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0021-972x/93/7703-0670$03.00/0
Journal of Clinical Endocrinology and Metabolism
Copyright Q 1993 by The Endocrine Society Vol. 77, No. 3
Printed in U.S.A.
A Role for Extracellular Calcium in the Regulation of
Placental Lactogen Release by Angiotensin-II and
Dopamine in Human Term Trophoblastic Cells*
ALAIN PETIT, NICOLE GALLO-PAYET, CATHY VAILLANCOURT,
DIEGO BELLABARBA, JEAN-GUY LEHOUX,
AND
SERGE Bl?LISLE
Department
of
Obstetrics and Gynecology, Faculty
of
Medicine, University
of
Montreal (A.P., C. V., S.B.),
Montreal, Quebec, Canada H3C 3J7; and the Faculty
of
Medicine, University
of
Sherbroohe (N.G.-P., D.B.,
J.-G. L.1, Sherbrooke, Quebec, Canada Jl H 5N4
ABSTRACT
We previously reported that angiotensin-II (AII) stimulated and
dopamine (DA) inhibited the release of human placental lactogen (hPL)
from trophoblastic cells. The mechanisms of action involved in these
endocrine regulations are poorly known. In this study, we investigated
the role of Ca*+ as a potential cellular mediator of the effects of AI1
and DA. Incubation of freshly isolated human term trophoblastic cells
with DA led to a dose-dependent inhibition of &Ca’+ influx, with a
maximum of 55 + 5% and an EC& of 10 f 3 pmol/L. This DA-inhibited
Ca’+ influx was reversed by spiperone, a Dz-dopamine receptor antag-
onist. Preincubation of cells with pertussis toxin completely blocked
the inhibitory effect of DA on placental *Ca*+ influx. Nifedipine (lo-’
mol/L), like DA, inhibited*Ca*+ influx (41 f 3% inhibition). Moreover,
nifedipine decreased hPL release (57 f 10%; E&,, 0.25 f 0.09 pmol/
L). Coincubation of DA and nifedipine did not enhance the inhibitory
effects of these agents on either “Ca*’ influx or hPL release. The
incubation of trophoblastic cells with [Sar’]AII, a potent agonist of
AII, led to a dose-dependent stimulation of ‘Y!a*+ influx. The maximal
stimulation was 221 f 37% of the control value, with an EC, of 50 *
15 nmol/L. This stimulation was inhibited by coincubation with the
AI1 antagonist [Sar’,Ala’]AII. [Sar’]AII-stimulated Ca” influx was
blocked by preincubation with pertussis toxin. Bay K 8644 also stim-
ulated &Ca” influx (238 f 41% of the control). Moreover, Bay K 8644
stimulated hPL release. The maximal stimulation was 180 + 22% of
the control value, with an EC& of 0.40 f 0.30 pmol/L. Coincubation of
Bay K 8644 and AI1 did not led to additional stimulation of either
“Ca*+ influx or hPL release. These results suggest that Ca*+ influx is
one mechanism that mediates AI1 and DA regulation of hPL release in
human term trophoblastic cells. (J Clin Endocrinol Metab 77: 670-
676, 1993)
T
HE HUMAN placenta synthesizes and secretes peptide
hormones structurally and biologically similar to those
secreted by the pituitary gland (1). However, mechanisms
regulating their production are poorly understood. We re-
cently described a stimulatory effect for angiotensin-II (AII)
on human placental lactogen (hPL) release through specific
receptors coupled to phosphatidylinositol breakdown (2, 3)
and an inhibitory effect for dopamine (DA) through D2
receptors (4, 5). However, the mechanism of action of DA is
still unknown. The DA and AI1 receptors are members of the
G-protein-coupled receptor superfamily (for review, see Ref.
6). The role of G-proteins in the coupling between receptors
and ionic channels is well known (7, 8). In the pituitary, DA
inhibits PRL release and calcium influx (9, lo), probably
through two types of voltage-dependent Ca*+ currents (11).
The effects of DA on these Ca2+ currents are mediated by at
least one G-protein (12). Recent experiments using pertussis
toxin (PTX) (4) suggest that similar G-protein(s) may also
mediate hPL release by the human placenta. However, the
role of Ca” in hPL release has yielded conflicting results;
inhibitory (13- 15) and stimulatory (16-l 8) effects of calcium
Received December 3, 1992. Accepted April 9, 1993.
Address requests for reprints to: Dr. Serge B&lisle, Centre de Re-
cherche, HBpital Ste-Justine, 3175 Cbte Ste-Catherine, Montreal, Que-
bec, Canada H3T lC5.
‘This work was supported by a grant from the Medical Research
Council of Canada.
and/or ionophores were reported. Using a specific voltage-
dependent Ca2+ channel agonist and antagonist, we dem-
onstrate in this study that the effects of both AI1 and DA on
hPL release may be mediated by Ca*+ influx.
