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Biochem. J. (1996) 316, 551–557 (Printed in Great Britain) 551
Regulation of proliferation of LLC-MK2cells by nucleosides and nucleotides:
the role of ecto-enzymes
Raf LEMMENS*, Luc VANDUFFEL, Henri TEUCHY and Ognjen CULIC
Limburgs Universitair Centrum, Department MBW, Biochemistry, Universitaire Campus, B-3590 Diepenbeek, Belgium
1. Using the incorporation of [methyl-$H]thymidine as a pro-
liferation marker, the effects of various nucleosides and nucleo-
tides on endothelial LLC-MK#cells were studied. We found that
ATP, ADP, AMP and adenosine in concentrations of 10 µM
or higher stimulate the proliferation of these cells. 2. Inhibition
of ecto-ATPase (EC 3.6.1.15), 5«-nucleotidase (EC 3.1.3.5) or
alkaline phosphatase (EC 3.1.3.1) significantly diminished the
stimulatory effect of ATP, indicating that the effect is primarily
caused by adenosine and not by adenine nucleotides. Also, the
effect depends only on extracellular nucleosides, since inhibition
of nucleoside uptake by dipyridamole has no influence on
proliferation. 3. Other purine nucleotides and nucleosides (ITP,
GTP, inosine and guanosine) also stimulate cell proliferation,
while pyrimidine nucleotides and nucleosides (CTP, UTP,
INTRODUCTION
Biological responses to extracellular ATP [1] and adenosine [2]
have been documented in virtually every major organ and}or
tissue system studied so far. There is a growing body of evidence
on the influence of extracellular adenosine and ATP on cell
proliferation. This effect is usually described as a phenomenon
mediated via P"-orP
#
-purinoceptors on the cell surface [3].
Evidence has been presented that extracellular ATP can
stimulate mitogenesis in mammalian cells by different signalling
pathways [4–6]. ATP stimulates the proliferation of endothelial
[7], smooth muscle [8–10], fibroblast 3T3 and 3T6, and tumour
A431 cells [11], glomerular mesangial cells [6,12] and macro-
phages [13]. In addition, various dinucleotide phosphates can
stimulate cell proliferation when present in the extracellular
medium [14,15].
Adenosine has been shown to stimulate the proliferation of
endothelial [7,16–18], neuroblastoma [19] and mouse fibroblast
3T3 cells [20], but also to inhibit the proliferation of fibroblasts
[21] and pericytes [22]. Interesting information about the potential
proliferative effect of extracellular adenosine came from a study
on thyroid cells in which cDNA for an A#-receptor was expressed,
causing stimulation of cell proliferation even in the absence of
externally added adenosine [23]. This finding indicates that
adenosine is normally released from the cells, either as such or in
the form of ATP and other adenine nucleotides as its precursors
[24].
Nucleotides present in all cells can be released through different
mechanisms: exocytosis [25,26], transport via intrinsic plasma
membrane proteins such as P-glycoprotein [27] and the cystic
fibrosis transmembrane conductance regulator [28,29], or cellular
Abbreviations used: IP3,D-myo-inositol 1,4,5-trisphosphate; RB-2, Reactive Blue 2; RO 20-1724, 4-(3-butoxy-4-methyoxybenzyl)-2-imidazolidione ;
DMEM, Dulbecco’s modified Eagle’s medium; NBCS, newborn-calf serum; AP4A, P1,P4-di(adenosine-5«) tetraphosphate; AP5A, P1,P5-di(adenosine-5«)
pentaphosphate.
* To whom correspondence should be addressed.
cytidine and uridine) inhibit proliferation. Furthermore, the
simultaneous presence of adenosine and any of the other purine
nucleosides is not entirely additive in its effect on cell pro-
liferation. At the same time any pyrimidine nucleoside, when
added together with adenosine, has the same inhibitory effect as
the pyrimidine nucleoside alone. 4. Apparently these proliferative
effects are neither caused by any pharmacologically known P"-
purinoceptor, nor are they mediated by cyclic AMP, cyclic GMP,
or -myo-inositol 1,4,5-trisphosphate as second messenger, nor
by extracellular Ca#+. 5. Therefore, we conclude that various
purine and pyrimidine nucleosides can influence the proliferation
of LLC-MK#cells by acting on putative purinergic and pyri-
midinergic receptors not previously described.
lysis. Mechanical forces applied on cells, like shear stress for
example, are also shown to cause release of intracellular ATP
[30]. Cells are able to release ATP in response to various agonists,
including ATP itself [24]. Therefore, the concept of nucleotides
as non-specific (universal) autocrine regulators of cell prolifer-
ation attracts a growing attention.
