serotonin and neuroendocrine peptides influence dna synthesis in rat and human small intestinal...

Serotonin and neuroendocrine peptides in¯uence DNA

synthesis in rat and human small intestinal cells in vitro

K . Z A C H R I S S O N 1 , 2 and A . U R I B E 1

1 Department of Medicine, Karolinska Institute, Danderyd Hospital, Stockholm, Sweden

2 Department of Clinical Pharmacology, Karolinska Institute, Danderyd Hospital, Stockholm, Sweden

ABSTRACT

Animal studies suggest a mediator role for neuroendocrine peptides and amines in regulating cell

proliferation in the gastrointestinal epithelium. Our aim was to examine the effect of serotonin and

selected gastrointestinal peptides on DNA synthesis in a rat and human small intestinal cell line

in vitro. IEC-6 and FHs-74 cells were incubated with epidermal growth factor (EGF), insulin-like

growth factor II, glucagon, substance P, neurokinin A, calcitonin gene-related peptide (GRP, CCGRP),

neurotensin and serotonin. The cells were labelled with [methyl-3H] thymidine and processed for

autoradiography. DNA synthesis was evaluated by the labelling index. Epidermal growth factor,

insulin-like growth factor II, glucagon, and substance P increased the labelling index in a dose-related

manner (P < 0.003). In contrast, a signi®cant dose-dependent reduction of the labelling index was

observed after administration of serotonin and neurokinin A (P < 0.0001). Neurotensin and CGRP did

not affect the labelling index. EGF, insulin-like growth factor II, glucagon, substance P, serotonin and

neurokinin A may be important physiological regulators of proliferation, of gastrointestinal cells.

Keywords DNA synthesis, IEC-6 cells, FHs-74 cells, neuroendocrine peptides, serotonin.

Received 6 June 1997, accepted 26 November 1997

The cell kinetics of the gastrointestinal epithelium is

regulated by extremely ef®cient and largely unknown

mechanisms that maintain a normal epithelial structure

and facilitate adaptation and repair. The regulation of

epithelial cell kinetics is probably multifactorial, partly

mediated by intraluminal factors, active substances

present in the mucosa and/or regulatory factors that

are distributed through the microcirculation.

The peptides and amines synthesized and/or re-

leased from different endocrine cells and nerve ®bres in

the gastrointestinal tract have many biological functions

that act in a paracrine, compartment-like manner

(Sundler et al. 1989) or may affect distant target cells via

the blood stream. Epidermal growth factor (EGF)

(Conteas & Majumdar 1989) and gastrin (Conteas &

Majumdar 1989) are peptides that stimulate cell pro-

liferation in epithelial cells, whereas somatostatin has

inhibitory actions (Lehy et al. 1979, Stange et al. 1984).

The long-term administration of the ulcerogenic

drug indomethacin to conventional rats increases DNA

synthesis and produces hypoplasia of the villi in the

small intestinal mucosa. These changes are associated

with increased tissue concentrations of glucagon, ne-

urotensin and neurokinin A (Uribe et al. 1997a,b). The

short-term administration of the cyclooxygenase in-

hibitor to germ-free animals reduces DNA synthesis in

the upper gastrointestinal epithelium and increases the

tissue concentrations of somatostatin, calcitonin gene-

related peptide (CGRP) and glucagon (Uribe et al.

1997a,b). Moreover, germ-free rats have high plasma

levels of glucagon (Uribe et al. 1991, 1994), large total

volumes of serotonin-immunoreactive cells and altered

tissue concentrations of somatostatin in the gastroin-

testinal tract associated with mucosal atrophy in the

colon (Uribe et al. 1994). However, indomethacin-

treated germ-free rats exposed to normal micro¯ora for

3 days develop a trophic reaction in the intestinal epi-

thelium which is associated with decreased tissue con-

centrations of the neuroendocrines mentioned above

and increased tissue concentrations of substance P

(Uribe et al. 1997a,b). Taken together, these ®ndings

suggest a causal relationship between the changes in

synthesis and/or the release of neuroendocrine pep-

tides and the observed cell-kinetic changes.