Materials and Methods
Hormones and chemicals
The AI1 analogs [Sar’]AII and [Sar’,Ala’]AII were purchased from
Peninsula Laboratories (Belmont, CA). DA, spiperone (8-[4-(4-fluoro-
phenyl)4-oxobutyl]l-phenyl-l,3,8-triazaspiro-[4,5]decan-4-one), nifedi-
pine, trypsin (type III), DNase-1 (type IV), bacitracin, phenylmethylsul-
fonylfluoride, and BSA were obtained from Sigma (St. Louis, MO). Fetal
bovine serum (FBS), penicillin (10,000 U/mL)-steptomycin (10,000 mg/
mL), Minimum Essential Medium with Earle’s salts, and Ham’s F-10
medium were purchased from Gibco (Burlington, Ontario, Canada).
Percoll and hPL RIA kits were obtained from Pharmacia (Dorval, Que-
bec, Canada). Bay K 8644 was purchased from Research Biochemicals,
Inc. (Natick, MA). Calcium-45 (“Ca’+) and PTX were obtained from
ICN (Mississauga, Ontario, Canada). All other chemicals were purchased
from Fisher Scientific (Montreal, Quebec, Canada).
Isolation
of
human trophoblustic cell
Human placentas freshly obtained from women who delivered nor-
mal infants at term (38-41 weeks gestation) were brought to the labo-
ratory immersed in cold 150 rnmol/L NaCl and processed within 30
min, as previously described (2). The placental tissue was separated
from the amnion and chorion, minced, washed with cold 150 mmol/L
NaCl, and digested for 8-10 periods (10 min each) at 37 C with
CALCIUM AND hPL RELEASE 671
Minimum Essential Medium supplemented with 0.25% trypsin, 150 U/
mL DNase-1, 200 U/mL penicillin, and 200 pg/mL streptomycin. The
supematant of each digestion was collected, filtered, pooled, and washed
using the same medium without enzyme. Erythrocytes were removed
by further centrifugation at 800 X g over a 60% Percoll barrier. Cell
viability, estimated before and after experiments by trypan blue exclu-
sion, was greater than 90%. Cells were used after a l-h resting period.
Measurement of 46Ca2+ influx
The rate of “Ca’+ influx was measured according to the method of
Mauger et al. (19). Briefly, the incubation (at 37 C) was started by adding
510 @L cell suspension [l-2 X lo6 cells in KRP (116 mmol/L NaCI, 3.5
mmol/L KCI, 1.8 mmol/L CaCh, 0.8 mmol/L MgSOr, and 1 mmol/L
NaH2P04, pH 7.4) supplemented with 5.55 mmol/L glucose, 2 mg/mL
BSA, 2.5 mmol/L NaHCOs)] to a tube containing 2.5 &i “Ca*+ and
stimuli (total volume, 600 pL). “Ca’+ uptake was determined by taking
IOO-pL samples at 15,30,45,60, and 75 s. Each sample was immediately
filtered through a Whatman GF/C glass fiber filter (Whatman, Clif-
ton, NJ) and washed four times with ice-cold washing buffer (144 mmol/
L NaCl, 5 mmol/L CaCl2, and 4 mmol/L Tris-HCl, pH 7.4). The rate of
Ca2* influx was calculated from the slope of regression lines obtained
during “Ca’+ uptake measurement.
PTX treatment
Freshly isolated trophoblastic cells were suspended (lo6 cells/ml) in
Ham’s F-10 medium supplemented with 10% FBS, 200 U/mL penicillin,
200 rg/mL streptomycin, and incubated 16-20 h at room temperature
without (control) or with 1 pg/mL PTX. The cells were centrifuged at
150 X g for 8 mm and washed by further centrifugation in KRP (pH 7.4)
supplemented with 5.55 mmol/L glucose, 2 mg/mL BSA, and 2.5 mmol/
L NaHC03.
Hormone assays and statistics
Placental hPL release was measured in the medium, as previously
described (2). Cells (lo6 cells/ml) were incubated for 2 h in triplicate at
37 C in PBS
(DH
7.4) SuDDJemented with 10 mmol/L Ma&. 1 mn/mL
BSA, 1 mg/mL ba&ra&, and 5.55 mmol/L glucose. ?he-cells were
centrifuged for 5 min at 150 x g, and the medium was collected and
kept frozen at -20 C until assayed. Medium hPL concentrations were
measured in duplicate by RIA using kits obtained from Pharmacia. The
cross-reactivity of the assay was l&s than 0.5% with human GH and
less than 0.06% with PRL. The results have been corrected for hPL
content at time zero (550 + 120 ng/106 cells; eight placentas) and were
analyzed by analysis of variance followed by the Fisher’s PLSD com-
parison test.