The extracellular metabolism of ATP and other nucleoside
phosphates is mainly controlled by enzymes like ecto-ATPase,
5«-nucleotidase and alkaline phosphatase, which are believed to
be ubiquitously present on the surface of many different cell
types [25]. These enzymes are capable of mediating a cascade
dephosphorylation of nucleoside phosphates, generating nucleo-
sides in the extracellular space [31–33]. Since the cellular effects
of adenosine and ATP are often very different, with only one of
these substances being biologically active, this nucleotide to
nucleoside conversion may be an important step in the regulation
of cell proliferation.
In this paper we describe the proliferative effects of adenosine
and ATP on five randomly chosen cell types. In particular, a
more detailed study was made of the effect of various naturally
occurring nucleotides and nucleosides on the proliferation of
LLC-MK#cells. The role of different ecto-nucleotidase enzymes
in the regulation of this process was also investigated.
EXPERIMENTAL
Materials
Reactive Blue 2 (RB-2), 8-bromo-cyclic AMP (sodium salt), 8-
bromo-cyclic GMP (sodium salt), receptor agonists and
552 R. Lemmens and others
antagonists, and toxins were purchased from RBI (Natick, MA,
U.S.A.). All nucleosides and nucleotides, 5-(N,N-dimethyl)-
amiloride (hydrochloride), dipyridamole, diadenosine poly-
phosphates, adenosine 5«-[α,β-methylene]-diphosphate and
forskolin were obtained from Sigma (St. Louis, MO, U.S.A.).
Oligomycin, levamisole, EGTA and ouabain (octahydrate) were
purchased from Janssen Chimica (Beerse, Belgium). Cerium
chloride was from Merck (Darmstadt, Germany). Glutar-
aldehyde was obtained from Fluka Chemie AG (Buchs,
Switzerland), and staurosporine from Calbiochem (CA, U.S.A.).
Cell culture products and 4-(3-butoxy-4-methyoxybenzyl)-2-
imidazolidione (RO 20-1724) were bought from Life
Technologies Inc. (New York, NY, U.S.A.). Radioactive sub-
stances came from DuPont de Nemours (Brussels, Belgium).
Suramin was kindly provided by Dr. J. A. Boutin (Institut de
Recherches Servier, Suresnes, France).
Cell culture
LLC-MK#(monkey kidney), HeLa S3 (human carcinoma), Cos-
7 (African green monkey kidney, fibroblast-like) and 293 (trans-
formed human kidney, embryonic) cell lines were obtained from
ATCC (Rockville, MD, U.S.A.). GM00637F cells (simian virus
40-transformed human skin fibroblasts) were purchased from
NIGMS (Camden, NY, U.S.A.). All cell cultures were tested for
mycoplasma using the Gibco Mycotect2kit (Life Technologies
Inc., New York, NY, U.S.A.). Cells were grown in Dulbecco’s
modified Eagle’s medium (DMEM), with penicillin–
streptomycin, -glutamate, sodium pyruvate, non-essential
amino acids and 10%(v}v) newborn-calf serum (NBCS). The
cells were trypsinized and plated on 96-well plates at a density of
2¬10%cells per well. After 16 h of incubation, the cells were
rinsed once with PBS, and once with serum-free medium, after
which the medium was replaced by DMEM without NBCS.
After another 48 h of incubation to induce starvation, pro-
liferative agents, inhibitors, agonists or antagonists were added.
Determination of cell number
The cells were left to grow for 48–72 h after addition of the
different nucleotides and nucleosides. To determine the cell
number, Alamar Blue (BioSource International, Camarillo, CA,
U.S.A.) was used. This reagent is an indicator dye, formulated to
measure quantitatively the proliferation of cells. It consists of an
oxidation–reduction indicator that yields a colorimetric change
in response to metabolic activity. The measurements were
performed as described in the general procedures provided by the
manufacturer.
[methyl-3H]Thymidine incorporation
Together with the different agents, [methyl-$H]thymidine (0.5 µCi
per well) was added. After 16 h of incubation, the medium was
removed and the cells were trypsinized. Using an automated 96-
well harvester, the cells were transported to a printed filtermat
and their radioactivity was determined by counting in a β-
scintillation counter. Except when stated otherwise, all results
are expressed as percentage stimulation: (incorporated radio-
activity with stimulus}incorporated radioactivity without stimu-
lus ®1)¬100.
