Nifedipine-activated Ca2+ permeability in newborn rat cortical collecting duct cells in primary culture

Laura Valencia1, Michel Bidet2, Sonia Martial2, Elsa Sanchez1, Estela Melendez1, Michel Tauc2, Chantal Poujeol2, Dolores Martin1, Maria Del Carmen Namorado1, Jose Luis Reyes1, and Philippe Poujeol2

1 Departamento de Fisiología, Centro de Investigación y de Estudios Avanzados del Institúto Politécnico Nacional, Mexico City DF 07000, Mexico; and 2 Unite Mixte de Recherche-Centre National de la Recherche Scientifique 6548, Université de Nice-Sophia Antipolis, O6108 Nice Cedex 2, France


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To characterize Ca2+ transport in newborn rat cortical collecting duct (CCD) cells, we used nifedipine, which in adult rat distal tubules inhibits the intracellular Ca2+ concentration ([Ca2+]i) increase in response to hormonal activation. We found that the dihydropyridine (DHP) nifedipine (20 µM) produced an increase in [Ca2+]i from 87.6 ± 3.3 nM to 389.9 ± 29.0 nM in 65% of the cells. Similar effects of other DHP (BAY K 8644, isradipine) were also observed. Conversely, DHPs did not induce any increase in [Ca2+]i in cells obtained from proximal convoluted tubule. In CCD cells, neither verapamil nor diltiazem induced any rise in [Ca2+]i. Experiments in the presence of EGTA showed that external Ca2+ was required for the nifedipine effect, while lanthanum (20 µM), gadolinium (100 µM), and diltiazem (20 µM) inhibited the effect. Experiments done in the presence of valinomycin resulted in the same nifedipine effect, showing that K+ channels were not involved in the nifedipine-induced [Ca2+]i rise. H2O2 also triggered [Ca2+]i rise. However, nifedipine-induced [Ca2+]i increase was not affected by protamine. In conclusion, the present results indicate that 1) primary cultures of cells from terminal nephron of newborn rats are a useful tool for investigating Ca2+ transport mechanisms during growth, and 2) newborn rat CCD cells in primary culture exhibit a new apical nifedipine-activated Ca2+ channel of capacitive type (either transient receptor potential or leak channel).

calcium channel; dihydropyridine; kidney; newborn


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CALCIUM IS ONE OF THE MOST IMPORTANT IONS involved in the regulation of many physiological functions in all mammalian species. Ca2+ homeostasis is thus vital to all living species and is generally achieved by the joint effects of intestine, bone, and kidney functions. In the kidney, the key role of distal nephron segments in the maintenance of Ca2+ balance has been clearly established (13). Indeed, in these renal segments, transepithelial Ca2+ reabsorption from the tubular fluid occurs, mainly by a transcellular pathway, thus reducing Ca2+ excretion in the final urine. The actual mechanism of this phenomenon has not been fully described thus far. However, it is known to be a two-step process, i.e., passive apical reabsorption of Ca2+ from the tubular lumen down the Ca2+ gradient (which involves cell membrane channels or transporters that are yet to be characterized), followed by active basolateral extrusion of the ion toward the internal medium. It has been proposed that L-type Ca2+ channels may be responsible for the apical reabsorption of Ca2+ in mouse and rabbit epithelial kidney cells (34), whereas basolateral Na+/Ca2+ exchangers may be responsible for Ca2+ transfer to the blood (45). Hormonal regulation of these processes has also been demonstrated, especially in distal nephron cells, which are the target of several hormones such as parathyroid hormone, calcitonin, calcitriol, and [Arg]8-vasopressin (AVP) (29).

Although it is clear that young animals and humans require large amounts of Ca2+ to support developmental processes during all stages of body growth, there are few studies and a limited number of publications relevant to the role played by the immature kidney in Ca2+ retention during the early stages of life. This lack of information is mainly due to the absence of a satisfactory model of study of the kidney in newborn and young animals. In fact, morphological and immunological characterization of immature nephron cells is poorly documented so far.

To clarify the mechanisms underlying Ca2+ reabsorption in the kidney, we designed a model of study using newborn rat kidney cells in primary culture. Epithelial cells derived from well-defined nephron segments, in our case, cortical collecting duct (CCD) cells, were grown to form monolayers of differentiated cells according to the technique previously described by us for the primary culture of adult rabbit proximal convoluted tubule (PCT) cells (25), bright distal convoluted tubule (DCTb) cells (28), and CCD cells (24). This model allows free access to the apical membrane of the cells and absolute control of the composition of the apical medium, thus making it easier to study the first step of Ca2+ entry (i.e., apical absorption).

We performed fluorescence video microscopy experiments, using fura 2 as an indicator of intracellular Ca2+ concentration ([Ca2+]i). We particularly focused on the physiological properties and pharmacological characteristics of the apical Ca2+ channels involved in Ca2+ reabsorption in CCD cells.