P
< 0.05 was considered significant.
Results
Effects of DA and AZZ on 45Ca2’ influx
Figures 1 and 2 demonstrate the regulation of 45Ca2’ influx
by DA and AI1 in human term trophoblastic cells. Unstimu-
lated trophoblastic cells showed a linear time-dependent
accumulation of 45Ca2+ for at least 75 s. This accumulation
of 45Ca2+ was inhibited in a dose-dependent manner by DA
(Fig. 1, A and B). The maximal inhibition reached 45 + 5%
of the control value (control, 10.6 + 1.2 dpm/s), with an
ECso of 10 + 3 pmol/L. This inhibition was completely
reversed by coincubation with spiperone, a specific DZ recep-
tor antagonist (Fig. 1C). Contrary to DA, AI1 specifically
stimulated 45Ca2+ influx in trophoblastic cells. The incuba-
tions were performed with the potent and stable AI1 agonist
[Sar’]AII, and the maximal stimulation was 221 f 37% of
the basal value with an ECso of 50 f 15 nmol/L (Fig. 2A).
This stimulation was completely reversed by coincubation
with the AI1 antagonist [Sar’,Ala’]AII (Fig. 2B). DA also
inhibited AII-stimulated 45Ca2+ influx to 91.8% of the control
value (not shown).
Effects of dihydropyridines (DHPs) on hPL release and 45Ca2’
influx
Figure 3 shows the effects of DHPs on hPL release in
human term trophoblastic cells. hPL release was stimulated
1.80 + 0.22-fold by Bay K 8644, with an ECso of 0.40 f 0.30
rmol/L, whereas nifedipine inhibited hPL release by 57 +
lo%, with an ECso of 0.25 + 0.09 pmol/L. The maximal
effects were obtained with 10m5 mol/L Bay K 8644 and lo-’
mol/L nifedipine without any effect on cell viability, as
demonstrated by trypan blue exclusion (results not showed).
Therefore, these concentrations of DHPs were used to char-
acterize the effect of extracellular Ca*+ on hPL release. Figure
4A shows that Bay K 8644 and AI1 stimulated 45Ca2’ influx
to a maximum of 238 + 41% and 221 f 37% over the control
value, respectively. Coincubation of Bay K 8644 and AI1 did
not result in a greater stimulation of 45Ca2’ influx (256 f
12% of the control value). Nifedipine alone inhibited Ca2+
influx by 43 + lo%, whereas DA inhibition attained 45 +
5%. Nifedipine significantly inhibited AII-stimulated 45Ca2+
influx; the influx decreased to 157 + 21% of the control
value. Coincubation of DA and nifedipine did not lead to
further inhibition of basal Ca2+ influx (50 + 11% inhibition).
DA significantly inhibited Bay K 8644-stimulated 45Ca2’ in-
flux to 106 + 10% of the control value. Figure 4B summarizes
the effects of these agents on hPL release. Thus, AI1 (180 +_
8% of the control value) and Bay K 8644 (160 + 15% of the
control value) stimulated hPL release, whereas DA (45 + 5%
of the control value) and nifedipine (42 f 14% of the control
value) inhibited this release. Coincubation of AI1 and Bay K
8644 did not further enhance hPL release (165 + 10% of the
control value), whereas nifedipine significantly inhibited AII-
stimulated hPL release (107 f 10% of the control value).
Coincubation of DA and nifedipine did not induce a greater
inhibition of hPL release (46 f 14% inhibition) compared to
DA or nifedipine alone. Yet, DA significantly inhibited Bay
K 8644-stimulated hPL release to 113 f 17% of the control
value.
Effects of PTX on Ya2+ influx
Figure 5 shows that pretreatment of human placental cells
with PTX completely blocked the effects of AII, DA, and
nifedipine on 45Ca2’ influx, whereas the effect of PTX on
Bay K 8644 was partial, but significant. However, PTX did
not affect basal 45Ca2+ influx (21.9 f 1.2 and 19.4 f 2.2
dpm/s . lo6 cells) for control and treated cells, respectively
(Fig. 5). We also observed that the 18-h preincubation time
reduced the 45Ca2+ influx response to AII; AI1 stimulation
was 155% of the control value after preincubation (Fig. 5)
compared to 221% of the control value without preincuba-
tion (Fig. 4).
672 PETIT ET AL.
JCEBrM.1993
Vol17.No3
Y
4
0
0 15 30 45 60 75
TIME (set)
25 ’
C -7 -6 -5 -4
Log CONCENTRATION (M)
FIG. 1.