Correspondence: AndreÂs Uribe, Department of Medicine, Karolinska Institute, Danderyd Hospital, 182 88 Danderyd, Sweden.

Acta Physiol Scand 1998, 163, 195±200

Ó 1998 Scandinavian Physiological Society 195

The aim of this study was to examine whether

serotonin and the neuroendocrine peptides mentioned

above in¯uence DNA synthesis in two different small

intestinal cell lines in vitro. IEC-6 cells, a well-charac-

terized (Quaroni & May 1980) non-tumour continuous

cell line derived from the mucosal crypts of the prox-

imal small intestine of germ-free Sprague-Dawley rats,

and FHs-74 cells, a normal, foetal, human, small in-

testinal cell line, were used. Both cell types have similar

morphological appearance, typical of epitheloid cells in

culture.

MATERIALS AND METHODS

Dulbeccos modi®ed Eagles medium (DMEM) with

glucose 4.5 L)1 and without sodium pyruvate, phosphate

buffered saline (PBS), trypsin±EDTA (0.5 g trypsin-

g L)1 salt solution and 0.2 g EDTA), foetal calf serum

(screened for virus and mycoplasma), bovine insulin,

penicillin/streptomycin (PEST), non-essential amino

acids and sodium pyruvate were purchased from GIBCO

BRL, Life Technologies, Paisley, Scotland.

The EGF from mouse submaxillary glands, insulin-

like growth factor II (IGF-II) ± human recombinant,

glucagon extracted from bovine and porcine pancreas,

substance P (SP) ± synthetic acetate salt, neurokinin A

(NKA) ± synthetic, CGRP from rat, neurotensin ±

synthetic acetate salt and serotonin ± hydrochloride

were obtained from SIGMA Chemical Company, St.

Louis, USA.

[Methyl-3H] thymidine (speci®c activity 92.5 ´1010 Bq mmol)1, aqueous solution) was purchased

from Amersham Sweden AB, Solna, Sweden.

Cell culture

IEC-6 cells and FHs-74 cells (American Type Culture

Collection, Rockville, USA), were propagated in 75 cm2

polystyrene cell culture ¯asks (Costar Europe Ltd,

Badhoevedorp, The Netherlands) in DMEM contain-

ing 5% foetal calf serum, bovine insulin 10 lg mL)1,

penicillin 50 E mL)1 and streptomycin 50 lg mL)1

(enriched DMEM), respectively. The medium of the

FHs-74 cells also contained 1 mL of a 100 mM non-

essential amino acid solution and 0.5 mM sodium

pyruvate. The cultures were maintained in a water-sat-

urated atmosphere with 5% CO2, at 37 °C (Revco-in-

cubator, Labora, Upplands VaÈsby, Sweden).

The cells were harvested by trypsination. After the

cells had been rinsed with PBS, 3 mL trypsin±EDTA

solution and 2 mL PBS were added and they were kept

in the incubator for 10 min. Then the trypsin was in-

activated by adding 5 mL enriched DMEM. The cell

suspension (10 mL) was placed in a sterile test tube and

centrifuged for 5 min at 20 °C and 685 ´ g.

The cells were reseeded in polystyrene wells pre-

pared with a circular cover glass, measuring 12 mm, at

the bottom of each well (24-well tissue culture cluster,

CostarÒ ). A density of 1.8±2.5 ´ 106 cells per well was

used.

The cells were allowed to thrive in the enriched

DMEM for 24 h and then starved in plain DMEM for an

additional 24 h to synchronize them in cell cycle.