We show here for the first time that newborn rat CCD cells in primary culture express apical Ca2+ channels that are activated by nifedipine and other dihydropyridines (DHP). The Ca2+ influx induced by nifedipine is strongly dependent on the extracellular Ca2+ concentration and is dramatically reduced by known Ca2+ channel inhibitors. So far, these channels have not been observed in adult rat CCD cells in primary culture, suggesting that they may be specific to the immature kidney. Therefore, their actual physiological role, especially their possible involvement in Ca2+ reabsorption in newborn and young animals, particularly in relation to their increased Ca2+ requirements, is now under close investigation.


    MATERIALS AND METHODS
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INTRODUCTION
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CCD Newborn Primary Cultures

Animals. All experiments were performed on Sprague-Dawley newborn rats (1 wk old). Animals were maintained with the dam until the day before the experimental procedure, and the kidneys were removed immediately before the study started.

Primary cultures. Kidneys were removed under sterile conditions from 6- to 8-day-old newborn rats anesthetized in a glass chamber with ether. Just before removal, both kidneys were perfused via the femoral artery with a Hanks' solution containing 600-700 U/ml collagenase (Worthington). The kidneys were then cut into small pyramids, incubated for 1 h at room temperature in aerated perfusion buffer containing 150 U/ml collagenase and 1 mM CaCl2, and then rinsed thoroughly in the same buffer devoid of collagenase. While still in the buffer, individual nephrons were hand dissected with the aid of a binocular microscope and stainless steel needles mounted on Pasteur pipettes. Tubules were transferred and seeded in collagen-coated 35-mm tissue culture dishes (6) filled with primary culture medium composed of an equal mixture of Dulbecco's modified Eagle's medium and Ham's F-12 (GIBCO BRL, Grand Island, NY) containing 15 mM NaHCO3, 20 mM HEPES, 2 mM glutamine (GIBCO BRL), 5 µg/ml insulin, 50 nM dexamethasone, 10 ng/ml epidermal growth factor (Promega), 5 µg/ml transferrin, 30 nM sodium selenite, 10 nM triiodothyronine, 100 U/ml penicillin, and 100 µg/ml streptomycin. The pH of the solution was 7.5. The insulin, dexamethasone, transferrin, and sodium selenite were purchased from Sigma Chemical (St. Louis, MO). The medium was changed 4 days after seeding and then every alternate day. Cultures were maintained at 37°C in a 5% CO2-95% air water-saturated atmosphere.

Fluorescence Experiments

Intracellular Ca2+ measurements. Monolayers of 1- to 5-day-old CCD cells grown on collagen-coated dishes were loaded for 45 min at 37°C with culture medium containing 5 µM fura 2-AM (Molecular Probes) and 0.01% Pluronic acid (Molecular Probes). The cells were then carefully rinsed three times with NaCl medium (in mM: 125 NaCl, 15 NaHCO3, 5 KCl, 1 CaCl2, 0.4 MgSO4, 5 glucose, 5 alanine, 2 glutamine, and 20 HEPES, pH 7.4). The culture dish was then mounted on the stage of the inverted microscope.

Experimental procedure. The optical system was the same as that described in our previous paper (3). Cells were successively excited at 350 and 380 nm, and each resulting image was digitized and stored on the hard disk of the computer. The acquisition rate used was one image every 5 s. Basal [Ca2+]i was determined from images recorded while cells were maintained in the NaCl medium. Test solutions containing nifedipine or other compounds as described below were then added to the apical surface of the culture and perfused over a 5-min period (at concentrations given in RESULTS). At the end of each experiment, the fluorescence signals relating to [Ca2+]i changes were calibrated as described by Cejka et al. (5). [Ca2+]i was calculated from the dual-wavelength fluorescence ratio using the Grynkiewicz equation (15).

Mn2+ influx measurements. Monolayers of 1- to 5-day-old cells were loaded with fura 2-AM as described above. Fluorescence-quenching experiments were started by addition of 50 µM MnCl2 to the NaCl medium. Fluorescent measurements were performed at 360 nm (isobestic point of fura 2, where the fluorescence signal is independent of the free Ca2+ concentration). The Mn2+ influx (representative of Ca2+ influx) was quantified by the slope of the quenching kinetics calculated over a period of 1 min in control and experimental conditions. Each raw image was the result of the integration of six frames averaged four times. The acquisition rate was one image every 5 s. Results were expressed as arbitrary fluorescence units (AFU), and the slopes were compared using paired Student's t-test.

Newborn Distal Tubules Isolation

Animals. Seven-day-old Wistar rats, both female and male, were used. They were grown in our animal house at room temperature (22-24°C) and 50-55% relative humidity. Animals were maintained with the dam until the day of the experiment. Dams were fed Purina chow (Purina Alief) and had access to tap water ad libitum. Care and handling of the animals were in agreement with internationally accepted procedures.