Cytochemical determination of adenosine production
The cells were trypsinized, seeded on chamber slides (Miles
Scientific, Naperville, IL, U.S.A.) and grown for at least 18 h
before being washed with Tris-buffered saline (TBS). Fixing
solution contained 2%(w}v) glutaraldehyde, 5%(w}v) sucrose
and 0.1 M cacodylate buffer (pH 7.4). Fixation time was
30–60 min. Subsequently, the cells were washed for 2–3 h with
0.1 M cacodylate buffer (pH 7.4) containing 5 %(w}v) sucrose
and used for cytochemical procedures. The cytochemical de-
tection of cells’ capability to produce adenosine from ATP as an
initial substrate was carried out in a 30 mM Tris}HCl buffer
(pH 7.4), containing 100 mM NaCl, 5 mM KCl, 1 mM ouabain,
5µg}ml oligomycin and 4 mM CaCl#. Additionally, the medium
also contained 0.2 mM CeCl$as a phosphate-capturing agent
and 40 nM [α-$#P]ATP (specific radioactivity was 800 Ci}mmol).
After incubation at 37 °C for 90 min, the medium was removed,
the upper parts of the chamber slides were detached and the
slides were washed three times for 5 min with TBS. The slides
were dried at room temperature and dipped in NTB2 auto-
radiographic emulsion (Eastman Chemical Company, Rochester,
NY, U.S.A.) diluted with distilled water in a 1:1 (v}v) ratio.
After exposure (typically for 2–3 days) at 4 °C in boxes containing
desiccant the slides were developed and inspected under the light
microscope.
Diadenosine polyphosphate breakdown
P",P%-di(adenosine-5«) tetraphosphate (AP%A) and P",P&-
di(adenosine-5«) pentaphosphate (AP&A) breakdown was carried
out in the same buffer as described for the cytochemical de-
termination of adenosine production. Substrate was 1 mM AP%A
or AP&A. ATP (1 mM) was used as a positive control. Typically,
10'cells were incubated for 1 h at 37 °C, the reaction was
stopped with a 1 %SDS solution and the phosphate released was
measured by the method of LeBel et al. [34], in which the cells are
lysed and the complete phosphate content in the reaction mixture
was photometrically determined. This method gives reliable
results up to phosphate concentrations of 20 µM.
Other determinations
For determination of the intracellular cyclic AMP and -myo-
inositol 1,4,5-trisphosphate (IP$) concentrations, the cells were
grown as described in the Cell culture section, using 24-well
plates at a density of 5¬10%cells per well. Before the different
agents were added, the cells were preincubated with RO 20-1724,
an inhibitor of cyclic AMP phosphodiesterase, for at least 1 h.
After 1, 3, 5 or 10 min of incubation with different agents, the
intracellular cyclic AMP was determined using the cyclic AMP
"#&I scintillation proximity assay (SPA) system (Amersham
International plc, Amersham, Bucks., U.K.).
To determine the intracellular amount of IP$, the cells were
preincubated with 10 mM LiCl for 1 h, and left to incubate with
the different agents for 30 s to 10 min, after which IP$was
determined, using the (IP$)[
$
H] assay system from Amersham
International plc, U.K.
In order to measure the protein content in the different wells,
the cells were first precipitated using ice-cold 10 %(w}v)
trichloroacetic acid for 20 min, and then solubilized with 0.2 M
NaOH, 1%(w}v) SDS at 95 °C for 15 min. A sample was taken
to determine the protein content with the BCA Protein Assay
(Pierce, Rockford, IL, U.S.A.).
Statistical calculations
Results were tested for significance using the two-tailed Student’s
t-test.
553The role of nucleosides in proliferation of LLC-MK2cells
RESULTS
First, we tested the effect of ATP and adenosine on five randomly
chosen cell lines: LLC-MK#, HeLa S3, Cos-7, 293 and
GM00637F (Figure 1). LLC-MK#, Cos-7 and GM00637F cells
showed a significant proliferative response to ATP and adenosine.
293 cells were significantly influenced only when ATP or
adenosine at a concentration of 100 µM was applied, while
neither of these substrates significantly influenced proliferation
of HeLa S3 cells. For a more detailed study of the influence of the
nucleosides and nucleotides on cell proliferation we used the
LLC-MK#cells.