Thereafter, they were incubated for 24 h with EGF,

which was used as a reference trophic factor, at 25, 50,

100, 200 and 400 ng mL)1 (4.1 ´ 10)9M±6.6 ´ 10)8

M),

IGF-II at 25, 50, 100, 150 and 200 ng mL)1

(3.3 ´ 10)9M±2.7 ´ 10)8

M), glucagon, substance P,

neurokinin A, CGRP, neurotensin and serotonin at

10)10±10)6M.

DNA synthesis:Autoradiography

In the last 4 h, 3.7 ´ 104 Bq mL)1 of [methyl-3H]

thymidine was added. Thereafter, the medium was re-

moved and the cells were rinsed with DMEM and ®xed

for 2 h at 4 °C in 1 mL of a solution containing 3%

glutaraldehyde, 0.1 M sodium cacodylate and 0.05 M

sucrose, pH 7.3. Then the cells were dehydrated for

5 min with 70 and 95% ethanol, respectively. Finally

the cover glasses were removed from the wells, dried

and mounted with xylene 60% and acrylharts (PertexÒ,

Histolab Products AB, VaÈstra FroÈlunda, Sweden) on

routine microscope slides. The preparations were

coated with GEL emulsion (Ilford 65, Nuclear Re-

search Emulsion, Basildon, England) and kept for 2

weeks in light-proof boxes at 4 °C. The ®lms were

developed in developer G 150 (Agfa Gevaert AB,

Kista, Sweden) for 15 min, ®xed in RP-F High Speed

Fixer (John Saxeby AB, SpaÊnga, Sweden) and ®nally

stained with toluidine blue.

DNA synthesis was evaluated by the labelling index

(LI%) using a light microscope (Nikon, Tokyo, Japan;

magni®cation ´ 400). For this purpose, an ocular grid

was used to de®ne the observation area. The total

number of labelled cell nuclei and the total number of

cells were recorded within 10 randomly chosen obser-

vation areas for each well. Cells with ®ve grains of

thymidine or more in their nuclei were regarded as la-

belled. The median value for each concentration was

obtained after examining six wells.

The LI% was calculated with the following formula:

LI% �X

labelledcells=X

cells

� �� 100

Viability test

Hundred microlitres of trypan blue was added to each

well for 10±15 min. Thereafter the cells were rinsed

Effect of neuroendocrine peptides and serotonin � K Zachrisson and A Uribe Acta Physiol Scand 1998, 163, 195±200

196 Ó 1998 Scandinavian Physiological Society

with PBS and the number of stained (nstained) and non-

stained (nnon-stained) cells were counted.

The cell viability (%) was calculated by the formula

nnonstained=�nnonstained � nstained�� 100

Statistical analysis

Results are calculated as mean �SD. The normal dis-

tribution was checked with the Anderson±Darling A2

test. The one-way analysis of variance was used to test

differences between groups. The level of signi®cance

was P < 0.05.

RESULTS

DNA synthesis

All labelled cells were strongly labelled and the back-

ground was negligible. The autoradiographs of IEC-6

cells incubated with 10)6M serotonin and of FHs-74

cells incubated with 10)6M SP were unsuccessful and

not evaluated.

Neuroendocrine peptides and serotonin in IEC-6 cells

EGF and IGF-II markedly increased the LI in a dose-

related fashion. The differences were apparent at con-

centrations of 8.2 ´ 10)9 and 3.3 ´ 10)9M, respec-

tively (P < 0.0001, Fig. 1).

Serotonin and NKA reduced the LI dose depen-

dently. The reduction of LI was statistically signi®cant

already at concentrations of 10)9 and 10)10M, respec-

tively (P < 0.0001, Table 1).

The labelling index was also signi®cantly increased

in IEC-6 cells incubated with glucagon and SP, starting

at concentrations of 10)7 and 10)8M, respectively

(P < 0.003, Table 1). CGRP and neurotensin did not

affect the labelling index (Table 1).

