(Received for publication, April 3, 1995; and in revised form, May 15, 1995)
From the
Treatment of confluent cultures of JAR human placental
choriocarcinoma cells with staurosporine caused a marked stimulation of
serotonin transport activity in these cells. The stimulatory effect was
noticeable at nanomolar concentrations of staurosporine, and a
treatment time of >4 h was required for staurosporine to elicit the
effect. At 40 nM and with a treatment time of 16 h, the
stimulation of the transport activity was 3.5-6.0-fold. None of
the several other protein kinase inhibitors tested had similar effect
except KT 5720, a protein kinase A inhibitor, which showed a small but
significant (
The Na
Figure 1:
Stimulation of serotonin transporter
activity in JAR cells by staurosporine. A, monolayer cultures
were treated with or without staurosporine (40 nM) for
indicated time periods (1-16 h) at 37 °C, following which
imipramine-sensitive uptake of serotonin was measured at a serotonin
concentration of 50 nM and with an incubation period of 3 min.
Values are means ± S.E. (n = 3). The control
uptake values for all treatment periods were similar (1.82 ±
0.06 pmol/mg of protein/3 min) and this value was taken as 100%. B, monolayer cultures were treated for 16 h at 37 °C with
varying concentrations of staurosporine (0-40 nM),
following which serotonin uptake was measured as described. Values are
means ± S.E. (n = 4). The control uptake (100%)
measured in cells treated in the absence of staurosporine was 1.41
± 0.08 pmol/mg of protein/3 min.
Figure 2:
Specificity of staurosporine effect.
Monolayer cultures were treated with or without staurosporine (40
nM) for 16 h at 37 °C, following which the uptake of
serotonin (the imipramine-sensitive component), taurine, and alanine
was measured. Final concentration of serotonin, taurine, and alanine
was 50, 30, and 5 nM, respectively, and the incubation period
for uptake measurement was 3 min. Values are means ± S.E. (n = 4).
Figure 3:
Influence of staurosporine on the kinetic
parameters of the serotonin transporter. Confluent cultures were
treated with (
Figure 4:
Serotonin uptake in plasma membrane
vesicles prepared from control and staurosporine-treated cells.
Monolayer cultures were treated with or without staurosporine (40
nM) for 16 h at 37 °C. Plasma membrane vesicles were
isolated from control and treated cells, and serotonin uptake was
measured in these membrane vesicles in the presence of an inwardly
directed NaCl gradient and an outwardly directed K
Figure 5:
Binding of RTI-55 to plasma membranes
isolated from the control and staurosporine-treated cells. Confluent
cultures were treated with (
Figure 6:
Influence of staurosporine on steady state
levels of serotonin transporter mRNAs and
This paper reports for the first time that staurosporine is a
potent inducer of the expression of serotonin transporter in the human
placental choriocarcinoma cells. The underlying mechanism appears to be
transcriptional activation of the serotonin transporter gene.
Supporting evidence for this mechanism includes (i) a
substantial lag period before the effect of staurosporine on the
serotonin transporter activity becomes noticeable, (ii) a marked
increase in the steady state levels of the serotonin
transporter-specific mRNAs, and (iii) effective
blockade of the staurosporine effect by actinomycin D, and inhibitor of
transcription. The increase in the steady state levels of the
transporter mRNAs resulting from the transcriptional activation is
accompanied by a parallel increase in the de novo synthesis of
the transporter protein. This is evidenced from (i) the blockade
of the staurosporine effect on the serotonin transport activity by
cycloheximide, an inhibitor of protein synthesis, (ii) the
staurosporine-mediated increase in the maximal velocity of the
transport system, (iii) the staurosporine-mediated increase in
the transporter density in the plasma membrane as seen from the
increase in the binding of RTI-55, a ligand specific for the serotonin
transporter in JAR cells, and (iv) the increased serotonin
transport in plasma membrane vesicles isolated from
staurosporine-treated cells versus control cells. These
effects of staurosporine are very similar to those of cholera toxin.