Isolation of renal tubules. Renal tubules from distal segments were isolated using the Percoll gradient technique described by Vinay et al. (43) and modified by Gonzalez-Mariscal et al. (14). Fifty rats aged seven days were decapitated. Both kidneys were rapidly removed and placed in 30 ml of ice-cold Krebs-bicarbonate solution (KB), at pH 7.4 and osmolality 290 ± 10 mosmol/kgH2O, in a 95% O2-5% CO2 atmosphere. The composition of KB was (in mM) 110 NaCl, 25 NaHCO3, 3 KCl, 1.12 CaCl2, 0.7 MgSO4, 2 KH2PO4, 10 sodium acetate, 5.5 glucose, and 5 alanine, plus 0.5 g/l bovine albumin. After decapsulation, the kidneys were halved, and the medulla was carefully dissected out. The resultant pieces of cortex were washed three times with 30 ml of the same ice-cold KB solution and resuspended in 25 ml of KB containing 0.15 g/100 ml collagenase (obtained from Clostridium histolyticum, type II; Sigma) and 0.5 ml of 10% (wt/vol) bovine albumin (fraction V). Digestion was carried out under shaking (40 rpm) at 37°C for 20 min. Tissue slices were then exposed for 20 min to a 95% O2-5% CO2 gas mixture. At the end of the digestion procedure, 30 ml of ice-cold KB were added, and the suspension was gently shaken to disperse the tissue fragments. The whole suspension was filtered through a metal strainer to remove the collagen fibers and then centrifuged at 12,200 g. The supernatant was discarded, and the tissue was rapidly resuspended in 30 ml of ice-cold KB. This washing procedure was repeated three times. After the last washing, the tissue pellet was resuspended in KB solution containing 5% bovine albumin for 5 min (4°C), spun again at 75 rpm for 1 min, and finally resuspended in 50% Percoll solution as described below.

Separation of nephron segments through a Percoll gradient. Percoll solution was freshly prepared before experiments. It was diluted to a final concentration of 50% with KB solution (final osmolality: 190 mosmol/kgH2O), exposed to a 95% O2-5% CO2 gas mixture, and then chilled to 4°C before use. Individual tissue pellets were prepared as described above and resuspended in 120 ml of ice-cold Percoll. This suspension was divided into four 30-ml aliquots, placed in four 50-ml polycarbonate centrifuge tubes, and spun for 30 min at 4°C and 15,000 rpm on a B-20 Beckman centrifuge equipped with a fixed-angle rotor head. This procedure separated the tissue content into four distinct bands enriched with different segments of the nephron (14).

Measurement of [Ca2+]i in fura 2-loaded tubules. Distal tubules were incubated with fura 2-AM (4 µM) for 30 min at room temperature. They were then rinsed three times with a modified Ringer solution that contained (in mM) 115 NaCl, 3 KCl, 1 MgSO4, 0.8 KH2PO4, 10 HEPES, 25 NaHCO3, 1 CaCl2, 5 glucose, and 5 leucine. Tubules were placed in a 3-ml quartz cuvette, and then fluorescence was measured with a dual-excitation spectrofluorimeter (Perkin Elmer LSB-50) equipped with a stirring device (23).

Effect of nifedipine on cytosolic Ca2+. To assess the effect of nifedipine on distal tubules of newborn and adult rats, we first recorded fluorescence under basal conditions for 3 min, and then nifedipine was added to the cuvette containing the tubules. [Ca2+]i was measured once the response was steady (plateau phase), and this concentration was used in the histogram. The steady-state time point was therefore variable from one experiment to another. Recording was followed for 40 min, and cytosolic Ca2+ was calculated (15).

Chemicals. Isradipine (racemic and enantiomers) was a kind gift of Dr. A. M. Suter (Novartis, Bern, Switzerland). All other products were obtained from Sigma.


    RESULTS
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Primary Cultures of Nephron Segment Microdissected From Newborn Rat Kidney

After microdissection and seeding, the tubules quickly adhere to the surface of the collagen, and cell growth occurs in the first 24 h. The cells proliferate as a monolayer for 7-10 days, after which the primary cultures start to regress. To check whether the monolayers contained apoptotic cells, we stained them with a 1% orcein solution (1 g of orcein, 100 ml of 70% ethanol, and 600 µl of 12 N HCl). In all 6- to 8-day-old primary cultures, the nuclear staining was diffuse and homogeneous, and chromatin was not condensed and fragmented, indicating the absence of any significant degree of apoptosis. All experiments were carried out in 3- to 7-day-old monolayers.

Effect of Vasopressin on Cytosolic Ca2+

V1 and V2 AVP receptors have been described in CCD cells of adult rat kidney (12). Therefore, we tested whether CCD cells of newborn rat were capable of preserving the same characteristics in primary culture. Addition of 100 nM AVP to the control solution evoked an increase in cytosolic Ca2+. However, in a given cell population, AVP induced two types of responses that exhibited different time courses (Fig. 1). The first type was observed in 30.7% of the cells (43/140 cells) and consisted of a gradual augmentation of [Ca2+]i (Fig. 1A). The second type of response was obtained in 27.1% of the cells (38/140 cells) and was characterized by an early transient increase in [Ca2+]i (Fig. 1B). The remaining 42.1% of the cells (59/140 cells) did not respond to AVP. These different AVP responses were observed in 11 separate experiments.


