Research Center, Hôpital Ste-Justine, and the Department of Obstetrics-Gynaecology, Université de Montréal, Montreal, QC, Canada H3T 1C5
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ABSTRACT |
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Our previous work
on aldosterone secretion suggested that dihydropyridine-sensitive
calcium channels, one type of voltage-dependent calcium channels
(VDCC), are functionally impaired in adrenal capsule preparations from
the pregnant rat. The aim of this study was to determine whether,
during pregnancy, the density and/or activity of these channels is
altered in the adrenal zona glomerulosa. These VDCC measured with
[3H]nitrendipine binding were not different
between membrane preparations of nonpregnant and pregnant rats. Western
blots were performed using two different antibodies, a polyclonal
(PcAb) directed against the 1-subunit of VDCC and a
monoclonal (McAb) that recognizes an intracellular domain of that
protein. McAb immunoreactivity showed a significant decrease in
preparations from pregnant rats, whereas no difference was observed
with PcAb. VDCC activity was estimated by
45Ca2+ uptake in isolated adrenal cortex and by
intracellular calcium concentration
([Ca2+]i) in adrenal glomerulosa
cells with the Ca2+ probe fura PE3. These measurements
revealed that KCl stimulation produced greater Ca2+ influx
in nonpregnant than in pregnant rats. Nifedipine (a blocker of VDCC)
inhibited this stimulation only in nonpregnant rats, whereas BAY K 8644 (an activator of VDCC) increased Ca2+ influx in pregnant
rats only. These data suggest that, during pregnancy, the altered
regulation of calcium homeostasis in adrenal glomerulosa is linked to a
conformational alteration of VDCC.
voltage-dependent calcium channels; pregnancy
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INTRODUCTION |
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VOLTAGE-DEPENDENT CALCIUM CHANNELS (VDCC) regulate the
entry of extracellular calcium ions (Ca2+) into the
cytoplasm, where they participate in a variety of calcium-dependent processes. VDCC are built up with five subunits, termed
1,
2,
,
, and
. The
1-subunit forms the conducting pore, is responsible for
voltage dependence, and is the site of action of different activators
and inhibitors (14). VDCC activation in response to depolarization
plays a crucial role in the generation of the calcium signal in many
cells. At least three types of VDCC have been identified in rat adrenal
glomerulosa cells: N-type (8), T-type, and L-type (8, 14,
24). Among these, the L-type is the most sensitive to
dihydropyridine (DHP). Adrenal glomerulosa cells are sensitive to small
variations of extracellular potassium (K+) (3-10 mM).
An elevation of K+ depolarizes the membrane and allows the
opening of VDCC, leading to increased 45Ca2+
uptake into glomerulosa cells and intracellular calcium concentration ([Ca2+]i) (3, 19). This sustained
Ca2+ influx has been a convenient target to study the
effect of pharmacological agents. Increased cytoplasmic
Ca2+ concentration can be inhibited by DHP
antagonists such as nifedipine or nitrendipine (1, 7, 12) and can be
potentiated by the DHP activator BAY K 8644 (12).
In a recent study (5), we reported that sensitivity of aldosterone secretion to potassium was reduced in whole adrenal cortex preparations from pregnant rats compared with nonpregnant animals. In addition, nifedipine shifted the concentration-response curve to K+ in adrenal preparations from nonpregnant rats toward that of term-pregnant rats. Conversely, BAY K 8644 produced a larger increase in sensitivity to K+ in the adrenal capsule from pregnant rats, resulting in superposition of the concentration-response curves to K+ of the two groups (pregnant and nonpregnant) (6). These observations led us to hypothesize that, during pregnancy, stimulated mobilization of extracellular calcium is reduced, and this is associated with functional impairment of the DHP-sensitive calcium channel. We wanted to characterize the nature of these changes by measuring the quantity and activity of these channels on, respectively, membrane preparations (binding of [3H]nitrendipine and Western blotting), isolated capsules (45Ca2+ uptake), and cells ([Ca2+]i) from the adrenal cortex of nonpregnant and term-pregnant rats.