As presented in Figure 2, adenosine, AMP, ADP and ATP
significantly stimulate cell proliferation of LLC-MK#cells in a
concentration-dependent manner. The presence of 10%(v}v)
NBCS stimulates cell proliferation to a lesser extent than 10 µM
of the adenine nucleotides or adenosine (21³3%).
We were able to demonstrate adenosine production from ATP
as initial substrate, using a cytochemical approach based on [α-
$#P]ATP for labelling and Ce$+ions as a phosphate-capturing
250
200
150
50
0
–50
100
stimulation (% of control)
LLC-MK2Cos-7 GM00637F 293 HeLaS3
*
**
*
*
*
*
*
*
**
**
*
*
** **
Figure 1 Effect of ATP and adenosine on the [methyl-3H]thymidine
incorporation in different cell lines
All cell lines were brought to starvation for 48 h in serum-free DMEM without NBCS, after which
ATP (100 µM, open bars ; 10 µM, closed bars) or adenosine (100 µM, hatched bars ; 10 µM,
cross-hatched bars) was added, together with [methyl-3H]thymidine. After 24 h the incorporated
radioactivity was determined. Results are mean values (³S.D.) for six independent measure-
ments from one typical experiment (*P!0.005 ; **P!0.05).
Figure 2 Influence of adenine nucleotides and adenosine on the [methyl-
3H]thymidine incorporation in LLC-MK2cells
Quiescence was induced by incubating the cells for 2 days in serum-free medium. Then
[methyl-3H]thymidine and the stimulatory agents (ATP, D; ADP, *; AMP, ^; adenosine,
E) were added in the appropiate concentrations. After 24 h, incorporated radioactivity was
determined by liquid scintillation counting. Presented data are mean values for six independent
measurements from one typical experiment ; standard deviation never exceeded 10% of the mean
value.
Figure 3 Light-microscopy photographs of extracellular adenosine pro-
duction of LLC-MK2cells as determined cytochemically
Cells were grown on chamber slides and incubated with 40 nM [α-32P]ATP (specific
radioactivity 800 Ci/mmol) as substrate. CeCl3in a final concentration of 0.2 mM was added
as a phosphate-capturing agent in the lower panel. Radioactive deposits were visualized using
autoradiographic emulsion. Magnification ¬500. Bar ¯20 µm.
agent (Figure 3). This method indirectly detects the adenosine
produced by complexing the radioactive phosphate released
from ATP at the cell surface with Ce$+ions [35].
In an attempt to discriminate between the effect of ATP and
the effect of adenosine on cell proliferation, we blocked ATP
hydrolysis using inhibitors of the different enzymes involved in
extracellular adenosine production. Blocking ecto-ATPase with
1 mM suramin or 1 mM RB-2 caused a significant reduction of
the ATP-mediated proliferative effect (Table 1). From these
results it can be calculated that the proliferation caused by
100 µM ATP is inhibited by 71%by suramin and by 84%by
RB-2. The inhibition of the ecto-ATPase activity by the same
concentrations of suramin and RB-2, as measured biochemically,
was 73%and 77%respectively [35]. As determined by Trypan
Blue stain cell counting, suramin and RB-2 were shown not to
influence the viability of the cells at the concentrations and time
periods used in the experiments (viability 105³21 %and
106³22%respectively, n¯16).
In line with these results, a concentration of 100 µM of non-
hydrolysable ATP-analogues, adenosine 5«-[α,β-methylene]-
triphosphate and 2-methylthio-ATP, which are in addition P#x-
and P#y-purinoceptor agonists, showed no influence on the
554 R. Lemmens and others
Table 1 Influence of enzyme inhibitors on the [methyl-3H]thymidine
incorporation in LLC-MK2cells
Cells were made quiescent by incubation in serum-free medium for 48 h, after which the
inhibitors were added at the indicated concentrations. ATP (100 µM) was used as a stimulating
agent and [methyl-3H]thymidine as a proliferation marker. After 24 h of incubation, the
incorporated radioactivity was counted. The results are expressed as percentage stimulation,
compared with the incorporation of radioactivity in the presence of the inhibitor without ATP.
Data are mean values (³S.D.) of six independent measurements from a typical experiment.