However, in contrast to cholera toxin treatment, staurosporine
treatment does not increase cAMP levels in JAR cells. Therefore, the
transcriptional activation of the serotonin transporter gene by
staurosporine is mediated by a cAMP-independent signaling pathway.
Interestingly, the effects of staurosporine and cholera toxin are not
additive, indicating that the signaling pathways, though one is
cAMP-dependent and the other is cAMP-independent, converge at some
point prior to the transcriptional activation of the transporter gene.
The physiological significance of such a dual pathway is not known. One
possibility is that it enables the cell to integrate the biological
effects of different external signals efficiently by providing a means
for cross-talk between the two pathways. The organization of the human
serotonin transporter gene has recently been reported(25) . The
gene is composed of 14 exons spanning
The precise nature of the signaling
pathway involved in the transcriptional activation of the serotonin
transporter gene in JAR cells by staurosporine remains to be
elucidated. The effect of staurosporine does not appear to be related
to its ability to block the protein kinases. There is precedence for
stimulation of gene expression by staurosporine which is not mediated
through inhibition of protein kinases. Staurosporine induces
differentiation and neurite outgrowth in PC12
cells(28, 29, 30) . In addition, it
potentiates markedly the ability of epidermal growth factor to cause
differentiation in these cells (31) . The underlying mechanism
for this potentiation has been shown to be the up-regulation of the
receptor for epidermal growth factor, resulting from induction of the
receptor gene expression(31) . Similarly, the receptor for
tumor necrosis factor in myeloid and epithelial cells is also
up-regulated by staurosporine(32) . However, the cellular
signaling pathway involved in staurosporine-mediated stimulation of
gene expression remains unknown.
The physiological functions of the
serotonin transporter expressed in the normal placental
syncytiotrophoblast have not been identified. It has been speculated
that the transporter is involved in the clearance of the vasoactive
monoamine serotonin from the intervillous space and in the
transplacental transfer of serotonin to the developing fetus (33, 34, 35) . These processes may be crucial
to the normal placental function and for optimal development of the
fetus. Therefore, a thorough understanding of the cellular mechanisms
underlying the regulation of the serotonin transporter in human
placenta is important. The choriocarcinoma cells offer an excellent
model system to study this phenomenon. Moreover, it is very likely that
the studies on the regulation of the serotonin transporter in these
cells may become relevant to understand the regulation of the serotonin
transporter in tissues other than the placenta (e.g. serotonergic neurons, endothelial cells, and astrocytes). To our
knowledge, choriocarcinoma cells are the only cultured cell lines of
human origin which possess endogenous serotonin transporter activity
and thus are valuable to studies involving the regulation of the human
serotonin transporter. The role of this transporter in the function of
the serotonergic neuronal pathways is well known. These neuronal
pathways are involved in a variety of physiological functions.
Alterations in the function of these neurons are believed to underlie
several pathological conditions. Therefore, unraveling the various cell
signaling mechanisms which participate in the regulation of the
serotonin transporter has immense clinical relevance.
We thank Sarah Taylor for expert secretarial
assistance.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
1.4-fold) stimulatory effect at a concentration of 5
µM. Blockade of RNA synthesis and protein synthesis in the
cells prevented completely the stimulation of the transport activity
induced by staurosporine. The stimulation was observed not only in
intact cells but also in plasma membrane vesicles prepared from
staurosporine-treated cells. The stimulation was accompanied by a
5-7-fold increase in the steady state levels of the
transporter-specific mRNAs, by a 7-fold increase in the maximal
velocity of the transport process, and by a 6-fold increase in the
transporter density in the plasma membrane. Even though both
staurosporine and cholera toxin had similar effects on the serotonin
transport activity in these cells, the effect was not additive when the
cells were treated with both reagents together. While treatment of the
cells with cholera toxin markedly elevated intracellular levels of
cAMP, staurosporine did not have any effect on the cellular levels of
this cyclic nucleotide. It is concluded that staurosporine up-regulates
the serotonin transport activity in JAR cells by increasing the steady
state levels of the serotonin transporter mRNA and by the consequent
increase in the transporter density in the plasma membrane and that the
process involves a cAMP-independent signaling pathway.