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MATERIALS AND METHODS |
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Animals. Female Sprague-Dawley rats (Charles River Canada, St-Constant, QC, Canada) weighing 225-250 g were mated with males. The morning when spermatozoa were found in vaginal smears was deemed to be day 1 of pregnancy. On day 22 of gestation (term), the animals were decapitated, and their adrenals were quickly removed. Nonpregnant rats picked randomly during the estrous cycle served as controls. All animals were housed under controlled light (lights on from 0600 to 1800) and temperature (21 ± 3°C). This study received approval from the local animal care committee, which is accredited by the Canadian Council on Animal Care.
Membrane preparation from adrenal capsules. Adrenal capsules containing zona glomerulosa (ZG) were isolated by manual compression. Preparations were made as described previously (10). In brief, the cortices of two rats were pooled, minced, and homogenized with a Polytron (Brinkman, Rexdale, ON, Canada) twice for 10 s at setting 8 in 0.25 M of sucrose. The homogenate was centrifuged at 1,500 g for 10 min at 4°C. This eliminates nuclei and unbroken cells, which sediment to form a pellet. The supernatant was then centrifuged at 50,000 g for 30 min at 4°C. The resulting particulate fraction was resuspended in binding buffer (50 mM Tris · HCl, 120 mM NaCl, 5 mM MgCl2, 0.02% NaN3, 5 mM EDTA, 1 µM leupeptin, 1 µM aprotinin, 1 µM pepstatin, and 100 µM phenylmethylsulfonyl fluoride). Membrane protein content was measured by the Bradford method (4).
Binding assays.
[3H]nitrendipine binding to membranes of the
adrenal cortex was studied in competition experiments. The ZG
preparations were assayed at concentrations of 125 µg of protein per
tube. The membranes were incubated in a final volume of 100 µl of
binding buffer (see above) supplemented with 0.4% BSA (Sigma, St.
Louis, MO) containing 1 nM [3H]nitrendipine
(73.5 Ci/mmol; Dupont Canada, Mississauga, ON, Canada). Nonspecific
binding was determined by competition with 1 µM nifedipine (RBI,
Natick, MA). [3H]nitrendipine was displaced
with BAY K 8644 (1010-10
5
M) (RBI), with nifedipine
(10
11-10
6
M) or with verapamil
(10
9-10
5
M; Sigma). Incubations were performed in the dark at room temperature for 60 min under constant agitation. Membrane-bound radioactivity was
separated from the unbound ligand on Whatman GF/C filters with a PHD
2000 Cell Harvester (Cambridge Technology, Watertown, MA).
Radioactivity was counted in a beta counter (LS 6000 IC model, Beckman
Instruments, Fullerton, CA). Specific binding was calculated by
subtracting nonspecific from total
[3H]nitrendipine binding. The binding results
were analyzed with the EBDA/Ligand program (16) to determine the
affinity (Kd) and density (Bmax) of
DHP-binding sites. Statistical analysis was done by Student's
t-test, and P < 0.05 was considered significant. The
results obtained from the displacement experiments were analyzed with
the ALLFIT 2.21 program for Windows. pD2 values
for pregnant and nonpregnant groups were compared by the F test
and Student's t-test.
Western blots.
Membrane fractions (prepared as above) at a concentration of 25 µg of
protein per well were heated at 100°C for 2 min in 62.5 mM
Tris · HCl (pH 6.8), 2% SDS, 10% glycerol and 5%
-mercaptoethanol. Proteins were separated by SDS-PAGE according to
the method of Laemmli (15). Briefly, SDS-PAGE was performed in a
1.5-mm-thick slab gel with a resolving gel consisting of 7.5%
acrylamide, 0.09% bis-acrylamide and 0.1% SDS in 370 mM
Tris · HCl (pH 8.8) and a stacking gel consisting of
5% acrylamide, 0.13% bis-acrylamide and 0.07% SDS in 125 mM
Tris · HCl (pH 6.8). The running buffer was 25 mM
Tris · HCl (pH 8.3), including 0.1% SDS and 0.2 M
glycine. Samples were loaded in duplicate on each gel. Heart
preparation was present on each gel as positive control.