Inhibitor Stimulation (%)
No inhibitor 154³16
1 mM Suramin 44³4
1 mM RB-2 24³8
100 µM Adenosine 5«-[α,β-methylene]-diphosphate 45³2
1 mM Levamisole 102³15
1 mM Levamisole100 µM adenosine 5«-[α,β-methylene]-diphosphate 11³1
Table 2 Influence of different nucleosides and nucleoside triphosphates on
the [methyl-3H]thymidine incorporation in LLC-MK2cells
Starvation of the cells was induced by incubating them for 48 h in serum-free medium. Then
100 µM of the nucleoside triphosphate, or the nucleoside, with or without 100 µM of adenosine
was added, together with [methyl-3H]thymidine. After 24 h at 37 °C, [methyl-3H]thymidine
incorporation into DNA was quantified by liquid scintillation counting. Presented data are mean
values (³S.D.) for six independent measurements from a typical experiment (P!0.001 for all
samples, as compared with control containing no stimulatory agent). In all cases when
adenosine was combined with ATP or other nucleosides, no statistically significant differences
could be shown when the effect of a combination was compared with the effect of at least one
of the agents used in the particular combination.
Stimulation (%)
Without adenosine With adenosine
ATP 172³15 204³4
Adenosine 100³4
GTP 55³3
Guanosine 66³3 124³16
ITP 70³8
Inosine 92³194³2
UTP ®11³0
Uridine ®40³1®41³9
CTP ®28³3
Cytidine ®51³6®49³11
cellular proliferation (stimulation 8³1%and ®1³0%re-
spectively, n¯6).
Blocking AMP hydrolysis by either adenosine 5«-[α,β-
methylene]-diphosphate (5«-nucleotidase) or levamisole (alkaline
phosphatase), caused partial inhibition of the proliferative effect
of ATP. When both inhibitors of AMP hydrolysis were applied
simultaneously, the stimulatory effect of 100 µM ATP on cell
proliferation was almost completely lost. In contrast, these
inhibitors did not influence the proliferative response to
adenosine (results not shown).
We also investigated whether the proliferative effect could be
achieved by other naturally occurring nucleosides and nucleo-
tides. All purine nucleosides and nucleotides tested, had
significant positive effects on cell proliferation, with the strongest
response being observed with adenosine and ATP. In contrast to
these findings, pyrimidine nucleosides and nucleotides signifi-
cantly inhibited cell proliferation (Table 2). Inhibition of ecto-
ATPase, 5«-nucleotidase and alkaline phosphatase markedly
diminished the effect of the pyrimidine as well as the purine
nucleoside triphosphates on cell proliferation (results not shown).
In addition, we studied the effects of the simultaneous presence
of different nucleotides and nucleosides. When ATP and
adenosine were present together in the cell culture medium, the
proliferative effect was not additive (Table 2). Similar results
were obtained when adenosine was added together with either
guanosine or inosine. However, the simultaneous presence of
equimolar concentrations of adenosine and pyrimidine nucleo-
sides (uridine or cytidine) resulted in an inhibitory effect, similar
to that observed when only the pyrimidine nucleoside was
present.
Since adenosine-containing dinucleotides also exert various
biological activities, including cell proliferation [14,15], we
examined a possible influence of AP%A and AP&A on LLC-MK#
cells. Both AP%A and AP&A showed a stimulatory proliferative
effect, similar to that of adenosine, but at much lower concen-
trations (Table 3). We were unable to show any inorganic
phosphate release, as measured spectrophotometrically, when
LLC-MK#cells were incubated with AP%A and AP&A, suggesting
that these diadenosine polyphosphates are not hydrolysed. With
the spectrophotometric method used, amounts of released
phosphate that are 20 times lower than those measured with
1 mM of ATP as substrate, can be detected.
In order to prove that the observed effects on cell proliferation
are exclusively due to extracellular nucleosides, we blocked the
nucleoside uptake with 10 µM dipyridamole, a specific nucleoside
transporter antagonist. Dipyridamole also inhibits the uptake of
[methyl-$H]thymidine (92³7%inhibition, n¯5). Therefore the
relative change in cell number was determined with a method
relying on the use of Alamar Blue as a redox indicator. We found
that dipyridamole did not reduce the proliferation caused by
either 100 µM adenosine or ATP. Also the inhibitory effect of
CTP was not reduced by dipyridamole (Table 4). These results
suggest that the extracellular presence of the nucleosides tested is
responsible for the observed effects on proliferation.
