/ml gentamycin.

Cells were seeded

in tissue culture Cluster Plates (24 wells, 2 cm 2 area/well) at

IxlOb/em2/

well and maintained standing in a warm room at 37°C. EU_E: a continuous epithelial cell llne derived from embryonal human epithelial tissue (Terni and Lo Monaco, 1958) was grown as monolayers in tissue culture Cluster Plates in Eagle's MEM on Hank's BSS plus Hepes 20 n~, 10% newborn bovine serum (Gibco), glutamine (5.84 mg/ml) and 50 ug/ml gentamycin.

About

i x lObcells/cm 2 were seeded in each culture well and

maintained standing in warm room (37°C). The 5HT uptake experiments were made when cell cultures were in the exponential growth phase (about 1.4-1.8 x 105 eells/om2); since FET and EUE cells have different lag and doubling times, 72 hours and 48 hours of culture respectively were required.

At the beginning of each experiment,

cell counts were made of both populations.

Cells from ~t least six

randomly chosen wells were detached from the surface with a trypsin tion,

0.25Z Difco

solu-

Trypsin in Phosphate Buffered Saline Ca ++ and Mg ++ free

(PBS). and Lzounted by J,eans of a Coulter Counter Mod. B.

The dye-exelu~on

test by ~'r~an blue was routinely made on cell cultures, giving a viab~ ~ t y index of 80190%.

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853

Uptake of 14C-5}{T Cells grown as previously descrlbed(about 3,5 x 105/~ample) were preincubated in the presence or absence of drugs for 20 min at 37=C in 0.6 ml PBS containing IO-5M pargyllne. addition of 14C-5HT.

Uptake was subsequently started by the

Control cells were incubated at 4=C to determine pas-

sive diffusion tbrough the membranes.

The difference between 14C-SHT accu-

mulated at 37~C and 4°C was taken as a measure of the active transport system. The reaction was stopped by cooling clusters on ice and washing twice with ice-cold PBS.

Cells were dissolved in 0.5 ml of sodlum-dodecyl-sulphate

(1% SDS) and 0.4 ml portions were counted in Bray's solution in a Packard Tri Carb Mod. 2450 liquid scintillation spectrometer.

Counting efficiency,

determined by internal standardization on randomly chosen samples, averaged 80%.

Control values were about 200 CPM at 37°C, and 50 CPM

at 4°C at I uM

14C-5HT. D~ugs_and Chemicals 14C-SHT (58 mC~/ggnol) was obtained from the RadiochemicalCentre, Fluoxetine (Lilly

Amersham.

110140) was obtained from the Lilly laboratories.

Chlorimlpramine HCI (Anafranil) was obtained from Ciba-Geigy. Ouabain (gStrophantin) was obtained from Boehringer Mannheim GmbH, W. Germany.

RESULTS The two cell populations showed a first period of rapid, linear uptake of 14C-SHT during the first hour of incubation, and a slower accumulation rate for 60 min thereafter (Fig. I).

The ~ime-course of 14C-SHT uptake was

similar for the two cell lines, and the tzme of linear uptake for subsequent studies was 20 minutes. Fig. 2 shows that 14C-5HT is actively taken up by both cell lines, as indicated by the significant reduction of 14C-5HT uptake when samples were cooled on ice or IO-3M Ouabain, a Na-K ATPase inhibitor; was added. As showu in Fig. 3, incubating FET cells at 37°C with different 14C-5HT concontra~ons results in a saturable uptake process, which reaches a plateau at a 5HT concentration of about 2 uM." The kinetic parameters for this high affinity uptake by FET are: k~ 1.3 uM, Vn~ax 8 pmols/20 mln/IO 6 cells), similar results were obtained for EUE cells (km 1.2 uM, V~ax 2 pmols/20 min/IO 6 cells).

Pharmacological Research Communications, Vol. 14, No. 9, 1982

854

Incubation of FET cells at 4°C with the same increasing concentrations of 14C-SHT results in non-saturable accumulation of 14C-SHT (Fig. 3), linear TIME COURSE OF I,&C.SHT UPTAKE EUE azFET

p m o l e s l l 0 6 celts

• =

i

&.