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Fig. 1.   Effect of 10-7 M [Arg]8-vasopressin (AVP) on cytoplasmic Ca2+ concentrations in cultured cortical collecting duct (CCD) cells. Representative results show the gradual increase in intracellular Ca2+ concentration ([Ca2+]i) in 7 of 18 individual cells (A), the transient increase in [Ca2+]i in 3 of 18 individual cells (B), and the means ± SE of the 7 cells in A () and the 3 cells in B (open circle ) as well as the 8 nonresponsive cells (black-triangle) of the same epithelium.

Effect of Nifedipine on Cytosolic Ca2+

Nifedipine is a well-known L-type Ca2+ channel blocker that has been demonstrated to strongly inhibit Ca2+ entry across the apical membrane of adult rat (unpublished results) and rabbit (33) distal tubules. Surprisingly, as shown in Fig. 2A, nifedipine resulted in an unusual effect in that it increased [Ca2+]i from 87.6 ± 3.3 nM to 389.9 ± 29.0 nM. This effect occurred in 65% of the cell population (n = 161/247 cells tested), whereas no significant [Ca2+]i variation was seen in the remaining 35% of cells ([Ca2+]i = 76.9 ± 3.1 nM; n = 86).


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Fig. 2.   Effect of nifedipine on [Ca2+]i in cultured CCD cells. A: representative results show the different profiles of responses observed after the addition of 20 µM nifedipine to CCD epithelium. Each plot represents the mean ± SE of 6 adult () or 13 newborn (open circle ) cells of the same epithelium. B: cytoplasmic Ca2+ variations (Delta [Ca]i) induced by the addition of 20 µM nifedipine to newborn and adult rat CCD cultures. Results represent the means ± SE of 161 of 247 cells from 13 newborn CCD primary cultures and 37 of 110 cells from 6 adult CCD primary cultures. *P < 0.01 vs. newborn.

Nifedipine effects were also assessed on primary cultures of proximal convoluted tubules from newborn rats. Nifedipine did not modify the cytoplasmic Ca2+ concentration in any of the 74 cells analyzed (data not shown).

It was of interest to demonstrate that the paradoxical nifedipine effect on CCD cells in primary culture was only observed in newborn rat renal tissue. For this purpose, further experiments were performed in primary cultures of CCD microdissected from adult rat kidney (2-mo-old rats). In these cultures, an increase in [Ca2+]i was also observed in the presence of nifedipine, but the percentage of responding cells and the amplitude of the response were significantly lower than those determined in newborn tissue. In this way, the Ca2+ response to nifedipine was observed in 37% of the cells, with a variation in [Ca2+]i (Delta [Ca2+]i) from 80.8 ± 4.2 nM to 213.3 ± 23.5 nM. This result is illustrated in Fig. 2B, which shows the Delta [Ca2+]i induced by nifedipine in both adult and newborn rat cells (CCD cells from adult rats: Delta [Ca2+]i = 132.5 ± 22.8 nM, n = 37; CCD from newborn rats: Delta [Ca2+]i = 302.3 ± 29.0 nM, n = 161; P < 0.01).

To compare the results obtained from cells in primary culture with those having an intact structure, we also performed fura 2 experiments on isolated distal tubules in suspension. As shown in Fig. 3A, the addition of nifedipine (20 µM) to newborn isolated rat distal tubules enhanced [Ca2+]i within 15 min but did not change [Ca2+]i in adult rat CCDs. Maximal [Ca2+]i was 214.0 ± 40.6 nM in newborn (n = 3) vs. 98.8 ± 7.9 nM in adult (n = 4) rats. Figure 3B shows that [Ca2+]i varied as a function of the concentrations of nifedipine used. Values for Delta [Ca2+]i obtained with 1, 10, and 20 µM nifedipine were 53.5, 104.7 ± 27.4, and 139.7 ± 32.3 nM, respectively. In contrast, no variation of [Ca2+]i was observed when nifedipine (10-50 µM) was added to isolated distal tubules from adult rat.


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Fig. 3.   Effect of nifedipine on [Ca2+]i in isolated newborn rat distal tubules in suspension. A: representative traces show the effect of 20 µM nifedipine on [Ca2+]i of newborn (top trace) and adult (bottom trace) distal tubules loaded with fura 2. Arrow indicates time of nifedipine addition. [Ca2+]i increase started 18 min after nifedipine was added to the suspension and reached the steady state 2 min later. Each trace is representative of 3 (top) or 4 (bottom) different experiments. B: Delta [Ca2+]i induced by the addition of 1, 10, or 20 µM nifedipine to fura 2-loaded newborn rat tubules. Results represent the means ± SE of 3 different tubule suspension preparations.