In an attempt to determine which type of purinoceptor is
involved in the proliferation of LLC-MK#cells, we tested N'-
cyclopentyladenosine and N'-[2-(3,5-dimethoxyphenyl)-2-(2-
methylphenyl)-ethyl]adenosine respectively as specific A"- and
A#-receptor agonists. No stimulating effect was observed when
these substances were tested at concentrations ranging from
1nMto10µM. Addition of 8-(p-sulphophenyl)theophylline, an
antagonist of adenosine receptors, at concentrations up to
100 µM, had no influence on the proliferative effect of either
100 µM ATP or adenosine (results not shown).
In most cases, adenosine receptors mediate their effect on cell
proliferation through a mechanism which involves cyclic AMP
as a second messenger. Therefore, we determined intracellular
cyclic AMP levels after stimulation with 100 µM adenosine or
cytidine. With 100 µM adenosine, no increase of the intracellular
cyclic AMP concentration could be found. Also, 100 µM cytidine
did not increase the cyclic AMP content of the cells. Inhibition
of cyclic AMP breakdown, by blocking phosphodiesterase with
20 µM RO 20-1724, did not alter the effect of adenosine or
cytidine. Furthermore the increase in cyclic AMP levels due to
10 µM forskolin, a stimulator of adenylate cyclase, was not
influenced by the additional presence of either adenosine or
cytidine. Cells were also incubated with 8-bromo-cyclic AMP,
which is taken up by the cells and transformed into cyclic
AMP, and therefore could potentially stimulate proliferation.
8-Bromo-cyclic AMP (100 µM) did not cause any effect on
the proliferation of the cells (stimulation 14³0.3 %,n¯7).
Similarly, 8-bromo-cyclic GMP (100 µM) was found to be
inactive in stimulating the proliferation (stimulation 10³1%,
555The role of nucleosides in proliferation of LLC-MK2cells
Table 3 Effect of AP4A and AP5A on the incorporation of [methyl-
3H]thymidine in LLC-MK2cells
Starvation of the cells was induced by growing them for 2 days in serum-free medium. AP4A
and AP5A were added (10 µM, 1 µM, 100 nM) together with [methyl-3H]thymidine and the
cells were incubated for one more day. The amount of [methyl-3H]thymidine incorporated in the
cellular DNA was quantified. Results are from one representative experiment in quadruplicate
(mean values ³S.D.; *P!0.05)
Stimulation (%)
10 µM1µM 0.1 µM
AP4A 104³15* 73³6* 32³7
AP5A98³37* 48³3* 21³4
Table 4 Effect of 10 µM dipyridamole on the number of LLC-MK2cells
Cells were grown in NBCS-free DMEM for 48 h. ATP (100 µM), CTP (100 µM), adenosine
(100 µM) and NBCS (10%) were added, without or with 10 µM dipyridamole. After 72 h, the
relative cell number was determined by the use of Alamar Blue. Results from a typical
experiment are given in percentage stimulation, compared with a blank containing no
stimulating agents.
Stimulation (%)
Without dipyridamole With dipyridamole
Adenosine 48³13 66³8
ATP 72³677³13
CTP ®14³6®5³2
n¯5). The possibility that IP$acts as a second messenger of the
observed proliferative effect was also checked. We found that the
IP$concentration was not influenced either by proliferation
stimulatory agents like 100 µM ATP or adenosine or by pro-
liferation inhibitory agents like 100 µM CTP or cytidine (con-
centrations of IP$were 1–5%lower than control). These findings
are strengthened by the fact that staurosporine, a protein kinase
C inhibitor, did not influence the proliferative effects of any of
the nucleosides (results not shown).
In order to find out whether influx of extracellular Ca#+ions
could be responsible for the proliferating stimulus in LLC-MK#
cells, we added EGTA to the medium. We found that EGTA, up
to 1 mM, was not capable of blocking any positive or negative
proliferative effect.
In an attempt to check for a possible participation of G-
proteins in a mechanism by which the nucleosides mediate cell
proliferation, we studied the effect of pertussis and cholera
toxins. Pre-incubation of the cells for 4 to 6 h with the toxins, did
not influence the proliferative effect of the nucleosides.
Finally we examined a possible involvement of the Na+}H+-
exchanger in the process of cell proliferation by extracellular
nucleosides. We compared the effect of various nucleosides in the
presence of 50 µM 5-(N,N-dimethyl)amiloride, an inhibitor of
the Na+}H+-exchanger. Blocking the exchanger significantly
decreased the proliferative effect of purine nucleosides
(adenosine, ®28³2%; guanosine, ®38³1%; and inosine,
®35³2%,n¯6; P!0.01). In the case of uridine and
cytidine, 5-(N,N-dimethyl)amiloride significantly increased the
inhibiting effect of the nucleosides (18³1%and 53³1%re-
spectively, n¯6; P!0.01).