In an attempt to reproduce the conditions previ-

ously observed in vivo (Uribe et al. 1997a,b) we exam-

ined the effect on DNA synthesis of the combined

administration of SP and NKA at a concentration of

10)6M and of glucagon and serotonin at a concentra-

tion of 10)7M, respectively. The LI of cells incubated

with both serotonin and glucagon was 8.2 � 0.7%,

which was slightly but signi®cantly lower than

9.9 � 1.5% as observed in the controls (P < 0.03). The

LI in cells incubated with SP and NKA was however,

not signi®cantly different from the control value

(11.1 � 2.1 vs. 12.5 � 3.0% in controls.

Neuroendocrine peptides and serotonin in FHs-74 cells

In FHs-74 cells, EGF, IGF-II, SP and glucagon stim-

ulated DNA synthesis (Fig. 1, Table 1) in a similar

fashion as in IEC-6 cells. The dose-dependent increase

in DNA synthesis following incubation with glucagon

Figure 1 DNA synthesis (labelling index, LI%) of IEC-6 cells (a, b) and FHs-74 cells (c, d), incubated in vitro with epidermal growth factor

(EGF) (a, c) and insulin-like growth factor II (IGF-II) (b, d). Values are given as median and interquartile range (*P < 0.001). Note that EGF

and IGF-II increase DNA synthesis dose dependently.


Acta Physiol Scand 1998, 163, 195±200 K Zachrisson and A Uribe � Effect of neuroendocrine peptides and serotonin

was even more marked in FHs-74 cells (P < 0.0001).

NKA and serotonin inhibited DNA synthesis dose

dependently, as in IEC-6 cells. (Table 1).

Cell viability

The viability of the cells, as estimated by exclusion of

typan blue, was almost 100% in all preparations.

DISCUSSION

Previous studies highlight the dif®culty of obtaining

unambiguous evidence of a direct growth-promoting

activity of neuropeptides in vivo (Uribe et al. 1997a,b) or

in heterogeneous collections of cells (Thoresen et al.

1990). Our results show that EGF, IGF-II, glucagon

and SP stimulate DNA synthesis in small intestinal cells

in vitro, whereas NKA and the amine serotonin inhibit

cell proliferation. Interestingly, the primary actions of

SP and NKA, as well as those of glucagon and sero-

tonin, were antagonized after incubation at equivalent

concentrations.

Serotonin appears to be a potent inhibitor of cell

proliferation in small intestinal cells. The inhibitory

action of serotonin on DNA synthesis was dose de-

pendent and this was apparent even in the presence of

trophic concentrations of glucagon. Various actions of

serotonin on cell proliferation have been reported,

depending on the species and cell types (Seuwen &

PouysseÂgur 1990). Serotonin seems to be mitogenic to

bovine smooth muscle cells (Nemeck et al. 1986), ®-

broblasts (Seuwen et al. 1988) and rat mesangial cells

(Takuwa et al. 1989) among others. In contrast to our

observations, the mitotic activity in rat jejunal crypts

was increased by a single dose of serotonin (Tutton

1974). However, serotonin inhibits cell growth in der-

mal and epidermal embryonic chick skin cells (de Rid-

der & Beele 1988), as well as in MOLT-4 T-cell

leukaemia and MCF-7 breast adenocarcinoma in culture

(Smith et al. 1992) which is in agreement with our

®ndings in IEC-6 cells. Stimulatory and inhibitory ef-

fects of serotonin on cell proliferation in smooth

muscle cells (Lee et al. 1991) suggest that the complex

actions of this amine are mediated by various mecha-

nisms. The stimulatory action seems to depend on

stimulation of intracellular events by serotonin, whereas

the inhibitory effect is a receptor-mediated, cell surface

inhibition of cell proliferation, associated with an in-

creased concentration of cyclic-AMP (Lee et al. 1991).