‘%a’+ influx in human term trophoblastic cells. A, Freshly isolated trophoblastic cells were incubated without (control) or with increasing
concentrations of DA and 2.5 &i %az+, as described in Materials and Methods. The incubation was stopped by filtration on Whatman GF/C
filters. The figure is representative of three different experiments, each performed in triplicate. B, ‘%a*+ influx (slopes of A) were presented as a
percentage of the control value (control, 10.6 f 1.2 dpm/s). C, Cells were incubated with lo-’ mol/L DA (C) or lo-’ mol/L DA and increasing
concentrations of the Dx dopamine antagonist spiperone. Values are the mean k
SE
of four experiments, each performed in duplicate. *, P < 0.05
us. control. H, Control; V, 10S7 mol/L DA, 0, 10” mol/L DA; A, lo-’ mol/L DA; 0, lo-’ mol/L DA.
Discussion
Our results demonstrate that Ca2+ is an important cellular
effector that mediates hPL release by DA and AI1 from
human placental cells. Furthermore, our data suggest that
these effects may be mediated by DHP-sensitive Ca*+ chan-
nels. We have shown that DA binds to specific D2 receptors
(5) and, via PTX-sensitive G-proteins, inhibits calcium influx.
The ECso of the effects of DA on 45Ca2+ influx (10 -I- 3 pmol/
L) and hPL release (1.0 f 0.8 pmol/L) (4) are in the same
concentration range; this could suggest that the DA-induced
inhibition of hPL release may be directly coupled to the
inhibition of calcium influx. Furthermore, the similarities
between the effects of DA and nifedipine on 45Ca2+ influx
and hPL release and their respective sensitivities to PTX
suggest a coupling between placental D2 receptors and DHP-
sensitive Ca*+ channels. This proposed mechanism is similar
to that of DA-inhibited PRL release in the pituitary (for
review, see Ref. 20). Even if Ca*+ currents have not been
recorded directly using patch clamp experiments, our results
obtained from 45Ca2+ influx studies using nifedipine and Bay
K 8644 also suggest the existence of L-type Ca*+ channels
on placental cells. The physiological relevance of DA to
placental Ca*+ influx and hPL release in human placental
cells needs to be addressed, because the concentrations of
DA used in this study appear relatively high. However,
similar concentrations have been used with somatomam-
motropic cell stain (GH,C) (21), adrenal glomerulosa cells
(22), adrenocortical cells (23), and anterior pituitary cells (24).
High concentrations of analogs were also necessary to obtain
a maximal effect with other amines, such as histamine (25).
The presence of DA (26,27) and D2 receptors (5) in placental
tissues agrees with a putative local paracrine modulation of
placental functions by DA and can explain the high concen-
trations needed for in vitro experiments.
The AII-stimulated effect implicates the binding of AI1 to
specific placental receptors (2,3) and the activation of phos-
CALCIUM AND hPL RELEASE
673
a “c -9 -8 -7 -6
0
C -9 -6 -7 -6
Log CONCENTRATION (M)
FIG. 2. Stimulation by AI1 of Was+ influx in human term trophoblastic cells. A, Freshly isolated trophoblastic cells were incubated for O-75 s
with increasing concentrations of AI1 agonist [Sari]AII. B, Cells were incubated with lo+ mol/L [Sar’]AII [control (C)] or with 10” mol/L AI1
and increasing concentrations of the AI1 antagonist [Sari,Ala*]AII. Results represent the mean f
SE
of four experiments, each performed in
duplicate. $I, P c 0.05 vs. control.
200
A 150
0
z
z
6
8
P
100
a
!!I
W
a
i
c 50
(
C
-8 -7 -6 -5
LOG CONCENTRATION (M)
FIG. 3. Dose-response curves for Bay K-stimulated and nifedipine-
inhibited hPL release. Freshly isolated trophoblastic cells were incu-
bated for 2 h at 37 C, as described in Materials and Methook, with
increasing concentrations of Bay K 8644 (0) or nifedipine (0). hPL
release calculated as a percentage of that observed in unstimulated
cells (control, 195 f 16 ng/lO’ cells), is expressed as the mean f
SE
of
four experiments, performed in triplicate. t, P < 0.05 vs. control.
phoinositide hydrolysis by phospholipaseC to produce ino-
sitol 1,4,5-triphosphate [Ins(1,4,5)P3] (3). The mechanism
leading to the activation of calcium influx by AI1 is complex.
The difference between the ECso for [Sar*]AII binding (0.4
nmol/L) (3) and inositol phosphate production (0.3 nmol/L)
(3) and the stimulation of 45Ca2+ influx (50 nmol/L) by [Sar’]
AI1 suggests an indirect effect of AI1 on Ca’+ influx. The
mediator could be Ins(1,4,5)P3 (28) or Ins(1,3,4,5)P4 (29) via
plasma membrane receptors. Moreover, as recently proposed,
the Ins(l,4,5)P3-emptied intracellular stores themselves could
provide the signal for the stimulation of calcium entry (30).