3

2,

,'5 3'0 Fig. i -

~'o

9'0

,~o ";;Me(r,,=n>

14C-5HT concentrations was i uM. Data represent mean values + S.D. of 6 replications. Fa~:tors infiuencin9

14C-5HTac-tige-transport

% 1&C-5HT UPTAKE

[~]FET: IIEUE

100-

50-

TREATMENT:NONE TEMPERATURE: 37°C

NONE &°C

OUAB~IN 10"3M 37 ° C

Fig. 2 - 14C-SHT concentration was I uM. Data zepresent mean values + S.D. of 6 replications. with the concentration of 5HT added, which represents passive diffusion through the membrane. We choose a 14C-SHT concentration about equal to the km for subsequent

855

Pharmacological Research Communications, I/ol. 14, No. 9, 1982

Kinetic of 1/'C-SHT uptake by fibroblast-likecells(FET) p m o l e s / l O 6 ce115/20

KM= 1.28 HM

.n

Vmax =7.9 p m o l / 2 0 rain/t06 coils • ,,, NET UPTAKE o = PASSIVE TRANSPORT

o'.s i ,,:-s.T [~"l

~Js

~,

Fig. 3 - The pmoles of 14C-bHT actively transported by 106 cells during 20 min of incubation are plotted against different concentrations of 14C-bHT. JThe curve represents net uptake (37°C -4°C) or passive transport (4°C). Kinetic parameters (km and Vmax) in the figure were calculated by Woolf and Lineweaver - Burk plot. experiments with serotonin uptake inhlbitors. Table I. Lilly.llOl40 and Chlorimipramine

The results are shown in

(CI-IMI), reported to be select-

ive potent inhibitors of 511T uptake in synaptosomes

(Wong et al., 1974;

Smith et al., 1978) and platelets (Wielosz et al., 1976; Wielosz et al., 1977), are equipotent (Table i) in inhibiting 14C-5HT uptake in FET cells, although at a concentration i00 times higher.

Drug concentrations higher

than 10 -4 are required to achieve 5HT uptake inhibition in EUE cells, indicating that these ceils are less sensitive to serotonin uptake inhibitors. Since EUE is a continuous cell llne, while FET used in the present experiment originated from a primary culture and were at early doublings, we reasoned that the'lower sensitivity of EUE cells to 5HT uptake inhlbitots might be due to changes in the 14C-bHT carrier mechanism occurring while the cells were kept in culture.

To check this we studied the effect

of Io-bM Lilly 110140 on FET at different doublings in culture. Fig. 4 shows that at early stages of FET culture Lilly lo-bM selectively inhibits 5HTuptake, tely ineffective.

while at later stages (19th~doubling)

Moreover,

it was comple-

the amount of 14C-51~ taken up by control cells

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Pharmaco/ogical Research Communications, Vol. 14, No. 9, 1982

TABLE I - Drug concentration giving half-maximal inhibition of 14C-5HT (I uM) uptake was obtained by a log dose-effect plot based on four concentrations at least, and represents the mean of three replications.

ICso r Mj Fibroblast-like cells

EUE cells

Lilly 110140

2.15 x I0-5

10-4

Chlorimipramine

2.29 x 10 -5

10 -4

14C-5HT

UPTAKE

el.

13 Controls II L i l l y 110 140 10"5M

14C-5HT UPTAKE

100]

FET 1

(fluoxetine)

FET19

EUE

Fig. ~ - 14C-5HTfconcentrations was I uM. Data shown are % of control active transport and represent mean values + S.D. of 4-6 replications. FET = i st doubling, FET 19 = 19th--doubling. p

O.001

different from controls (Student's t test).

at this time was significantly lower than in the early stage of eulture~ and very similar to that found in EUE cells whereas Lilly 110140, at this concentration, wa~s ineffective in inhibiting 14C-'5HT uptake.

Pharmacological Research Communications, I/ol. 14, No. 8, I 9 8 2 DISCUSSION Much of our knowledge of 5HT uptake has come from studies with nerve cells and platelets (Narotzky and Bondareff, 1974; Suddlth et al., 1978; Wielosz et al., 1976; de Caetano G., 1978) which have specialized cell membrane structures involved in the mechanism of active transport of serotonin.

The 5HT uptake by nerve cells is the most important factor of

inactivation of neurotransmitters released at the synaptic cleft. It has been recently suggested that 5HT uptake by platelets is a reliable ~mdel of this neuronal uptake process (de Gaetano, 1978; Drummond and Gordon$ 1975). The present data suggesting that other non-specialized ceil types such as fibroblasts and epithelial cells may be able to take up 5HT via a high affinity uptake system demonstrate that different cell lines, FET and EUE, have this particular membrane cell function.