- and Cl
-coupled
serotonin transporter that is expressed in the serotonergic neurons,
platelets, and placenta is a target for cocaine and antidepressants.
Molecular cloning studies have demonstrated that a single gene codes
for the transporter in neuronal and non-neuronal
cells(1, 2, 3) . Human placental
choriocarcinoma cells express a serotonin transporter which is
identical to the transporter present in normal tissues(4) .
These cells have proved to be very useful in studies involving the
regulation of the human serotonin
transporter(4, 5, 6) . In these cells, the
serotonin transporter is up-regulated by cAMP(4) , and the
regulation involves transcriptional activation(5) .
Calmodulin-dependent processes also participate in the regulation of
the transporter, presumably involving phosphorylation/dephosphorylation
of the transporter protein(6) . There is evidence in other cell
types for regulation of the serotonin transporter by additional
cellular signaling pathways such as Ca
(7) ,
protein kinase C(8, 9) ,
cGMP(10, 11) , and oxidants(12) . However,
these signaling pathways modulate the serotonin transporter activity
rapidly and the underlying mechanism apparently involves covalent
modification of the transporter protein without altering the
transporter density. Therefore, cAMP is the only cellular signal thus
far known which has been shown to regulate the serotonin transporter by
transcriptional activation. The influence of cAMP on the serotonin
transporter is not selective for the choriocarcinoma cells since a
similar role for cAMP in the regulation of the serotonin transporter
has been demonstrated in the brain(13) . The present study
identifies a second cellular signaling pathway involved in the
transcriptional activation of the serotonin transporter in
choriocarcinoma cells, and this pathway is mediated by staurosporine.
Materials
The JAR and BeWo human placental
choriocarcinoma cell lines were purchased from the American Type
Culture Collection. Culture media (RPMI 1640 and F-12), penicillin,
streptomycin, and trypsin were obtained from Life Technologies, Inc.
Fetal bovine serum, cholera toxin, iproniazid, imipramine, serotonin,
staurosporine, prostaglandin E, apotransferrin,
hydrocortisone, thyroxine, phorbol esters, cycloheximide, and
actinomycin D were from Sigma. Human recombinant insulin was from Novo
Nordisk Pharmaceuticals, Inc. (Princeton, NJ). Protein kinase
inhibitors were from Research Biochemicals, Inc. (Natick, MA) and from
LC Laboratories (Woburn, MA). Paroxetine was obtained from Beecham
Pharmaceuticals (Betchworth, Surrey, United Kingdom).
5-[1,2-
H]Hydroxytryptamine (serotonin) binoxalate
(specific radioactivity, 25.4 Ci/mmol), L-[3-
H]alanine (specific radioactivity,
76.9 Ci/mmol), and [
I]RTI-55
(
)(specific radioactivity, 2200 Ci/mmol) were
purchased from DuPont NEN. [2-
H]Taurine (specific
radioactivity, 30.0 Ci/mmol) was obtained from American Radiolabeled
Chemicals, Inc. (St Louis, MO).
Culture of JAR and BeWo Cells
The cells were
cultured with either RPMI 1640 medium (JAR) or F-12 nutrient mixture
(BeWo) as described previously(14) . The media were
supplemented with 10% fetal bovine serum, penicillin (100 units/ml),
and streptomycin (100 µg/ml). Trypsin-released cells were seeded in
35-mm Petri dishes at a density of 1.5 10
cells/dish and allowed to grow as a monolayer. Twenty-four h
after subculturing, the medium was replaced with a hormonally defined
medium which did not contain fetal bovine serum. The defined medium
consisted of either RPMI 1640 or F-12 nutrient mixture, supplemented
with insulin (5 µg/ml), apotransferrin (5 µg/ml), prostaglandin
E
(2.5
10
mg/ml), hydrocortisone
(5
10
M), and thyroxine (5
10
M). Treatment with different reagents
was carried out for indicated time periods in the defined media prior
to uptake measurements.