However, the PTX sensitivity of the effects of AI1 and Bay K
8644 on Ca2+ influx suggests a direct stimulation of Ca2+
influx via DHP-sensitive Ca’+ channels coupled to PTX-
sensitive G-proteins. The AR-stimulated Ca2+ influx in ad-
renal glands (31) was also PTX sensitive. Moreover, AI1
induced the activation of DHP-sensitive calcium channels
sensitive to PTX in an adrenal cortical cell line (32). As AI1
and Bay K 8644 similarly stimulated Ca*+ influx and hPL
release, it appears that Ca2+
may be the principal mediator
of hPL release stimulation by AII. However, we reported
that, contrary to the AR-stimulated Ca’+ influx, this effect of
AI1 on hPL release was not sensitive to PTX (4). These results
suggest that the calcium channels implicated in AII-stimu-
lated hPL release may also be modulated by a second mes-
senger pathway insensitive to PTX [i.e. Ins(1,4,5)P3 produc-
tion]. The relative importance of each signal may be related
to incubation time (initiation or maintenance of the effect).
Nonetheless, the effects of AI1 on Ca2+ influx (this study)
and InsP production (3) suggest that in the human placenta,
AI1 binds to the AT1 AI1 receptor subtype, which is known
to be linked to inositol phosphate production, and Ca*+ influx
(for review, see Ref. 33). This hypothesis is supported by
recent data demonstrating that the human placenta contains
only this AI1 receptor subtype (34). Thus, the results suggest
that, as observed in other tissues (35), the human placenta
AI1 receptor may be linked to two G-proteins: one insensitive
to PTX (which is coupled to phospholipase-C), and the
second linked to DHP-sensitive calcium channels.
The mechanism by which PTX switches off the effects of
the G-protein linked receptors (such as DA and AI1 receptors)
674 PETIT ET AL.
JCE&M*1993
Vol77.No3
FIG. 4. Effects of DHPs on ‘%a*+ influx and hPL release in human term trophohlastic cells. A, Freshly isolated trophoblastic cells were incubated
for O-75 s at 37 C, as described in A4aterkz.k and Methods, with lo-’ mol/L DA, lo-’ mol/L AII, lo-’ mol/L nifedipine (NIFE), or lo-” mol/L Bay
K 3644 (Bay K) and combinations of these molecules (control, 10.6 f 1.2 dpm/s). B, Cells were incubated for 2 h at 37 C, as described in
Materials
and Methods, with the same agents. hPL release, calculated as a percentage of that observed in unstimulated cells (control, 195 + 16 ng/lO’ cells),
is expressed as the mean f
SE
of four experiments, performed in triplicate. *, P < 0.05 us. control; **, P
C
0.05 us. AII; ***, P < 0.05 vs. BAY
K.
is well known and requires the uncoupling of the receptor
from the G-proteins by ADP-ribosylation of the a-subunit
of these G-proteins (36). However, to our knowledge, this is
the first report concerning the inhibition of the effects of
nifedipine and Bay K 8644 on Ca2+ influx by PTX in the
human placenta. The effect of PTX, even if present for all of
the drugs used in the Ca2+ influx experiments, is probably
specific because the toxin did not affect AII-stimulated ino-
sit01 phosphate production under comparable experimental
conditions (4). The PTX sensitivity of the effects of nifedipine
and Bay K 8644 suggests a direct interaction of G-protein
with DHP-sensitive calcium channels. Similar interactions of
PTX with L-type calcium channel activity were observed
using the patch-clamp technique in chromaffin (37) and
neuronal (38, 39) cells; the results of these studies suggested
that G-protein coupled to Ca*+ channels may modify the
conformation of nifedipine- and Bay K 8644-binding sites.
The ADP-ribosylation of the channels by PTX is possible;
ADP-ribosylation of placental membrane proteins by PTX
(4) suggests the ADP-ribosylation of proteins with mol wwt
of 40 and 41 kilodaltons (kDa). Sodium dodecyl sulfate-
polyacrylamide gel electrophoresis analysis of DHP-sensitive
calcium channels from other tissues (40) indicated that the 0
(50 kDa)-, 6 (24-30 kDa)-, and y (30 kDa)-subunits are
possible substrates, considering a putative difference in the
mol wt of the subunits in different tissues. However, no
ADP-ribosylation site was suggested by the amino acid se-
quences of these proteins. The present study did not allow
us to clearly define the mechanism responsible for the effect
of PTX on both nifedipine- and Bay K 8644-mediated Ca*+
influx in human placenta.