DISCUSSION
In this study, we investigated the effect of extracellular nucleosides
and nucleotides on five randomly chosen cell lines. Adenosine
and ATP were shown to positively affect proliferation of three
cell lines: LLC-MK#, Cos-7 and GM00637F. We focused our
attention on LLC-MK#cells for two reasons. First, these cells
showed a high proliferative response to externally added ATP
and adenosine, and secondly, they express a high activity of ecto-
enzymes with nucleotide-dephosphorylating capacities [35].
When adenosine, AMP, ADP and ATP were tested for pro-
liferation effects, ATP was shown to influence the cellular
proliferation to the highest extent, followed by ADP, AMP and
adenosine.
The proliferative effect we observed is caused by extracellular
adenine nucleotides or adenosine, since blocking the nucleoside
transporter had no effect on the stimulation caused by the
nucleosides or nucleotides. This conclusion is further supported
by the fact that we observed a fully developed proliferative effect
after exposure of the cells to adenosine or ATP for only 10 min
(results not shown).
Our results do not correspond with the known pharmacological
properties of P#-purinoceptors [1,3,36]. These receptors bind
ATP and ADP as the active ligands, whereas AMP and adenosine
are pharmacologically inactive. Evidence against the involvement
of P#-purinoceptors in the observed proliferative effect comes
from the experiment in which different steps of the extracellular
ATP dephosphorylation were blocked. Together with the
ineffectiveness of adenosine 5«-[α,β-methylene]-triphosphate and
2-methylthio-ATP in influencing the proliferation of LLC-MK#
cells, these results rule out the possibility of a major involvement
of P#-purinoceptors and clearly show that adenosine is causing
the proliferation of LLC-MK#cells. Our results are partially in
disagreement with the study of Van Daele et al. [7], who found
evidence for the presence of a P#-purinoceptor, as well as for an
unusual class of adenosine receptors being involved in the
proliferation of aortic endothelial cells. Our present results point
out the possible importance of ecto-nucleotidase enzymes in the
control of cell proliferation mediated by extracellular nucleotides,
as also suggested by Slivinskii et al. [37]. These authors show that
alkaline phosphatase, in addition to 5«-nucleotidase, can be an
important pathway for the extracellular production of adenosine.
Also, it was shown that the presence of ecto-ATPase activity
has a marked effect on the apparent potency of ATP on
P#-purinoceptors, indicating that ecto-ATPase could be a key
element in regulation (termination) of the cellular response to
ATP [38].
It is interesting to note that adenosine itself stimulates 5«-
nucleotidase in rat mesangial cells [39]. In different cells its
activity increases during the exponential growth phase of the
cells in culture and is significantly down-regulated once the cells
reach confluency [40].
We found that all observed effects were due to the nucleosides ;
while the purine nucleosides stimulated, the pyrimidine nucleo-
sides inhibited cell proliferation. Similar to our results, Kartha
and Toback [41] showed that purine nucleotides, but not
pyrimidine nucleotides, stimulate cell proliferation of BSC-1
cells. Furthermore GTP and guanosine have been reported to
cause proliferation of different cell types [42]. In contrast to these
findings, guanosine induces necrosis of cultured aortic endothelial
cells [43]. UTP and uridine inhibit the proliferation of LLC-MK#
cells and of endothelial cells [7]. Uridine also inhibits the growth
of neuroblastoma cells [19]. Furthermore, purine nucleosides and
nucleotides stimulate chick astrocyte proliferation in itro, while
pyrimidine nucleosides and nucleotides are inactive [44].
556 R. Lemmens and others
The lack of a fully additive effect for adenosine and ATP
argues against two separate modes of action for ATP and
adenosine. This is in agreement with the experiments where
inhibition of ATP hydrolysis blocked the proliferative effect of
ATP. The greater response to ATP can be explained by a
stimulatory effect of ATP on the proliferation due to adenosine.
Also, no additive or synergistic effect could be seen with
combinations of two purine nucleosides, suggesting that all
purine nucleosides may act on a common putative purinoceptor.
When purine nucleosides were combined with pyrimidine nucleo-
sides, the inhibitory effect of the pyrimidine nucleosides com-
pletely overruled the stimulating effect of the purine nucleosides.