Uptake Measurements in Cells
The dishes containing
monolayer cultures of the cells were taken out of the incubator and let
stand at room temperature for 2 h. The culture medium was then
aspirated, and the cells were washed once with the uptake buffer. One
ml of uptake buffer containing radiolabeled substrate (serotonin,
alanine, or taurine) was added to the cells and incubated for 3 min.
Uptake was terminated by aspirating the buffer and subsequently washing
the cells three times with fresh uptake buffer. The cells were lysed
with 1 ml of 0.2 N NaOH, 1% SDS, and the lysate was
transferred to scintillation vials for quantitation of radioactivity.
The composition of the uptake buffer was 25 mM HEPES-Tris, pH
7.5, 140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl, 0.8 mM MgSO
, 5 mM
glucose, and 0.1 mM iproniazid, an inhibitor of monoamine
oxidases. Serotonin uptake that occurred independent of the serotonin
transporter (i.e. diffusion) was determined by measuring the
uptake in the presence of imipramine (0.1 mM), and this
component was always <10% of total uptake measured in the absence of
imipramine.
Determination of cAMP and cGMP
JAR cells were
cultured in 35-mm dishes for 24 h in RPMI 1640 medium supplemented with
10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml
streptomycin. At the end of 24 h, the medium was removed and replaced
with a hormonally defined medium. When present, staurosporine and
cholera toxin were added to this medium at a concentration of 40
nM and 100 ng/ml, respectively. The incubation was continued
for 16 h. In cases where the treatment of the cells with staurosporine
or cholera toxin was only for 2 h, the cells were incubated with the
defined medium for 14 h, following which staurosporine or cholera toxin
was added, and the incubation was continued for 2 h. At the end of the
incubation, cell cultures were washed with ice-cold buffer and lysed in
1 ml of 0.1 N HCl. After 30 min, the acid medium was collected
and stored frozen until use. Measurement of cAMP and cGMP in these
samples was done by radioimmunoassay as described previously (15) .
Preparation of Plasma Membrane Vesicles from Cultured JAR
Cells
The cells were cultured in 225-cm flasks for
24 h in RPMI 1640 medium, supplemented with penicillin (100 units/ml),
streptomycin (100 µg/ml), and fetal bovine serum (10%), after which
the medium was replaced with the hormonally defined medium containing
either dimethyl sulfoxide (control) or staurosporine (40 nM)
in dimethyl sulfoxide. The cells were then incubated for 16 h,
following which plasma membrane vesicles were prepared from these cells
as described previously(6) . Membrane vesicles were suspended
in 10 mM HEPES-Tris, pH 7.5, containing 75 mM potassium gluconate, 130 mM mannitol, 2 mM EGTA,
and 10 mM KF for serotonin uptake measurements and 10 mM Tris-HCl, pH 7.5, containing 230 mM mannitol, 1
mM MgSO
, 2 mM EGTA, and 10 mM KF
for RTI-55 binding measurements. Protein concentration of the final
membrane suspension was determined and adjusted to 1 mg/ml (RTI-55
binding) or 2.5 mg/ml (serotonin uptake).
Uptake Measurements in Membrane Vesicles
Uptake of
serotonin in JAR cell plasma membrane vesicles was measured by the
rapid filtration method as described earlier(16, 17) .
The uptake medium was 150 mM NaCl buffered with 10 mM HEPES-Tris, pH 7.5. Uptake was measured with a 15-s incubation and
at a serotonin concentration of 0.2 µM. Serotonin uptake
that occurred by diffusion was determined by measuring the uptake in
the presence of 0.1 mM imipramine. This value was subtracted
from total uptake to determine the transporter-mediated uptake.
RTI-55 Binding
Equilibrium binding of
[I]RTI-55 to the JAR cell plasma membranes was
assayed (6) by incubating the membranes (50 µg of membrane
protein) with different concentrations of the ligand (0.025-1
nM) at room temperature for 1 h in a total volume of 0.5 ml.
The binding buffer was 10 mM Tris-HCl, pH 9.5, containing 300
mM NaCl and 1 mM MgSO
. The binding was
terminated by addition of 3 ml of ice-cold stop buffer (10 mM Tris-HCl, 200 mM NaCl, pH 7.5) followed by filtration of
the mixture on a GF/F glass fiber filter (0.7-µm pore size) which
had been presoaked in 0.3% polyethylenimine. The filter was washed with
3
5 ml of the ice-cold stop buffer, and the radioactivity
associated with the filter was determined using a gamma counter.