Role of Intracellular and Extracellular Ca2+ in the Nifedipine Response

To determine the mechanisms involved in the nifedipine-induced Ca2+ increase, we investigated the role of intracellular Ca2+ stores in CCD cells by using thapsigargin (TG), an irreversible sarcoplasmic reticulum (SR) Ca2+-ATPase inhibitor. TG was thus used to empty the intracellular pools. In the presence of 0.5 µM TG (Fig. 4), [Ca2+]i increased from a basal value of 62.3 ± 5.1 nM to a peak value of 246 ± 19.2 nM within 45 s. [Ca2+]i returned to its basal value within 12-15 min. This rise and fall of Ca2+ was consistent with the known action of TG in various cell types. Under these experimental conditions, the addition of nifedipine (20 µM) provoked a rise in [Ca2+]i up to 439.1 ± 39.0 nM (n = 14). This data strongly suggests that the response to nifedipine was not due to Ca2+ release from internal stores. Consequently, the influence of external Ca2+ on the nifedipine-induced response was studied. Figure 5A shows that removal of external Ca2+ (NaCl buffer plus 0.5 mM EGTA in the absence of added Ca2+) totally blocked the nifedipine-dependent Ca2+ increase, since this maneuver led to a [Ca2+]i decrease of 24.8 ± 5.9 nM (n = 3), while returning the cells to a normal Ca2+ buffer restored the nifedipine response. This shows the dependence of nifedipine-induced [Ca2+]i increase upon exposure to external Ca2+.


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Fig. 4.   Effect of thapsigargin on nifedipine-induced increase in [Ca2+]i. Newborn CCD monolayers were first incubated for 15 min with 10 µM thapsigargin (TG). Nifedipine (Nif, 20 µM) was then added in the presence of TG. The trace represents the means ± SE of 15 cells from the same CCD culture and is representative of 3 experiments performed on different monolayers.



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Fig. 5.   Effect of extracellular Ca2+ on the nifedipine-induced increase in [Ca2+]i. A: representative results show the response of the cells after 20 µM nifedipine was added in the presence and absence of external Ca2+. During nifedipine exposure, the NaCl buffer was replaced by a Ca2+-free NaCl buffer containing 0.5 mM EGTA (horizontal bar). The trace represents the means ± SE of 4 cells from the same CCD culture and is representative of 3 experiments performed on different monolayers. B: effect of 20 µM nifedipine on Mn2+ influx. Mn2+ entry into the cytoplasm was monitored by fluorescence-quenching measurements, using an excitation wavelength of 360 nm, in fura 2-loaded CCD cultures. The initial slope was the result of the perfusion of NaCl buffer containing 50 µM MnCl2 (arrow). Nifedipine addition increased the slope of the curve, demonstrating an increased Ca2+ influx into the cells. The trace represents the means ± SE of 9 () and 5 (open circle ) cells of the same epithelium and is representative of 3 experiments performed on different monolayers.

To provide evidence that nifedipine induced an increase in Ca2+ entry, we took advantage of the property of fura 2 fluorescence being quenched by Mn2+, a commonly used substitute for Ca2+. The fluorescence at 360 nm (isobestic point for fura 2) is independent of cytosolic Ca2+. Thus only fura 2 quenching due to Mn2+ uptake by the cells is able to modify the fluorescence signal at this wavelength. When cells were perfused with a NaCl solution containing 50 µM MnCl2, an immediate decrease in fluorescence was observed in newborn rat CCD cells in primary culture. As shown in Fig. 5B, nifedipine (20 µM) accelerated the rate of quenching in 20 of 24 cells, thus indicating an enhanced influx of divalent cations (control: 0.13 ± 0.01 AFU/s; nifedipine: 0.20 ± 0.04 AFU/s; P < 0.01).

Pharmacology of the Nifedipine Response

To investigate the pharmacological properties of the nifedipine-induced Ca2+ entry, we then tested the effect of BAY K 8644, a DHP compound known to activate L-type Ca2+ channels. As observed with nifedipine, the addition of BAY K 8644 (20 µM) produced an increase in [Ca]i from 76.7 ± 7.7 nM to 580.3 ± 71.3 nM in 25 of 47 cells. This effect was significantly higher than that induced by nifedipine for the same experimental conditions (Delta [Ca]i = 503.5 ± 69.3 nM, n = 25 cells, vs. 302.3 ± 29.0 nM, n = 161 cells; P < 0.001).

Similarly to the DHP, phenylalkylamines (PAs) and benzothiazepines (BTZs) act on the alpha 1C-subunit of the L-type Ca2+ channels (37). In the present study, the addition of either verapamil (20 µM) or diltiazem (20 µM) to the bathing medium did not modify [Ca2+]i (n = 4 experiments).