Electrophoresis was conducted at room temperature under a constant
voltage of 185 V for ~40 min (Mini Protean II Electrophoresis Cell,
Bio-Rad Laboratories, Mississauga, ON, Canada). Electrophoretic
transfer of proteins from the polyacrylamide gels to the nitrocellulose
membranes (Bio-Rad Laboratories) was carried out at 50 V for 2.5 h in
25 mM Tris · HCl (pH 6.8) containing 0.2 M glycine,
20% methanol and 0.037% SDS. Transfers were made in a Bio-Rad
Trans-Blot Electrophoretic Transfer Cell, with use of supercooling
coil to prevent the buffer temperature from increasing.
Measurement of
45Ca2+ uptake.
The method used was adapted from the one developed in our laboratory
for isolated blood vessels (21). Adrenal capsules from pregnant and
nonpregnant rats were equilibrated at 37°C for 45 min in F12 medium
(GIBCO, Burlington, ON, Canada) and for another 45 min in physiological
Krebs solution (118 mM NaCl, 5 mM HEPES, 3 mM KCl, 1.25 mM
MgCl2 , 5.55 mM glucose and 1.5 mM CaCl2). The two cortexes of one rat were then cut in half, and each of the four
pieces was placed in a tube and preincubated in Krebs solution for 30 min at 37°C. 45Ca2+ (0.5 µCi) was added
to the four groups for 10 min in Krebs solution at 37°C. The piece
used for nonspecific uptake contained 50 mM La3+, a second
one served for basal measurement, and the two other pieces were
stimulated with KCl (6 mM) in the presence or absence of nifedipine (1 µM) or BAY K 8644 (1 µM). The pieces of cortex were then washed in
ice-cold Ca2+-free Krebs solution supplemented with 50 mM
La3+ at 4°C for 20 min, and then they were weighed and
incubated with PCA-H2O2 (1:1) at 37°C for 1 h to dissolve all tissues. Radioactivity counted by scintillation
spectrometry in a -counter (LS 6000 IC model, Beckman Instruments)
was related to the apparent tissue content of calcium (mmol/kg tissue).
Nonspecific 45Ca2+ uptake was subtracted from
the total to obtain the specific value. The results were expressed as
means ± SE. Data were compared by ANOVA (1-way or 2-way, according to
protocol), and P < 0.05 was considered significant.
Isolation of glomerulosa cells. Glomerulosa cells were obtained from adrenal capsular tissue of pregnant and nonpregnant rats by collagenase digestion and mechanical dispersion. After dispersion, the cells were filtered on a nylon (250 µm) filter and washed twice with F12 medium (GIBCO) supplemented with 0.2% BSA and 1.25 mM Ca2+. They were then counted and resuspended at a concentration of 3 × 105 cells/ml in the same medium and allowed to equilibrate for 1 h at 37°C before the loading procedure was started. Contamination by zona fasciculata/reticularis was minimal. The relative purity of the glomerulosa cell preparation was >90%; the viability of the cells was >90%, as was shown by the trypan blue method.