Nonspecific binding was determined in the presence of 5 µM paroxetine.
Isolation of Poly(A)
Poly(A) RNA and Northern
Analysis
RNA was isolated from JAR
cells using FastTrack mRNA isolation kit (Invitrogen). Northern
hybridization was carried out as described earlier (5, 18) under high stringency conditions using
P-labeled human serotonin transporter cDNA as the probe.
The same membrane blot was used for probing with
P-labeled
human
-actin cDNA as an internal control for RNA loading and
transfer efficiency. This was done by stripping of the blot followed by
rehybridization with the
-actin cDNA probe.
Influence of Phorbol-12-myristate-13-acetate (PMA) and
Staurosporine on Serotonin Uptake in JAR Cells
This
investigation was initially started to determine whether the serotonin
transporter expressed in JAR cells is regulated by protein kinase C as
it has been shown for the serotonin transporter present in platelets (9) and in endothelial cells(8) . Treatment of JAR
cells for 2 h with 1 µM PMA, an activator of protein
kinase C, was found to cause a significant inhibition of the serotonin
transporter activity, measured as the imipramine-sensitive serotonin
uptake (Table 1). The inhibition was small (15%), but the
effect was consistent in three different experiments. Treatment of the
cells with staurosporine, a nonspecific but very potent inhibitor of
protein kinases, for 2 h did not have any significant effect on the
transporter activity. However, when treatment of the cells with PMA was
done in the presence of staurosporine, PMA failed to inhibit the
transporter. These experiments indicated that the serotonin transporter
expressed in JAR cells is inhibited by protein kinase C, even though
the effect is much smaller than found in platelets (40%; (9) )
and in endothelial cells (30%, (8) ).
We then investigated
the long term regulation of the serotonin transporter in JAR cells by
PMA. Treatment of the cells with 1 µM PMA for 16 h caused
a significant increase in the transporter activity (Table 1).
This stimulation could be either due to the PMA-induced down-regulation
of protein kinase C (which could be expected to relieve the transporter
from the inhibition caused by protein kinase C that may be
constitutively active to a significant extent even in the absence of
PMA) or due to protein kinase C-dependent cellular effects that are
unique to the long term treatment with PMA. We studied the influence of
staurosporine on the long term stimulatory effect of PMA (Table 1). These experiments led to a surprising and unexpected
finding that treatment of the cells with 40 nM staurosporine
alone for 16 h caused a 5-fold increase in the transporter activity.
The magnitude of increase was not altered by PMA. The stimulatory
effect of staurosporine does not appear to be due to the inhibition of
protein kinase C. If this were to be the case, one would have expected
staurosporine to have either no effect or an inhibitory effect on the
transporter activity, based on the long term effect of PMA. Moreover,
the stimulation of the transporter activity by staurosporine requires
long term treatment because such an effect was not noticed when the
cells were treated with staurosporine only for 2 h. This interesting
observation prompted us to investigate in more detail the long term
regulatory effect of staurosporine on the serotonin transporter.
Stimulation of Serotonin Uptake by Staurosporine in JAR
and BeWo Cells
Imipramine-sensitive serotonin uptake in JAR
cells was measured after the cells were treated with 40 nM
staurosporine for various time periods (1-16 h) at 37 °C (Fig. 1A). There was no noticeable change in the uptake
up to 4 h of treatment. With longer periods of treatment, there was
marked stimulation of serotonin uptake, 30% at 6 h, 90% at 8 h, and
265% at 16 h. Dose-response studies carried out using a 16-h treatment
time revealed that staurosporine is a very potent stimulator of
serotonin uptake (Fig. 1B). A significant stimulation
was noticeable at a staurosporine concentration as low as 2.5
nM. The magnitude of stimulation increased as the
concentration of staurosporine increased. The uptake in cells treated
with 40 nM staurosporine was 3.4-fold compared to the uptake
in control cells. Similar results were obtained with BeWo cells,
another human choriocarcinoma cell line (data not shown). Staurosporine
at concentrations greater than 40 nM was found to affect cell
viability to a significant extent.