The present study proposes an important role for extracel-
lular Ca*+ in the regulation of hPL release, because the effects
of DA and AI1 on both hPL release and Ca*+ influx are
receptor specific, and the molecules known to reduce (nife-
dipine) or increase (Bay K 8644) the intracellular calcium
concentration mimic the effects of DA and AIL respectively,
on hPL release. However, we did not investigate the effect
of the extracellular Ca*+ concentration on hPL release. Pre-
vious reports (4) showed the limited application of this
protocol with freshly isolated trophoblastic cells, as a low
extracellular Ca*+ concentration led to a diminution of cell
membrane integrity. Our results are in good agreement with
those of previous studies using cuhured placental explants
CALCIUM AND hPL RELEASE
675
*
i
Cl without PTX
n with PTX
CONTROL All BAY K DA NIFE
FIG. 5. Effect of PTX on “Ca*+ influx in human term trophoblastic
cells. Freshly isolated trophoblastic cells were suspended (lo6 cells/mL)
in Ham’s F-10 medium supplemented with 10% FBS and incubated for
16-20 h at room temperature with or without 1 pg/mL PTX. Control
and treated cells were then incubated for O-75 s at 37 C without
stimulus (control) or with lo-’ mol/L DA, lo- mol/L nifedipine
(NIFE), 10e6 mol/L Bay K 8644 (Bay K), or lO+ mol/L [Sar’JAII in
the presence of 2.5 &i ?a*+. Results are the mean f
SE
of triplicate
determinations with three different placentas. *, P < 0.05 us. control.
(17, 18), perifused placental cells (16), or perfused placental
fragments (41), which demonstrated a stimulatory effect of
extracellular Ca2+ and/or ionophores on hPL release. Our
results are also in agreement with the regulatory role of Ca2+
in other endocrine/paracrine systems, such as pituitary lac-
totrophs (20), gonadotrophs (42), and somatotrophs (43).
Although most studies suggest a stimulatory role for Ca*+ on
hPL release, other studies, using placental fragments (13) or
cultured trophoblastic cells (14,15,44), showed an inhibitory
effect of Ca2+ on hPL release. An inhibitory effect of Ca2’
was also reported in parathyroid cells for the secretion of
PTH (45) and in juxtaglomerular cells for the secretion of
renin (46). These two types of cells are implicated in the
regulation of bodily Ca2+ and electrolyte homeostasis,
whereas trophoblast cells are known to be the principal
constituent of an endocrine organ (47). The conflicting find-
ings concerning the role of Ca2+ in the regulation of hPL
release might be explained by the diversity of protocols used.
In particular, most of the interpretations of the role of Ca2+
in the modulation of hPL release were based on the used of
Ca2+ chelators
(i.e.
EGTA) and ionophores. However, the use
of chelators and ionophores can lead to disparate interpre-
tations of the role of Ca2+. Thus, addition of an ionophore
does not guarantee a physiological change in the intracellular
Ca*+ concentration (48). Depending on pH, ionic strength,
temperature, and cells, intracellular Ca2+ could easily raise
to toxic levels or not change at all. Furthermore, some cells,
including freshly isolated trophoblastic cells (4), may tolerate
low Ca2+ (or EGTA-containing) buffer only for short periods.
Moreover, incubations with EGTA can slowly deplete Ca2+
stores, so that cellular responses may vary with the duration
of exposure to Ca’+-free medium (48).
In conclusion, the results presented in this manuscript
provide more information on the role of extracellular calcium
by measuring Ca2+ influx and correlating this influx with
hPL release. However, the modulatory effects of AI1 and DA
on the intracellular calcium concentration were not demon-
strated. The use of fluorescent probes (fura-2) to correlate
the hormonal regulation of hPL release with the variations
in the intracellular Ca2+ concentration would be useful.
Nonetheless, our results suggest that calcium influx, via
DHP-sensitive calcium channels, is one mechanism that me-
diates AI1 stimulation and DA inhibition of hPL release by
human term trophoblastic cells.
Acknowledgments
We thank Paulette Mercier and Hugues Beaudry for technical assist-
ance, and Suzanne Beaudet for typing the manuscript.
References
1. Chard T. 1986 Placental synthesis. Clin Obstet Gynecol. 13:447-
467.
2. Tence M, Petit A. 1989 Characterization of angiotensin II biding
sites in the human term placenta. Mol Cell Endocrinol. 63:111-119.
3. Petit A, Guillon G, Tence M, et al. 1989 Angiotensin II stimulates
both inositol phosphate production and human placental lactogen
release from human trophoblastic cells. J Clin Endocrinol Metab.
69:280-286.
4. Petit A, Guillon G, Pantaloni C, et al. 1990 An islet-activating
protein-sensitive G-protein is involved in dopamine inhibition of
both andotensin-stimulated inositol uhosnhate nroduction and hu-
man placental lactogen release in human’trophbblastic cells. J Clin
Endocrinol Metab. 71:1573-1580.