Measurement of [Ca2+]i. Freshly isolated glomerulosa cells were incubated for 30 min at 37°C in F12 medium with 20 µM fura PE3 AM (Teflabs, Austin, TX) and 0.04% pluronic acid F127 (Sigma). They were then washed by centrifugation (800 g for 10 min), and the pellet was resuspended in one volume of F12 medium and incubated for 1 h at room temperature to ensure full hydrolysis of the AM group. Aliquots of 500 µl of cell suspension were washed with Hanks buffer (130 mM NaCl, 3 mM KCl, 20 mM HEPES, 0.1 mM MgCl2, 5.0 mM NaHCO3, 1.1 mM CaCl2, and 1% glucose). The final pellet was resuspended in 400 µl Hanks buffer (150,000 cells). Stimulation with KCl was done in the absence or presence of BAY K 8644 or nifedipine. Fura PE3 fluorescence (excitation at 340 and 380 nm and emission at 510 nm) was recorded in a Perkin-Elmer LS 50 luminescence spectrometer (Perkin-Elmer, Norwalk, CT). [Ca2+]i was estimated from the ratio of fluorescence at 340 and 380 nm from Rmax, obtained by treatment with ionomycin, and from Rmin by adding excess EGTA, using a value of 204 (20°C) for the Kd of fura PE3 (25). After addition of 1.5 mM of KCl, [Ca2+]i at the plateau was subtracted from the plateau value obtained at the basal level. Data were then plotted as increase in [Ca 2+]i vs. K+ concentration, and the area under the curve was calculated for each experiment (in the absence or presence of BAY K 8644 or nifedipine) using the Sigma Plot for Windows program (26). This method permits evaluation of the overall difference between treatments of cell suspensions. The data were compared by one-way ANOVA and considered significant at P < 0.05.
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RESULTS |
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Binding assays. In preliminary experiments, we determined the optimal conditions for reliable measurement of binding to calcium channels on adrenal glomerulosa from nonpregnant and pregnant rats. Binding experiments using increasing concentrations of capsule membrane, ranging from 25 to 300 µg of protein, showed linearity in preparations derived from nonpregnant rats (r = 0.96). Similar results were obtained from adrenal membrane preparations of pregnant rats on day 22 of gestation. The optimal incubation time of [3H]nitrendipine was fixed at 60 min, determined by varying the duration of the reaction from 15 to 90 min. [3H]nitrendipine binding increased linearly (r = 0.96) with rising radioactivity concentrations between 0.05 and 3.75 nM. Consequently, we chose to perform our binding experiments with 1 nM of [3H]nitrendipine and 125 µg of protein per tube for 60 min. Linearization of data from the competition curves by Scatchard analysis revealed a single binding site of DHP on this channel (data not shown).
When the results of the saturation curve were analyzed by EBDA-Ligand, the Kd obtained with [3H]nitrendipine on glomerulosa membranes was 0.23 nM (confidence interval from 0.19 to 0.28 nM) in the nonpregnant group and 0.25 nM (confidence interval from 0.22 to 0.27 nM) in the pregnant group [not significant (NS)]. The Bmax was 59.1 fmol/mg (confidence interval from 50.9 to 67.3 fmol/mg) and 64.6 fmol/mg (confidence interval from 54.8 to 74.4 fmol/mg) of protein, respectively (NS). Figure 1 shows the displacement curves in membrane preparations of adrenal ZG derived from nonpregnant and pregnant rats. There was no significant difference between the two groups when using nifedipine (Fig. 1A), BAY K 8644 (Fig. 1B), or verapamil (Fig. 1C). Nifedipine and BAY K 8644 were more potent than verapamil in displacing [3H]nitrendipine in both membrane preparations. The DHP antagonist nifedipine at 10
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Western blots.
Western blots were performed on adrenal glomerulosa membranes derived
from nonpregnant and pregnant rats to confirm the binding study
results. By use of a monoclonal antibody against the intracellular loop
of the 1-subunit of VDCC, a single band at 175 kDa was
detected, as seen in Fig. 2A
(top). Estimation of band density from Western blots (n = 3) showed a significantly lower concentration of
1-subunits in the cortex of pregnant rats. However, by
use of a polyclonal anti-DHP receptor
1-subunit, the
relative density of the bands on membrane preparations derived from
both groups did not show any statistical difference (n = 5;
Fig. 2B). The heart was used as a positive control (Fig.
2B, top).
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Measurement of
45Ca2+ uptake.