The Mn2+-quenching technique was then used to study the rate of Ca2+ entry induced by different DHP compounds. Results for these experiments are reported in Fig. 6A. BAY K 8644 (20 µM) was more efficient than nifedipine in increasing Ca2+ entry into newborn rat kidney CCD cells in culture (control: 0.19 ± 0.02 AFU/s; BAY K 8644: 0.72 ± 0.08 AFU/s; n = 39/43 cells; P < 0.001). Isradipine (PN-200-110) is used as a reference molecule for the DHP receptor because of its high affinity for the DHP binding site (30). The addition of 20 µM isradipine evoked an increase in Ca2+ entry similar to that induced by 20 µM BAY K 8644 (control: 0.12 ± 0.01 AFU/s; isradipine: 0.86 ± 0.05 AFU/s; n = 42/42 cells; P < 0.001). We also compared the sensitivity of the cells to the S (+) and R (-) purified isoforms of isradipine to the racemic one (Fig. 6B). At 1 µM, both enantiomers evoked the same typical augmentation of Ca2+ entry into the cell [0.15 ± 0.01 (control) vs. 0.26 ± 0.02 AFU/s (S-isradipine); n = 16/27 cells; P < 0.001; and 0.13 ± 0.02 vs. 0.26 ± 0.02 AFU/s (R-isradipine); n = 17/25 cells; P < 0.001].These two values were similar to the racemic one (control: 0.13 ± 0.02 AFU/s; isradipine: 0.23 ± 0.02 AFU/s; n = 15/24 cells; P < 0.01).


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Fig. 6.   Effect of various dihydropyridines (DHPs) on Mn2+ influx into CCD cells. A: Mn2+ influx into the cells was initiated by 50 µM MnCl2 and monitored as described in Fig. 5. The slope obtained after the addition of 20 µM nifedipine, 20 µM BAY K 8644, or 20 µM isradipine (shaded bars) was compared with the initial slope (open bars). Results are expressed as means ± SE for 20 (nifedipine), 39 (BAY K 8644), or 42 (isradipine) cells from 3-5 experiments performed on different CCD cultures. *P < 0.01, **P < 0.001. B: effect of both enantiomers of isradipine on Mn2+ influx into CCD cells. The slopes of the fura 2 quenching obtained before (open bars) and after (shaded bars) addition of the racemic and both enantiomer forms of isradipine (1 µM). Results are expressed as means ± SE for 15 (isradipine), 16 [S(+)-isradipine], and 17 [R(-)-isradipine] cells from 3 experiments performed on different CCD cultures. *P < 0.01.

Effect of Ca2+ Channel Blockers on the Nifedipine-Dependent [Ca2+]i Increase

To further characterize the nifedipine-sensitive [Ca2+]i increase, we tested the effect of different Ca2+ channel blockers. As shown in Fig. 7, the trivalent cations lanthanum (La3+) and gadolinium (Gd3+) significantly reduced the increase in [Ca2+]i induced by nifedipine in a dose-dependent manner. However, La3+ was only active at very high concentrations, i.e., 100 µM La3+ was necessary to achieve significant (82%) inhibition. More specific L-type Ca2+ channel inhibitors belonging to the PA and BZT families were also tested. In contrast to verapamil, diltiazem (20 µM) significantly inhibited the nifedipine effect (62%).


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Fig. 7.   Effect of Ca2+ channel inhibitors on the nifedipine-induced increase in [Ca2+]i. Histogram represents the effect of nifedipine on Delta [Ca]i in the absence (open bars) or presence of different Ca2+ channel inhibitors. Results are expressed as means ± SE for n cells from 3-5 experiments performed on different CCD cultures; n is indicated in parentheses above each bar. *P < 0.05, **P < 0.01, ***P < 0.001.

Role of K+ movements in the Nifedipine-Dependent [Ca2+]i Increase

To investigate the role of K+ on the [Ca2+]i increase induced by nifedipine, we performed experiments on newborn rat CCD monolayers previously treated with the antibiotic valinomycin to abolish the apical gradient of K+ while totally depolarizing the cells. Results for these experiments are shown in Fig. 8A. As observed with rabbit DCT in primary culture (34), valinomycin (10 µM) induced a small rise in [Ca2+]i from 77.9 ± 5.2 nM to 110.7 ± 11.2 nM in 21/28 cells immediately after valinomycin application. The subsequent application of nifedipine always produced an increase in [Ca2+]i (to 481.1 ± 96.7 nM; n = 21/28 cells). As shown in Fig. 8B, the variation in [Ca2+]i induced by nifedipine in the presence of valinomycin (Delta [Ca2+]i = 296.0 ± 69.3 nM; n = 21) was not significantly different from that measured in the absence of the antibiotic (302.3 ± 29.0 nM; n = 161). Hence, nifedipine-induced [Ca2+]i increase was not related to K+ flux or transmembrane potential. To confirm this result, we blocked K+ channels by using BaCl2 (5 mM). This maneuver did not affect the nifedipine-induced [Ca2+]i increase from 128.9 ± 18.2 nM to 406.5 ± 46.5 nM in 25 of 32 cells {Delta [Ca2+]i = 277.6 ± 40.6 nM (nifedipine + Ba2+; n = 25) vs. 302.3 ± 29.0 nM (nifedipine alone; n = 161); P = not significant; Fig. 8B}.


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Fig. 8.   Effect of K+ permeability on the nifedipine-induced increase in [Ca2+]i. A: representative results show the response observed after 20 µM nifedipine was added (arrow) in the presence of 10 µM valinomycin (horizontal bar). The trace represents the means ± SE for 14 cells of the same epithelium and is representative of 3 experiments performed on different monolayers. B: histogram compares the effect of nifedipine on Delta [Ca]i in the presence of valinomycin (Valino, 10 µM) or BaCl2 (Ba2+, 5 mM) with the effect of nifedipine alone (control). Results are expressed as means ± SE for 160 (control), 21 (valinomycin), and 25 (Ba2+) cells from 3-16 experiments performed on different CCD cultures.