Specificity of Staurosporine Effect
This was
assessed by measuring the uptake of taurine and alanine in control and
staurosporine (40 nM, 16 h)-treated JAR cells and by comparing
the results with serotonin uptake (Fig. 2). These experiments
showed that under the conditions in which the uptake of serotonin was
stimulated 3.6-fold by staurosporine, the uptake of taurine and the
uptake of alanine remained unaffected.
Influence of Staurosporine on Kinetic Parameters of the
Serotonin Transporter
The serotonin transporter activity in
control and in staurosporine (40 nM, 16 h)-treated JAR cells
was kinetically analyzed to determine the influence of staurosporine on
the kinetic parameters, Michaelis-Menten constant (K) and maximal velocity (V
) of the transport system. The results, given
in Fig. 3as Eadie-Hofstee plots, show that the
staurosporine-induced stimulation of serotonin uptake is accompanied by
changes in K
as well as in V
. Treatment of the cells with staurosporine (40
nM, 16 h) increased the K
value
from 0.31 ± 0.01 to 0.60 ± 0.03 µM. The V
was also markedly affected by staurosporine.
The V
in control cells was 9.1 ± 0.2
pmol/3 min/mg of protein which increased to 66.6 ± 2.4 pmol/3
min/mg of protein in staurosporine-treated cells.
) or without (
) staurosporine (40 nM)
for 16 h at 37 °C. Imipramine-sensitive serotonin uptake was
measured in these cells over a serotonin concentration range of
0.05-1 µM and with an incubation period of 3 min.
Results (means ± S.E., n = 4) are given as
Eadie-Hofstee plots (i.e.v/sversusv). v, serotonin uptake rate in pmol/mg of
protein/3 min; s, serotonin concentration in µM.
Correlation coefficient (r
) for linear plots was
>0.99.
Influence of Cycloheximide and Actinomycin D on the
Staurosporine-induced Stimulation of Serotonin Uptake
The lag
period observed in the stimulation of serotonin uptake by staurosporine (Fig. 1A) indicated that de novo synthesis of
the serotonin transporter protein might be involved in the process.
Therefore, we assessed the influence of cycloheximide, an inhibitor of
protein synthesis, and actinomycin D, an inhibitor of RNA synthesis, on
the staurosporine-induced stimulation of serotonin uptake in JAR cells (Table 2). For this purpose, the cells were treated with or
without staurosporine (20 nM) in the absence or in the
presence of cycloheximide (10 µM) or actinomycin D (0.02
µg/ml) for 16 h, following which the activity of the serotonin
transporter was measured by determining the imipramine-sensitive
serotonin uptake. Cycloheximide and actinomycin D have been shown under
these conditions to block the de novo synthesis of protein and
RNA, respectively(5, 19) . The results of the
experiments on serotonin uptake revealed that the stimulatory effect of
staurosporine was completely abolished by cycloheximide and actinomycin
D, strongly suggesting that transcriptional activation might underlie
the observed stimulatory effect of staurosporine.
Effects of Protein Kinase Inhibitors on Serotonin Uptake
in JAR Cells
Staurosporine is one of the most potent inhibitors
of protein kinases, but is rather non-selective. It inhibits protein
kinase C as well as cAMP- and cGMP-dependent protein kinases (protein
kinase A and protein kinase G). In addition, tyrosine kinases and
Ca/calmodulin-dependent kinases are also inhibited by
staurosporine. We screened a variety of protein kinase inhibitors for
their ability to stimulate serotonin uptake in JAR cells in order to
find out whether inhibition of any of these protein kinases underlies
the staurosporine-induced stimulation of serotonin uptake (Table 3). The results of the experiment showed that
staurosporine is the only protein kinase inhibitor with the ability to
enhance serotonin uptake. KT 5720, a selective inhibitor of PKA, showed
a small but significant ability to stimulate the uptake. However, H-9,
HA-1004, and K-252b which are expected to inhibit PKA at concentrations
used in the experiment failed to show any effect. Similarly,
chelerythrine which is a selective inhibitor of protein kinase C also
had no effect on the uptake. H-89, an inhibitor of PKA and PKG, did not
stimulate but instead markedly inhibited the uptake. Genistein and
tyrphostin, inhibitors of tyrosine kinases, had no effect. CGS 9343B, a
selective calmodulin antagonist, inhibited the uptake. The inability of
any of these protein kinase inhibitors to simulate the staurosporine
effect strongly suggests that the inhibition of protein kinases by
staurosporine may not be related to the stimulation of serotonin uptake
induced by staurosporine.