DHP-sensitive calcium channel activity in adrenal capsules of
nonpregnant and pregnant rats was estimated by
45Ca2+ uptake. In preliminary experiments, we
measured basal conditions to work with. Basal measurements were taken
at different times; after 10 min, 45Ca2+ uptake
reached a plateau (data not shown). As seen in Fig.
3, basal 45Ca2+
uptake in adrenal capsules from pregnant rats (0.32 ± 0.02 mmol 45Ca2+/kg tissue) was significantly greater
(P < 0.05) than from nonpregnant animals (0.23 ± 0.02 mmol
45Ca2+/kg tissue). The addition of 6 mM of KCl
increased 45Ca2+ uptake in nonpregnant rats
only. This figure also shows that the basal
45Ca2+ uptake of pregnant rats was as high as
the KCl-stimulated 45Ca2+ uptake of the
nonpregnant animals. There was no statistical difference in
KCl-stimulated 45Ca2+ between the two groups.
To test DHP-sensitive calcium channel activity, the effect of potassium
on 45Ca2+ uptake was studied in the presence
and absence of BAY K 8644, an activator, or nifedipine, an inhibitor of
these channels, in adrenal glomerulosa from nonpregnant and pregnant
rats. As shown in Fig. 4A, BAY K
8644 (1 µM) did not affect 45Ca2+ uptake in
nonpregnant rats; the uptake rate in KCl-stimulated adrenal glomerulosa
from pregnant rats was significantly enhanced from 0.29 ± 0.03 to
0.41 ± 0.03 mmol 45Ca2+/kg tissue. Figure
4B shows that nifedipine (1 µM) significantly inhibited the
45Ca2+ uptake in nonpregnant tissue from 0.28 ± 0.03 to 0.19 ± 0.03 mmol
45Ca2+/kg tissue (P < 0.05), whereas
this inhibitor did not have a significant effect in the pregnant one.
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Measurement of
[Ca2+]i.
Glomerulosa cells were loaded with the fluorescent Ca2+
probe fura PE3 to measure the effects of KCl stimulation. The addition of KCl rapidly increased [Ca2+]i in
nonpregnant and pregnant rats (Fig. 5). We
observed that the [Ca2+]i response
was concentration dependent, and this response was higher in
glomerulosa cells from nonpregnant than from pregnant animals. In each
experiment, the increase in [Ca2+]i
after each addition of 1.5 mM of KCl was calculated (steady-state response minus basal level) and plotted (Fig.
6A) against the final
[K+]. To test the effect of BAY K 8644 (a
DHP-sensitive calcium channel activator) and nifedipine (a
DHP-sensitive calcium channel inhibitor), fura PE3-loaded glomerulosa
cells were exposed to these pharmacological agents before being
gradually depolarized by the consecutive addition of KCl to the cell
suspension. The two modulators did not have an effect on basal
[Ca2+]i. The area under the curve,
obtained from Fig. 6A, was calculated for each experiment in an
attempt to evaluate the overall difference in the change of
[Ca2+]i, instead of focusing on the
increase seen with each dose of KCl added. Figure 6B displays
the mean area under the curves for each set of experiments in
nonpregnant and pregnant rats. We observed that BAY K 8644 had no
effect on the [Ca2+]i response to
KCl stimulation in adrenal ZG cells from nonpregnant rats, whereas
nifedipine significantly reduced it. In pregnant rats, the magnitude of
[Ca2+]i increase by addition of KCl
was smaller than that in nonpregnant rats. BAY K 8644 at 1 µM
markedly potentiated the response to KCl addition in pregnant rats,
whereas nifedipine (1 µM) did not have a significant effect. The
outcomes of these DHP-sensitive calcium channel modulators in cells of
pregnant rats were opposite to the results obtained in nonpregnant
rats.