The inhibitory action of serotonin on DNA synthesis

reported in our study support, however, previous ob-

servation in rats shows that prolonged administration

of prostaglandin E2 (PGE2) increases the total mucosal

volume of serotonin-immunoreactive cells (Uribe et al.

1989, 1992, Kapraali et al. 1994) and secondarily re-

duces the mitotic activity in the antral glands (Uribe

et al. 1989).

In this study, concentrations of NKA similar to that

observed in the small intestinal tissue of rats (Uribe

et al. 1997a,b) inhibited DNA synthesis and antago-

nized the stimulatory actions of SP. However, other

studies reported no effect of NKA on cell proliferation

in a similar cell line (BjoÈrk et al. 1994) and that NKA

stimulates proliferation of human ®broblasts in vitro

(Nilsson et al. 1985) as well as of smooth muscle cells

(Nilsson et al. 1985) planarian cells (BagunaÁ et al. 1989)

and murine thymocytes (SoÈder & HellstroÈm 1989).

Differences in species and cell types might, to some

extent, account for the reported mitogenic and anti-

mitogenic actions of NKA.

SP stimulated DNA synthesis in IEC-6 and FHs-74

cells, which is in accordance with in vitro studies

showing that SP stimulates the cell proliferation of

cultured arterial smooth muscle cells (Nilsson et al.

Table 1 DNA synthesis (labelling index, LI%) of IEC-6 and FHs-74 cells incubated in vitro with different concentrations of serotonin,

neurokinin A (NKA), glucagon, substance P, calcitonin gene-related peptide (CGRP) and neurotensin

Cell line and substance Control 10)10M 10)9

M 10)8M 10)7

M 10)6M

IEC-6 cells

Serotonin 22.2 + 6.7 19.4 + 6.7 16.9 + 4.8* 15.6 + 5.1* 9.2 + 7.3*

NKA 15.6 + 2.6 12.7 + 2.1* 11.2 + 8.8* 6.7 + 8.1* 5.9 + 4.3* 3.9 + 6.8*

Glucagon 13.4 + 3.0 12.0 + 1.0 11.4 + 0.6 14.6 + 3.8 17.7 + 1.6* 21.3 + 2.6*

Substance P 16.4 + 5.0 11.0 + 5.8 18.4 + 4.5 25.1 + 8.1* 25.5 + 5.1* 26.0 + 5.1*

CGRP 9.9 + 1.5 10.4 + 2.1 11.6 + 1.0 12.0 + 1.0 12.0 + 2.6 11.6 + 1.8

Neurotensin 14.6 + 0.9 14.4 + 1.9 14.5 + 1.5 13.2 + 1.4 13.4 + 0.6 13.2 + 0.2

FHs-74

Serotonin 13.6 + 1.1 7.7 + 1.1* 6.9 + 0.5* 4.3 + 1.0* 3.3 + 0.3* 3.0 + 0.6*

NKA 18.8 + 1.2 8.3 + 0.8* 8.4 + 0.8* 6.0 + 0.8* 4.9 + 0.9* 3.3 + 0.6*

Glucagon 13.6 + 1.5 17.5 + 1.5 24.3 + 1.5* 27.9 + 1.5* 31.2 + 1.7* 34.7 + 1.7*

Substance P 15.2 + 4.0 22.0 + 4.0 27.8 + 2.1* 3.7 + 5.3* 37.6 + 5.2*

Values are expressed as mean �SD.

P < 0.05 for differences from controls



1985) and human ®broblasts (Nilsson et al. 1985).

Moreover, high levels of SP were observed in the ileum

of ex-germ-free rats exposed to micro¯ora associated

with an increased DNA synthesis (Uribe et al. 1997a,b).