Presence of Ca2+ Leak Channels

It has been reported for different types of myocytes that a class of leak channels permeable to Ca2+ could be activated by nifedipine (21, 44). These Ca2+ channels can also be activated by free radicals. Therefore, the technique of Mn2+ quenching of fluorescence was used to study the effect of H2O2 (as a representative free radical) on Ca2+ entry in cultured CCD cells. Figure 9A clearly shows that the addition of H2O2 (100 µM) strongly activated Ca2+ influx into the cells (control: 0.14 ± 0.01 AFU/s; H2O2: 0.42 ± 0.02 AFU/s; n = 34/54 cells; P < 0.001). Ratiometric fura 2 experiments were also performed to measure the effect of H2O2 on [Ca2+]i. As shown in Fig. 9B, exposure of cells to H2O2 led to a significant increase in [Ca2+]i only in the absence of the polycationic compound protamine (100 µM), an inhibitor of the Ca2+ leak channel (7). This demonstrates the presence of Ca2+ leak channels in our CCD cell preparation. It is of interest though to note that protamine did not inhibit the nifedipine-induced increase in [Ca2+]i measured by fura 2 fluorescence (data not shown), which leaves open the question of whether the newborn rat CCD cell nifedipine-activated Ca2+ channel is really a Ca2+ leak channel.


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Fig. 9.   Effect of H2O2 on CCD cells. A: effect of H2O2 on Mn2+ influx. Mn2+ influx into the cells was initiated by 50 µM MnCl2 and monitored as described in Fig. 5. The initial slope was the result of the perfusion of the NaCl buffer containing 50 µM MnCl2 (arrow). Subsequent addition of 100 µM H2O2 increased the slope of the curve, demonstrating an increased rate of Mn2+ entry into the cell. The trace represents the means ± SE for 9 () and 5 (open circle ) cells of the same epithelium and is representative of 3 experiments performed on different monolayers. B: effect of H2O2 on [Ca2+]i. Representative results show the response observed after the addition of 100 µM H2O2 (horizontal bars) in the presence and absence of 100 µM protamine. Values are means ± SE.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To study the transport of Ca2+ in CCD cells from newborn rat, we established primary cultures of CCD cells. Among the techniques commonly used for the preparation of monolayers, we chose to culture defined segments of CCD obtained by microdissection. The main advantage of this technique is the accurate identification of the nephron segment inoculated on the collagen-coated culture dish. Primary cultures of microdissected tubules from rabbit form monolayers that retain several characteristics of the in situ epithelia (24, 40). To ensure that rat CCD primary cultures resembled the original epithelium, we tested the effect of AVP on intracellular Ca2+ levels. Indeed, CCD is a major target of AVP, which stimulates net Na+, K+, and H2O transport in this segment. Moreover, we showed (unpublished observations) that apical AVP triggered [Ca2+]i increase in newborn rat CCD cells in culture at concentrations of the hormone close to, or lower than, regular luminal concentrations (10-10-10-6 M). The AVP-dependent Ca2+ increase we obtained in the present paper clearly shows that CCD cells in primary culture retain functional vasopressin receptors. Because both V1 and V2 receptors have been reported to exist in rat CCD (16, 22, 31, 36, 41), the heterogeneity of Ca2+ responses that we observed can be explained by either the expression of both V1a and V2 receptors in our primary cultures or the cell heterogeneity of the preparation.