Serotonin Uptake in Plasma Membrane Vesicles Prepared
from Control and Staurosporine-treated JAR Cells
The experiments
with cycloheximide and actinomycin D indicated that the stimulatory
effect of staurosporine might involve de novo synthesis of the
serotonin transporter, thus increasing the transporter density in the
plasma membrane. An additional supporting evidence for this mechanism
would be to demonstrate the staurosporine-induced increase in serotonin
uptake in plasma membrane vesicles isolated from these cells. For this
purpose, we prepared plasma membrane vesicles from control and
staurosporine (40 nM, 16 h)-treated JAR cells and measured in
these vesicles imipramine-sensitive serotonin uptake in the presence of
appropriate ionic gradients (inwardly directed Na and
C1
gradients and outwardly directed K
gradient). The results are given in Fig. 4. The value for
imipramine-sensitive serotonin uptake at 0.2 µM concentration was 0.97 ± 0.06 pmol/15 s/mg of protein in
plasma membrane vesicles prepared from control cells. The corresponding
value in vesicles prepared from staurosporine-treated cells was 8.13
± 0.15 pmol/15 s/mg of protein, a 7-fold increase compared to
the control value. Thus, the stimulatory effect of staurosporine on
serotonin uptake could be demonstrated at the plasma membrane level.
gradient. Concentrations of serotonin during uptake measurement
was 0.2 µM, membrane protein used for each assay was 100
µg, and the incubation period for uptake measurement was 15 s.
Uptake was measured in the presence as well as in the absence of
imipramine (0.1 mM). Values are means ± S.E. (n = 6).
Binding of [
We estimated the serotonin transporter density in the
plasma membranes derived from control and staurosporine-treated cells
by analyzing the binding of [I]RTI-55 to Plasma
Membranes Isolated from Control and Staurosporine-treated JAR
Cells
I]RTI-55 to the
membranes. RTI-55 is a cocaine analog which is a high affinity ligand
to the serotonin transporter and also to the dopamine and
norepinephrine
transporters(20, 21, 22, 23) .
However, since the JAR cells do not express the dopamine and
norepinephrine transporters(18, 24) , RTI-55 can be
considered as a selective ligand for the serotonin transporter in these
cells. We determined the kinetic parameters for RTI-55 binding in JAR
cell plasma membranes by measuring equilibrium binding over a
concentration range of 0.025-1 nM. The results given in Fig. 5as Scatchard plots revealed that the apparent dissociation
constant (K
) was 0.30 ± 0.01
nM and the maximal binding capacity (B
)
was 195 ± 3 fmol/mg of protein in control membranes. The
corresponding values for the binding in membranes from
staurosporine-treated cells were 0.29 ± 0.01 nM and
1127 ± 21 fmol/mg of protein. These data show that treatment of
the cells with staurosporine results in an increase (approximately
6-fold) in the B
for RTI-55 binding, without
altering the K
.
) or without (
) staurosporine
(40 nM) for 16 h at 37 °C. Plasma membranes were isolated
from control and treated cells and used for measurement of
[
I]RTI-55 binding as described under
``Experimental Procedures.'' Concentration range for RTI-55
was 0.025-1.0 nM. Nonspecific binding was measured in
the presence of 5 µM paroxetine, and this component
constituted <5% of total binding. Results (means ± S.E., n = 3) are given as Scatchard plots (i.e.BversusB/F). B, RTI-55 bound
in fmol/mg of protein; F, concentration of free RTI-55 in
nM. Correlation coefficient (r
) for the
linear plots was >0.99.
