Functional alteration of dihydropyridine-sensitive Ca2+ channels in the adrenal glomerulosa of pregnant rats

May Simaan, Serge Picard, Jean St-Louis, and Michèle Brochu

Research Center, Hôpital Ste-Justine, and the Department of Obstetrics-Gynaecology, Université de Montréal, Montreal, QC, Canada H3T 1C5


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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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 alpha 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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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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 alpha 1, alpha 2, beta , gamma , and delta . The alpha 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.


    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 (10-10-10-5 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% beta -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.

After transfer, the nitrocellulose sheets (on average, 8.5 × 5 cm) were incubated for 24 h at 4°C in 15 ml of 1% blocking solution (Boehringer Mannheim, Laval, QC, Canada). The membranes were afterward incubated either with an anti-rabbit DHP receptor Ca2+ channel alpha 1-subunit (diluted 1:2,000, mouse monoclonal IgG; Upstate Biotechnology, Lake Placid, NY) for 1 h or with an anti-DHP receptor Ca2+ channel alpha 1- subunit (diluted 1:5,000, sheep polyclonal IgG; Upstate Biotechnology) for 4 h at room temperature with gentle shaking. The monoclonal antibody recognizes an intracellular domain of the alpha 1-subunit. The antibodies were diluted in 0.5% blocking solution. After incubation with antiserum, the nitrocellulose membranes were washed twice with 50 mM Tris-buffered saline (pH 7.5) containing 0.1% Tween 20 (TBS-Tween, Bio-Rad Laboratories) and twice with 0.5% blocking solution. Next, immunoblottings of the Ca2+ channel alpha 1-subunit were undertaken with a BM Chemiluminescence Western blotting kit POD-conjugated goat anti-mouse IgG for the monoclonal antibody (Boehringer Mannheim) and POD-conjugated rabbit anti-sheep IgG for the polyclonal antibody (Jackson ImmunoResearch, West Grove, PA). The membranes were washed four times in TBS-Tween and were incubated with chemiluminescent detection substrate (20 µl/cm2; Boehringer Mannheim). Autoradiography was performed with Kodak X-Omat AR5 film (Eastman Kodak, Rochester, NY). The intensity of the bands was quantified with an LKB Ultrascan XL apparatus (Pharmacia, Dorval, QC, Canada). VDCC protein levels were expressed as arbitrary densitometric units, and the values of the nonpregnant and pregnant membrane preparations were divided by the nonpregnant value on each gel. The value of the pregnant preparation was relative to that of the nonpregnant one.

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 beta -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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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 10-6 M displaced almost 60% of [3H]nitrendipine-total binding in both nonpregnant and pregnant rats, whereas verapamil, a phenylalkylamine, at 10-5 M, produced 50% displacement of this binding. Statistical analysis for pD2 values did not reveal significant differences between the two groups. With the F test, the variances were equal in the three groups, with P = 0.224 for nifedipine, P = 0.438 for BAY K 8644, and P = 0.210 for verapamil. The Student's t-test for two observations of equal variance showed that the mean pD2 values were not significant, with P = 0.841 for nifedipine, P = 0.552 for BAY K 8644, and P = 0.795 for verapamil.


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Fig. 1.   Displacement of total [3H]nitrendipine binding by nifedipine (A), BAY K 8644 (B), and verapamil (C) (n = 5) in membrane preparations of adrenal zona glomerulosa (ZG) derived from nonpregnant and pregnant rats. Results are expressed as means ± SE.

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 alpha 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 alpha 1-subunits in the cortex of pregnant rats. However, by use of a polyclonal anti-DHP receptor alpha 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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Fig. 2.   Effect of pregnancy on density of dihydropyridine (DHP)-sensitive calcium channel alpha 1-subunit in membrane preparations of rat adrenal ZG: (A) using a monoclonal antibody (n = 3), (B) using a polyclonal antibody (n = 5). Representative immunoblot of alpha 1-subunit (25 µg protein) (top panels); relative density of alpha 1-subunits (bottom panels). Membrane preparations of heart were used as a positive control in (B). Bars are expressed as means ± SE. ** P < 0.01 vs. nonpregnant rats.

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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Fig. 3.   Basal and 6 mM KCl-stimulated 45Ca2+ uptake rate in adrenal ZG derived from nonpregnant (n = 18) and pregnant (n = 12) rats. Basal as well as KCl-induced uptake was measured after 10 min of incubation with 45Ca2+. Results are expressed as means ± SE. + P < 0.05 vs. nonpregnant rats. * P < 0.05 vs. basal uptake.



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Fig. 4.   Effects of BAY K 8644, a voltage-dependent calcium channels (VDCC) activator (A), and nifedipine, a VDCC inhibitor (B), on Ca2+ uptake in adrenal ZG stimulated with 6 mM KCl. Adrenal capsules from nonpregnant and pregnant rats were incubated for 10 min with 45Ca2+ (basal) or with 45Ca2+ and KCl in absence or presence of BAY K 8644 (1 µM; n = 7) or nifedipine (1 µM; n = 5). Results are expressed as means ± SE. * P < 0.05 vs. basal; black-triangle P < 0.05, black-triangleblack-triangle P < 0.01 vs. KCl.

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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Fig. 5.   Effect of potassium on intracellular Ca2+ concentration ([Ca2+]i) in adrenal glomerulosa cell suspension derived from nonpregnant and pregnant rats. Representative curve of [Ca2+]i response to stimulation with potassium.



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Fig. 6.   Effect of potassium on [Ca2+]i in absence or presence of BAY K 8644 or nifedipine in adrenal glomerulosa cell suspension derived from nonpregnant and pregnant rats. A: representation of [Ca2+]i increase expressed as a function of actual K+ concentration in the medium; increase in [Ca2+]i was calculated after each addition of KCl, and steady-state response was subtracted by basal level. B: mean of area under the curve obtained in A for each set of experiments. Results are expressed as means ± SE. *** P < 0.001 vs. KCl (nonpregnant: n = 17 for KCl, n = 6 for BAY K 8644, and n = 7 for nifedipine; pregnant: n = 11 for KCl, n = 6 for BAY K 8644, and n = 5 for nifedipine).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha 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 alpha 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 (Delta ) 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.


    ACKNOWLEDGEMENTS

The authors thank Marie-Claude Gauthier for technical assistance and Sylvie Julien for secretarial work.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Endocrinol Metab 278(5):E925-E932
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