5. Petit A, Vaillancourt C, Bellabarba D, Lehoux J-G, Gallo-Payet
N, Belisle S. 1990 Presence of Ds-dopamine receptors in human
term placenta. J Receptor Res. 10:205-215.
6. Lameh j, Cone RI, Maeda S, et al. 1990 Structure and function of
G protein coupled receptors. Pharmaceut Res. 7~1213-1221.
7. Birnbaumer L, Abramowitz J, Yatani A, et al. 1991 Roles of G
proteins in coupling of receptors to ionic channels and other effector
systems. Crit Rev Biochem Mol Biol. 25:225-244.
8. Birnbaumer L, Perez-Reyes E, Bertrand P, et al. 1991 Molecular
diversitv and function of G nroteins and calcium channels. Biol
Reprod: 44:207-224. a
9. Enjalbert A. 1989 Multiple transduction mechanisms of dopamine,
somatostatin, and anaiotensin II recenters in anterior nituitary cells.
Horm Res. 31:6-12. ” A 1
10. Vallar L, Meldolesi J. 1989 Mechanisms of signal transduction at
the donamine DZ recentor. Trends Phannacol Sci. 10:74-77.
11. Lledo P-M, LeRendre P, Israel J-M, Vincent J-D. 1990 Dopamine
inhibits two characterized voltage-dependent calcium currents in
identified rat lactotronh cells. Endocrinoloev. 127:990-1001.
12. Lledo P-M, Israel J&I, Vincent J-D. 199: A guanine nucleotide-
binding protein mediates the inhibition of voltage-dependent cal-
cium currents by dopamine in rat lactotrophs. Brain Res. 528:143-
147.
676 PETIT ET AL.
13. Choy VJ, Watking WB. 1976 Effects of ionic environment on the
release of human placental lactogen in vitro. J Endocrinol. 69:349-
358.
31.
14. Handwerger S, Conn PM, Barrett J, Barry S, Golander A. 1981 32.
Hausdorff WP, Sekura RD, Aguilera G, Catt KJ. 1987 Control of
aldosterone production by angiotensin II is mediated by two guanine
nucleotide regulatory proteins. Endocrinology. 120:1668-1678.
Hescheler J, Rosenthal W, Hinsch KD, Wulfem M, Trautein W,
Schultz G. 1988 Angiotensin II-induced stimulation of voltage-
dependent Ca2+ currents in adrenal cortical cell line. EMBO J. 7:619-
624.
Human placental lactogen release in vitro: paradoxical effects of
calcium. Am I Phvsiol. 240:E550-E555.
15. Hochberg Z,-Bi& T, Perlman R, Lahav M, Barzilai D. 1984 The
modulatik of placental lactogen secretion by calcium: studies with
cultured human term Dlacenta. Mol Cell Endocrinol. 37~359-362.
Zeitler P, Murphy E,‘Handwerger S. 1986 Arachidonic acid stim-
ulates ‘5calcium efflux and HPL release in isolated trophoblast cells.
Life Sci. 38:99-107.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
Polliotti B, Meuris S, Lebrun P, Robyn C. 1990 Stimulatory effects
of extracellular calcium on chorionic gonatrophin and placental
lactogen release by human placental explants. Placenta. 11:181-
190.
Polliotti 8, Meuris S, Lebrun P, Robyn C. 1991 Calcium influx
similarly stimulates the release of chorionic gonadotrophin and
placental lactogen from human placental explants. Trophoblast Res.
b:189-198. -
Mauger JF’, Poggioli J, Guesdan F, Claret N. 1984 Noradrenaline
vasopressin and angiotensin increase Ca2+ influx by opening a
con&on pool of Ca+> channels in isolated rat liver cellk B&hem J.
221:121-127.
Lamberts SWJ, MacLeod RM. 1990 Remlation of prolactin secre-
tion at the level of the lactotroph. PhysiGl Rev. 70:2?9-318.
Albert PR. Neve KA. Bunzow JR, Civelli 0.1990 Couulinp; of a
cloned rat.dopaminelD2 receptor to inhibition of adenyiyl &lase
and prolactln secretion J Biol Chem. 265:2098-2104.
Gallo-Payet N, Chouinard L, Bale&e
M-N,
Guillon G. 1990 Dual
effects of dopamine in rat adrenal glomemlosa cells. Biochem Bio-
phys Res Commun. 172:1100-1108.
Morra M, Leboulenger F, Desrues L, Tonan
M-C,
Vaudry H. 1991
Dopamine inhibits inositol phosphate production arachidonic acid
formation and corticosteroid release by frog adrenal gland through
a pertussis toxin-sensitive G-protein. Endocrinology. 128:2625-
2632.