SP and NKA act through the same receptors although,

as reported in this study, they had opposite actions on

cell proliferation in small intestinal cells, and the com-

bined administration of these peptides did not affect

DNA synthesis. Both tachykinins are present in neu-

rones of the submucosal plexus and they have a com-

mon precursor, beta-preprotachykinin, which contains

amino acid sequences of both substance P and ne-

urokinin A that can be liberated by proteolysis (Nawa

et al. 1983). Thus, we suggest that changes in the

breakdown of the precursor due to different proteolytic

processing may give either stimulatory or inhibitory

signals to the epithelial cells.

Glucagon increased DNA synthesis and the maxi-

mal value of LI was, in FHs-74 cells, 140% higher than

that of the controls. These ®ndings are in agreement

with previous reports showing an enhanced DNA

synthesis in cultured guinea-pig jejunal cells (Uttenthal

et al. 1982) and rat ileal cells (Watanabe et al. 1988) in-

cubated with enteroglucagon. In contrast, studies in vivo

failed to show any trophic actions of glucagon on cell

proliferation (Goodlad et al. 1991). The actions of

glucagon on cell proliferation are complex, as shown by

the reported stimulatory (Watanabe et al. 1988) and

inhibitory (Watanabe et al. 1988) actions of glucagon 1±

21 on cell proliferation in small intestinal cells. These

dual actions of glucagon are probably due to a growth-

promoting effect early in the pre-replicative period (G0

or early G1), as shown when low doses are used, and an

inhibitory effect, at a point shortly before the G1 to S

transition, which becomes apparent at higher dose

levels (Thoresen et al. 1990). Moreover, complex in-

teractions occur in vivo, affecting the local availability of

glucagon and other peptides that may in¯uence cell

proliferation. Neurotensin (Brubaker 1991) and CGRP

release glucagon in vivo (Brubaker 1991) and, in turn,

glucagon may release the antitrophic peptide, somato-

statin (Bado et al. 1988).

An interesting ®nding was the dose-related stimula-

tion of DNA synthesis by IGF-II, which was of the same

magnitude as that of EGF. The latter is in accordance

with a recent report (Chao & Donovan 1996). IGF-II

receptors, identi®ed throughout the gastrointestinal ep-

ithelium (Labuthe & Amiranoff 1989) are markedly in-

creased in intestinal adaptation, e.g. following small

intestinal resection (Grey et al. 1991) which suggests that

this peptide may be an important physiological regulator

of gastrointestinal cell proliferation.

Neurotensin and CGRP did not affect DNA syn-

thesis in our system. It should be kept in mind how-

ever, that these neuropeptides can in¯uence cell

proliferation in vivo (Uribe et al. 1997a,b) by releasing

glucagon (Brubaker 1991) and somatostatin (Bado et al.

1988), respectively. Thus, indirect effects on cell pro-

liferation may explain discrepancies concerning the

action of neurotensin on cell proliferation in vivo (Feurle

et al. 1985, Feurle & Niestroj 1991, Evers et al. 1992).

In summary, serotonin and NKA inhibit DNA

synthesis in two different small intestinal cell lines dose

dependently, whereas EGF, IGF-II, glucagon and SP

stimulate cell proliferation. Our ®ndings suggest that

these growth factors may be important regulators of

cell proliferation in vivo and that they contribute to

modulate the changes in cell proliferation observed in

humans following extensive small bowel resection

(Grey et al. 1991) as well as in germ-free rats exposed to

micro¯ora (Uribe et al. 1997a,b) and in conventional

animals given indomethacin and prostaglandin E2

(Uribe et al. 1997a,b).

The study was supported by grants from the Foundations of the

Karolinska Institute, Danderyd Hospital Development Funds (176),

Anders Otto SwaÈrds Foundation for Medical Research, the Sera®mer

Foundation, the Swedish Board of Health and Welfare, the Lisa and

Johan GroÈnberg Foundation and the Ruth and Richard Juhlin

Foundation.

We are most grateful to Dr Carl-Erik Elwin for providing labo-

ratory facilities and for generous support.

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serotonin and neuroendocrine peptides influence dna synthesis in rat and human small intestinal...

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