Diadenosine polyphosphates are involved in the physiological
control of blood pressure [45] and regulation of neutrophil
function [46]. We found that AP%A and AP&A were more potent
than adenosine or ATP in stimulating cell proliferation. AP%A
and AP&A presumably do not penetrate normal, intact cell
membranes, suggesting that the proliferative effect we observed
could be due to direct binding of these compounds to receptors
of the P#dtype [3]. It is also possible that in some other cell types
AP%A and AP&A influence cell proliferation through a dual
effect, binding to P#d-purinoceptors and binding of ATP, ADP,
AMP or adenosine, produced as degradative products of
diadenosine polyphosphates, to other classes of purinoceptors
[10,47,48]. With the analytical method used, we obtained no
evidence that LLC-MK#cells can hydrolyse these diadenosine
phosphates. Therefore they cannot act as precursors for
adenosine. Due to the structural differences between the
diadenosine polyphosphates and adenosine, it seems highly
unlikely that they exert their proliferative effect through binding
to a common receptor. A receptor for diadenosine phosphates
has been identified and biochemically characterized in different
cell types [49,50]. Our results suggest the presence of such a
receptor on LLC-MK#cells, making it likely for AP%A and AP&A
to act as regulators of cell proliferation.
The question remains as to what kind of receptors mediate the
proliferative effect of nucleosides. We found no evidence that the
effect of adenosine is mediated by either A"-orA
#
-receptors, as
was described previously by Kartha and Toback [41] for BSC-1
cells. According to our results, it seems unlikely that cyclic AMP,
cyclic GMP, IP$, protein kinase C or Ca#+mediates the pro-
liferation triggered by extracellular adenosine. It is also
interesting to note that, in monkey kidney epithelial cells, cyclic
AMP does not stimulate proliferation [41]. The conclusion that
the effect of adenosine may be mediated by an unusual class of
low-affinity nucleoside receptor is in good agreement with the
study of Van Daele et al. [7] with endothelial cells. Our
observation that both pertussis and cholera toxins did not alter
the proliferative effects of the various nucleosides or nucleotides,
supports this conclusion and suggests that the signal transduction
is probably not linked to pertussis or cholera toxin-sensitive G-
proteins.
According to our results, it is possible to propose that all
purine nucleosides act on one class of putative receptors.
Characterization of the inhibiting proliferative effects of the
pyrimidine nucleosides suggests that they also might regulate
this function upon binding to a new class of putative
pyrimidinergic receptors. Currently it has been accepted that
nucleosides other than adenosine are ineffective on P"-
purinoceptors, while nucleoside triphosphates other then ATP,
UTP [3] and exceptionally GTP [51] are inactive on P#-
purinoceptors.
Activation of the Na+}H+-exchanger is an early event generally
considered to be necessary for stimulation of protein synthesis
and cell-cycle progression. Our results show that in LLC-MK#
cells the Na+}H+-exchanger might indeed be involved in the
regulation of cell proliferation by nucleosides. Since the in-
volvement of cyclic AMP, cyclic GMP, IP$, protein kinase C or
Ca#+is excluded, the dependence of the effects on a functional
Na+}H+-exchanger supports the possibility that the putative
receptors may belong to the protein tyrosine kinase or a related
receptor group [52]. It was shown that endothelial cells stimulated
by extracellular ATP undergo intracellular alkalinization as a
result of subsequent activation of the Na+}H+-exchanger [53].
Recently it was shown that ATP and UTP trigger the activation
of mitogen-activated protein kinase cascade and that this may be
responsible for the potent mitogenic activity of both nucleotides
[6].
In summary, we have presented evidence that the proliferation
of LLC-MK#cells is influenced by nucleosides formed through
dephosphorylation of extracellular nucleotides by ecto-enzymes,
which can therefore act as regulators of cellular proliferation.
The proliferative effect of nucleosides is of extracellular origin
with purine nucleosides causing stimulation of cell proliferation
and the pyrimidine nucleosides causing inhibition of cell pro-
liferation. The Na+}H+-exchanger plays a role in both pathways
for regulating cell proliferation. These pathways are not com-
pletely independent of each other, since the effect of pyrimidine
nucleosides overrules the effect of purine nucleosides. Further-
more, this control seems not to be mediated by any of the known
P"-orP
#
-purinoceptors.
We wish to thank Dr. L. Kupers and Dr. P. Stinissen for the use of laboratory facilities
and the staff of the Dr. Willems Institute for their co-operation. We also thank Dr. P.
Janssen for his valuable advice with the statistical calculations.
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