Enjalbert A, Guillon G, Mouillac B, et al. 1990 Dual mechanisms
of inhibition by dopamine of basal and thyrotropin-releasing hor-
mone-stimulated inositol phosphate production in anterior pituitary
cells. J Biol Chem. 265:18816-18822.
Hill SJ. 1990 Distribution properties and functional characteristics
of three classes of histamine receptor. Pharmacol Rev. 42:45-83.
Philippe M, Ryan KJ. 1981 Catecholamines ln human amniotic
fluid. Am J Obstet Gynecol. 139:204-208.
Divers WA, Wilkes MM, Babaknia A, Yen SSC. 1981 An increase
in catecholamlnes and metabolites in the amniotic fluid compart-
ment from middle to late gestation. Am J Obstet Gynecol. 139:483-
486.
Kuno M, Gardner P. 1987 Ion channels activated by inositol1,4,5-
triphosphate in plasma membrane of human T-lymphocytes. Na-
ture. 326:301-304.
Irvine RF, Moore RM. 1986 Micro-injection of inositol 1,3,4,5-
tetrakisphosphate activates sea urchin eggs by a mechanism de-
pendent on external Ca’+. Biochem J 240:914-920.
Taylor CW. 1990 Receptor-regulated Ca’+ entry-secret pathway or
secret messenger? Trends Pharmacol Sci. 11:269-271.
JCEikM.1993
Vol77.No3
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
Timmermans PMWM, Wong PC, Chiu AT, Herblin WF. 1991
Nonpeptide angiotensin II receptor antagonists. Trends Pharmacol
Sci. 12:55-62.
Kalenga MK, Degasparo M, Dehertogh R, Whitebread S, Vank-
rieken L, Thomas K. 1991 Human placenta contains only AT1 AII-
receptor subtype. Reprod Nutr Dev.*31:257-268. -
Valotton MB. Caoooni AM, lohnson EIM, Lana U. 1990 Mode of
. II
action of angiotensin II and vasopressin on their-target cells. Horm
Res. 34:105-110.
Ui M, Katada T. 1990 Bacterial toxins as probe for receptor-Gi
coupling. Adv Second Messenger Phosphoprot Res. 24:63-69.
Cena V, Brocklehurst KW, Pollard HB, Rojas E. 1991 Pertussis
toxin stimulation of catecholamine release from adrenal medullary
chromaffin cells: mechanism may be by direct activation of L-type
and G-type calcium channels. J h;lembr-Biol. 122:23-31. _ -
Scott RH. D01~hin AC. 1987 Activation of a G urotein oromotes
agonist r&pokes to calcium channel ligand. Na&e. 330:?60-762.
Bergamaschi S, Govoni S, Cominetti P, Parenti M, Trabucchi M.
1988 Direct coupling of a G protein to dihydropyridine binding
sites. Biochem Biophys Res Commun. 156:1279-1286.
Catteral WA, Nunoki K, Lai Y, De Joungh K, Thomsen W, Rossie
S. 1990 Structure and modulation of voltage sensitive sodium and
calcium channels. Adv Second Messenger Phosphoprot Res. 24:30-
35.
__.
Welsch F. 1979 Release of human chorionic somatomammotrophin
from perfused lobules and superfused fragments of term placenta:
spontaneous liberation and the effects of choline@ drugs dibutyr-
ylcyclic adenosine monophosphate and calcium. Res Commun
Chem Path01 Pharmacol. 24:211-222.
Catt KJ, Stojilkovic SS. 1989 Calcium signalling and gonadotropin
secretion. Trends Endocrinol Metab. 1:15-20.
Kraicer J, Spence JW. 1981 Release of growth hormone from
purified somatotrophs: use of high K+ and the ionophore A23187
to elucidate interactions among Ca’+, adenosine 3’,5’-monophos-
phate and somatostatin. Endocrinology. 108:651-657.
Hochberg Z, Perlman R, Bick T. 1987 Interrelated calcium ion and
cyclic AMP inhibition of placental lactogen secretion by cultured
human term trophoblast. Acta Endoainol (Copenh). 114:68-73.
Nemeth EF, Carafoli E. 1990 The role of extracellular calcium in
the regulation of intracellular calcium and cell function. Cell Cal-
cium. 11:319-321.
Kurtz A, Penner R. 1989 Angiotensin II induces oscillations of
intracellular calcium and blocks anomalous inward rectifying potas-
sium current in mouse renal juxtaglomerular cells. Proc Natl Acad
Sci USA. 86~3423-3427. _
47. Talamantes F, Ogren L. 1988 The placenta as an endocrine organ:
polypeptides. In: Knobil E, Neil D, Ewing L, et al, eds. The physi-
ology of reproduction. New York: Raven Press; 2093-2144.
48. Poenie M. 1992 Measurement of intracellular calcium with fluores-
cent calcium indicators. Neuromethods. 20:129-174.