Influence of Staurosporine on Steady State Levels of the
Serotonin Transporter mRNAs
Since co-treatment with actinomycin
D completely blocks the stimulatory effect of staurosporine on
serotonin uptake, it was inferred that staurosporine most likely causes
transcriptional activation of the serotonin transporter gene.
Therefore, poly(A) RNA was isolated from control and
staurosporine (40 nM, 16 h)-treated JAR cells and analyzed by
Northern hybridization using the human serotonin transporter cDNA as
the probe(1) . The same membrane blot was reprobed with the
-actin cDNA probe as an internal control for RNA loading and
transfer efficiency. JAR cells as well as normal placenta express three
different serotonin transporter mRNA transcripts, 6.8, 4.9, and 3.0
kilobases in size. Treatment of the JAR cells with staurosporine
increased the steady state levels of all three transcripts (Fig. 6) Taking into consideration the small difference in the
levels of the
-actin mRNA between the two lanes containing the RNA
samples from control and staurosporine-treated cells, the increase in
the 6.8-, 4.9-, and 3.0-kilobase serotonin transporter mRNA transcripts
was 4.5-, 7.1-, and 6.5-fold, respectively.
-actin mRNA. Confluent
cultures were treated with (lane B) or without (lane
A) staurosporine (40 nM) for 16 h at 37 °C.
Poly(A)
RNA was isolated from control and treated
cells. The size-fractionated mRNA was probed with
P-labeled human placental serotonin transporter cDNA and
with
P-labeled human
-actin cDNA by sequential
hybridization. Results are from a representative experiment. Comparable
results were obtained in three different
experiments.
Relationship between Staurosporine and Cholera Toxin in
Inducing the Serotonin Transporter Activity in JAR Cells
We have
shown previously that treatment of JAR cells with cholera toxin
enhances the serotonin transporter activity (4) and that this
effect is accompanied by increased serotonin transporter mRNA levels
and also by increased binding of RTI-55 to the membranes(5) .
Since the increase in serotonin transporter activity induced by
staurosporine also showed a similar phenomenon by being accompanied by
increases in mRNA levels and RTI-55 binding, it was of interest to
determine whether or not cholera toxin and staurosporine can act
synergistically. The results given in Table 4show that when the
cells were treated independently with either cholera toxin (100 ng/ml)
or staurosporine (40 nM) for 16 h, the increase in
imipramine-sensitive serotonin uptake was 3.3- and 6.0-fold,
respectively. However, when the cells were treated with cholera toxin
and staurosporine together, the increase in the uptake was 6.6-fold.
This value was almost the same as the value obtained with staurosporine
alone, indicating that the effect of cholera toxin and staurosporine
were not additive.
Influence of Staurosporine on Intracellular Levels of
cAMP and cGMP
Cholera toxin treatment leads to elevation of
intracellular levels of cAMP in JAR cells, and this effect is
accompanied by an increase in the activity of the serotonin
transporter(4) . Since the influence of cholera toxin and the
influence of staurosporine on serotonin uptake are not additive, it was
important to determine whether treatment of the cells with
staurosporine causes elevation of intracellular cAMP levels as
treatment with cholera toxin does. Therefore, we measured cAMP levels
in control cells and in cells treated with either staurosporine or
cholera toxin (Table 5). It was found that staurosporine did not
alter the cellular content of cAMP, whether the treatment time was 2 or
16 h. In contrast, treatment with cholera toxin for 2 h increased the
cAMP levels 22-fold. Even after treatment with cholera toxin for 16 h,
the cAMP levels remained elevated (8-fold) compared with control
cells. The cellular content of cGMP was not affected by staurosporine
and by cholera toxin.
31 kilobases, and the
promotor region contains cAMP response element which is the binding
site for the cAMP-dependent transcription factor,
CREB(26, 27) .
-carbomethoxy-3
-(4-iodophenyl)tropane; PMA,
4
-phorbol-12-myristate-13-acetate.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.