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DISCUSSION |
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In a previous study on aldosterone secretion (6), we observed that
sensitivity to K+ is reduced in adrenal ZG derived from
22-day pregnant compared with nonpregnant rats. Our
concentration-response curves to K+ in the presence of
DHP-sensitive calcium channel modulators led us to propose that, during
pregnancy, DHP-sensitive calcium channels are functionally impaired. We
thus became interested in confirming that, during pregnancy, the
density and/or activity of DHP-sensitive calcium channels in adrenal
glomerulosa are modified. The major findings of this paper are
1) the quantity of binding sites for DHP in adrenal membranes
is not modified during pregnancy; 2) there appears to be
alteration in the L-type channel in the adrenal cortex of pregnant
rats, as revealed by the diminished immunoreactivity of the monoclonal
antibody that recognizes the intracellular loop of the
1-subunit that is not confirmed by the use of polyclonal antibody; 3) basal 45Ca2+ uptake is
significantly larger in adrenal glomerulosa derived from pregnant rats;
4) KCl induces an increase in 45Ca2+
uptake only in nonpregnant rats; 5) nifedipine significantly depresses the KCl-induced 45Ca2+ uptake and
[Ca2+]i elevation in nonpregnant
rats only; and 6) BAY K 8644 potentiates the KCl-induced
45Ca2+ uptake as well as the
[Ca2+]i rise in pregnant rats.
Binding studies were performed to compare the affinity and number of DHP-binding sites. In these experiments, a single class of high-affinity DHP-binding sites was identified in adrenal ZG derived from nonpregnant and pregnant rats. We reported that the Kd for [3H]nitrendipine was 0.23-0.25 nM in both groups. This is in good agreement with the results of Finkel et al. (9), who obtained a value of 0.26 ± 0.04 nM in adrenal ZG membranes. Godfraind et al. (11) reported that the dissociation constant of [3H]nitrendipine for the majority of tissues was between 0.01 and 1 nM. The Bmax that we measured was 59.1 fmol/mg of proteins in the ZG of nonpregnant rats. Finkel et al. (9) obtained a value in the same range as ours (105 ± 5.7 fmol/mg of proteins), albeit a little higher. Our results show that binding of [3H]nitrendipine was not modified during pregnancy in the rat ZG. The absence of difference in Bmax and Kd values for [3H]nitrendipine between tissues from nonpregnant and pregnant rats has also been observed in membrane preparations of aorta (20) and mesenteric arteries (23). Nifedipine and BAY K 8644 had a greater affinity to adrenal ZG than verapamil did in both groups, as shown by the partial displacement of [3H]nitrendipine by verapamil. VDCC are modulated by distinct classes of Ca2+ modulators such as DHPs (nifedipine and BAY K 8644), phenylalkylamines (verapamil), and benzothiazepines (diltiazem). There is a structural difference between diverse classes of VDCC antagonists, and their respective binding sites are distinct (1). Our radioligand binding assays support the idea about the heterogeneity of the calcium channel antagonists. DHP-binding capacity generally reflects the total number of sensitive calcium channels present in the membrane, although they are not necessarily functional or active channels (2). However, our results suggest that DHP-sensitive calcium channel density is not modified during pregnancy.
To support the radioligand binding data, protein immunoreactivity was
also examined. Western blot analysis of adrenal glomerulosa membrane
preparations revealed a marked decrease in total DHP-sensitive calcium
channel protein when using the monoclonal antibody, which recognizes
the carboxy terminal (intracellular portion) of the 1-subunit of VDCC. However, no difference was detected
between the two groups with the polyclonal antibody. These observations suggest that the epitope recognized by the monoclonal antibody is
modified in adrenal ZG of pregnant rats. It has been reported by
Perez-Reyes et al. (18) that genetic regulation of Ca2+
channel expression is the object of structural diversity due to
alternative splicing. The latter affects regions encoding transmembrane segments as well as the intracellular COOH-terminal tail (22). We thus
propose that alternative splicing or different isoforms of the channel
could appear during pregnancy. This will not affect DHP binding but may
alter the function of the channel.