This transport system for 5HT

found in cultured EUE and FET ceil is energy and temperature-dependent. It shows a high affinity carrier similar to that found for platelets, glial cells, neuroblastoma and endothelial cells (Narotzky and Bondareff, 1974; Suddith et al., 1978; Wielosz et al., 1976; Pearson et al., 1977) (km approx I uM), but very d~fferent from brain and spinal cord synaptosomes (km approx O.i uM) (Menuini et al., 1978). Another interesting point is that 5HT uptake is modified during cellular aging in fibroblast cell culture.

Our results show that 5HTuptakelshlgher

at the first subcultures declining at subsequent passages until the 19 th doubling,

The 5HT uptake process in the

cultured cells seems to be less

sensitive to specific 5HT inhibitors than platelets and synaptosomes; in fact, concentrations of Lilly 110140 and CMI about IOO times stronger are r~quired to inhibit 14C-bHT uptake by FET. Lilly 110140 (IO-5M), a selective, specific inhibitor of'bHT uptake, preferentially blocks the indolamine uptake at early passages, but becomes progressively less active as the cells age,

Our results are in line in

this respect with other studies of cells aging in culture (Makman et al., 1979). Another explanation - for the reduced uptake by FET at the late stage of culture and the lack of effect of Lilly 110140 could be that the cell population at the beginning of culture may be very heterogeneous, including neuronal cells from the spinal cord.

These could be responsible for the

greater accumulation of 5HT and for the sensitivity to uptake inhibition by Lilly 110140.

However, this hypothesis is weakened by the fact that we

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Pharmaco/og(cal Research Communications, VoL !4, No. 9, ! 982

found only one uptake system, with km about I uM, suggesting the existence of a cell population with the same uptake characteristics (Drunm~ond and Gordon, 1975),

Our previous studies showed that the llfespan of FET is lim-

ited to II doublings, and that at this age cells lose their capacity to induce retraction in a platelet-free plasma clot (Curatolo et al., 1980). The limit of the 11 doublings can,. however, be passed by modifying the feeding regimen of cultures so that FET cells become capable of indefinite growth, like continuous lines. The lack of specifically inhibited 5HT uptake in FET cells at the 19 th doubling suggests that the cell aging could modify some parameters of 5HT uptake.

In our opinion the transformation of a cell line (FET) with a nor-

mally finite life-span into a continuous line could induce the loss of a specific receptor-like system present at the earliest stages of normal f~broblasts.

ACKNOWLEDGEMENT This work was supported by Contract no. 79.02168.04 international Program.

II year from C.N.R.

Judith Baggot, Antonietta Di Bitetto, Vineenzo de

Ceglie, Vanna P~stotti, helped to prepare this manuscript.

REFERENCES Curatolo, L., Balconl, G., Borgia, R., Morasca, L. and Donati, M.B. (1980) In vitro ]6, 731. de Gaeta~lo, C. (1978) in: Platelets: A MultidlscipliDary Approach (G. de Caetano and S. Garattini, eds.), Raven Press, New York, p. 373. Drummond, A.H. and Gordon, J.L. (1975) Biochem. J. 150, 129. Makman, M.H., Ahn, H.S.~ Thal, L.J., Sharpless, N.S., Dvorkin, B., Horowitz, S.G. and Rosenfeld, M. (1979) Fedn. Proe. 3,B, 1922. Mennlnl, T., Pataccini, R. and Samanln, R. (1978) Br. J. Pharmac. 64, 75. Narotzky, R. and Bondareff, W. (1974) J. cell Biol. 63, 64. Pearson, J.D., Olberman, H.J. and Gordon, J.L. (1977) Biochem. Soc. Tran.~. 5, 1181. Smith, L.T., Hanson, D.R. and Omenn, G.S. (1978) Brain Res. _146, 400. SuddJth, R.L., Hutchison , H.T. and Haber, B. (1978) Life Sci. 22, 2179.

Pharmacological Research Communications, Vol. 14, No. 9, 1982

Terni, M. and Lo Monaco, G.B. (1958) Lo Sperimentatore IO8, 177. Wielosz, M., Dall'Olio, A., de Caetano, G. and Garattini, S. (1977) J. Pharm. Pharmac. 29, 546. Wielosz, M., Salmona, M., de Gaetano, G. and Garattini, S. (1976) Naunyn-Schmiedeberg's Arch. Pharmac. 296, 59. Wong, D.T., Horng, J.S., Bynaster, F.P., Hauser, K.L. an~ Motloy, B.B. (1974) Life Sci. 15, 471.

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