Consequently we looked at the activity of DHP-sensitive calcium channels by measuring 45Ca2+ uptake and [Ca2+]i. In adrenal glomerulosa cells, elevated extracellular K+ depolarizes the plasma membrane and increases Ca2+ influx, which is inhibited by Ca2+ channel blockers and activated by Ca2+ channel activators. In the present study, KCl was able to stimulate 45Ca2+ uptake and [Ca2+]i significantly in nonpregnant rats. In the pregnant group, KCl was less efficient in stimulating [Ca2+]i influx. Meyer et al. (17) reported a blunted response to K+ depolarization in mesenteric resistance arteries from late-gestation rats. They suggest that this may involve an increase in basal membrane potential (hyperpolarization). If the adrenal behaves like vascular smooth muscle, a putative gestation-induced hyperpolarization of the ZG cells would decrease DHP-sensitive calcium channel activity. Our results in ZG cells are compatible with such a mechanism. In this study, the effect of nifedipine was pronounced in nonpregnant rats, where it significantly inhibited 45Ca2+ uptake and [Ca2+]i induced by KCl. BAY K 8644 potentiated KCl-induced 45Ca2+ uptake and [Ca2+]i increase in pregnant rats. Decreased reactivity to vasoconstrictor agents was also observed in isolated aortic rings from pregnant rats by Roy et al. (20, 21). These authors reported decreased sensitivity to calcium channel blockers and reduced calcium uptake in vascular smooth muscle cells, which were not due to lower binding of the tritiated calcium channel blockers (21). Calcium homeostasis is an important aspect of maternal and fetal physiology during gestation, and evidence from the literature is suggestive of alterations in calcium metabolism in the pathogenesis of hypertension during pregnancy (13). Deficiencies in calcium intake have been linked to preeclampsia and/or eclampsia. However, additional investigation is needed with respect to the mechanism of calcium's effects in human pregnancy.
We have already observed that basal secretion of aldosterone is higher
in adrenal capsules from pregnant compared with nonpregnant rats (5).
This agrees with present observation of higher basal 45Ca2+ uptake (Fig. 3), although with the
present state of knowledge these observations remain difficult to
explain and need more investigation. However, in the present work, we
observed that basal [Ca2+]i is
smaller in glomerulosa cells from pregnant compared with nonpregnant
rats. Therefore, both basal calcium uptake and
[Ca2+]i are interpreted to be
modified in opposite ways during pregnancy, suggesting that calcium is
highly sequestered intracellularly (in pools or bound to protein)
during pregnancy. On the other hand, the stimulated aldosterone
secretion in response to 6 mM of KCl was similar between adrenal
capsules of nonpregnant and pregnant rats (5). This earlier observation
correlates with the similar uptake of 45Ca2+
reported here. These observations suggest that the aldosterone secretion in response to KCl is related to the variation () in [Ca2+]i rather than to the absolute
intracellular calcium concentration.
In conclusion, our study demonstrates alteration in the activity but not in the density of DHP-sensitive calcium channels in the adrenal ZG during rat pregnancy. The expression of a different isoform of DHP-sensitive calcium channel has been suggested in the present report. Its implication in the reduction of DHP-sensitive calcium channel function remains to be elucidated.
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ACKNOWLEDGEMENTS |
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The authors thank Marie-Claude Gauthier for technical assistance and Sylvie Julien for secretarial work.
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FOOTNOTES |
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This study was supported by a grant from Conseil de Recherches Médicales du Canada and Conseil de Recherches en Sciences Naturelles et en Génie du Canada. May Simaan received a studentship from La Fondation de l'Hôpital Ste-Justine. Michèle Brochu is a scholar from Fonds de la Recherche en Santé du Québec.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: M. Brochu, Centre de Recherche, Hôpital Ste-Justine, 3175 Côte Sainte-Catherine, Montreal, QC, Canada H3T 1C5 (E-mail: brochum{at}ere.umontreal.ca).
Received 10 June 1999; accepted in final form 8 December 1999.
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