In our experiments, CCD cells showed two types of response after AVP application. The transient increase in [Ca2+]i is in agreement with the binding of AVP to V1a receptors, inducing the activation of phospholipase C and inositol 1,4,5-trisphosphate (IP3), and finally leading to the release of Ca2+ from intracellular stores (1). The slow increase in [Ca2+]i could be due to the stimulation by AVP of an apical membrane Ca2+ permeability via a presently unidentified signaling pathway. The presence of DHP-sensitive apical Ca2+ channels (L-type Ca2+ channels) in the distal nephron of the mammalian kidney is now well established (26, 27, 33). We thus tested the hypothesis that these channels might be involved in the AVP-dependent slow increase in [Ca2+]i and attempted to block them with nifedipine, a known inhibitor of L-type Ca2+ channels. Surprisingly, nifedipine induced a large increase in [Ca2+]i, an unusual behavior for this drug that is normally used as a Ca2+ flux inhibitor in several epithelia and particularly in renal tissue (33, 34). This nifedipine-induced [Ca2+]i increase appears to be specific of DCT tissue, because it did not occur in primary cultures of proximal cells. Moreover, this phenomenon is drastically reduced, both in frequency and quantitatively, in CCD monolayers obtained from adult rat. Similar results obtained with distal tubule suspensions confirm that Ca2+ permeability activated by nifedipine is a characteristic of newborn rat. It can be pointed out that, for primary culture experiments, we worked with very young adults (8 wk old) because the dissection is easier. However, although these rats can already breed (and therefore can be considered adults), their kidney development is not yet complete. It is likely that they still retains some features of newborn rat kidney, among which is the nifedipine-activated Ca2+ channel, although in lesser amount, thus explaining the weak increase in [Ca2+]i observed with adult rat primary culture. This result also attests to the fact that the nifedipine effect is not a consequence of cell differentiation during the growth of the culture. We cannot be sure that the nifedipine response in tubule suspension is the same as that obtained with the CCD primary culture. However, nifedipine response was either strongly reduced (CCD primary cultures) or totally absent (tubule suspensions) in adult vs. newborn renal preparations, which in both cases suggests the absence of nifedipine-related Ca2+ changes in adult CCD. The different time courses in Ca2+ increase between the two models can be explained by the likely apical localization of the nifedipine binding site. Indeed, in culture experiments, nifedipine is directly applied onto the apical membrane of the cells, thus triggering a quick response. By contrast, in the tubule suspension model, the drug needs to gain access to the apical membrane, which may be more difficult because, as previously described, isolated tubules tend to collapse in suspension. This, rather than a major difference in differentiation state, seems to be the most reasonable explanation, because, as described by Jamous et al. (24), the 3- to 5-day-old CCD cell culture that we used already possesses most of the features of intact CCD epithelium, especially the vasopressin receptors. However, we cannot totally discard the latter hypothesis, and we cannot neglect the fact that distal tubule suspensions may contain more than CCD cells (i.e., DCT cells), which may alter their characteristics compared with those of purified CCD cells.

Experiments performed on CCD cells pretreated with TG gave us initial proof in favor of a major role for external Ca2+ in the increase of [Ca2+]i induced by nifedipine. Preventing the Ca2+ increase by removing external free Ca2+ and adding EGTA confirmed the importance of external Ca2+ and, thus, the action of nifedipine on the Ca2+ permeability of the membrane. We then obtained direct evidence for Ca2+ entry into CCD cells by using Mn2+. In this way, increased Mn2+ uptake after the addition of nifedipine indicates increased Ca2+ uptake. These results suggest that the target of nifedipine could be a Ca2+ channel present on the apical membrane of CCD cells from newborn rat in primary culture. We also investigated the role of other DHP compounds. The most specific agonist of L-type Ca2+ channels, BAY K 8644 (48), stimulated both an increase in [Ca2+]i and an influx of Mn2+, as does nifedipine. To further confirm the DHP specificity, we used isradipine, a highly potent Ca2+ channel antagonist, commonly used as a reference because of its high affinity for the DHP receptor (30). In our experiments, isradipine induced a strong increase in [Ca2+]i.

Many studies have been carried out that have attempted to correlate the affinity of DHP for their receptor, along with their effects on the Ca2+ channel. Most of these were done using [3H]isradipine and led to the conclusion that multiple binding sites with different affinities for DHP exist on L-type Ca2+ channels (9, 39). According to this latter characteristic, we studied the stereoselectivity of the DHP-induced Ca2+ response in CCD cells by using the two enantiomers of isradipine. Despite their different affinities for the DHP binding site on the L-type Ca2+ channel (19), S- and R-isradipine had similar effects on the stimulation of Mn2+ influx.

The trivalent cations Ld3+ and Gd3+ significantly inhibited the nifedipine effect on [Ca2+]i, but only at concentrations ranging between 10 and 100 µM. This could be due to a nonspecific form of inhibition. The wide-ranging pharmacological characterization of the L-type Ca2+ channel has resulted in the classification of agonist/antagonist drugs into three main, yet structurally dissimilar, groups: DHPs, PAs, and BTZs. It is well established that PA and BTZ binding sites are different from the DHP receptor site on Ca2+ channels. Nevertheless, it has been proven that the DHP binding site and especially DHP affinities can be affected after the binding of PA or BTZ to their own receptor site (30, 37). We thus investigated such a possibility in the newborn rat CCD cells in culture. To do so, we tested the effects of verapamil (a PA) and diltiazem (a BTZ) on the nifedipine-induced [Ca2+]i increase. Only diltiazem significantly blocked the rise in [Ca2+]i, suggesting that the rise may result from Ca2+ influx via channels structurally analogous to voltage-gated Ca2+ channels. The identity of adult (inhibition) and newborn (stimulation) nifedipine binding sites on Ca2+ channels remains to be elucidated.

The concentrations of nifedipine generally used to test its inhibitory effects on Ca2+ channels are in the 0.1-1 µM range. It has been shown that the use of nifedipine concentrations of 10 µM or more could interfere with K+ channel activity (10, 11). However, given that the nifedipine-dependent [Ca2+]i increase was maintained after valinomycin or Ba2+ application, even though 20 µM nifedipine was used here, we can conclude that K+ channels were probably not implicated in the DHP-sensitive influx of Ca2+.

An increase in Ca2+ entry after the application of various DHPs has previously been shown by Coulombe et al. (8) using rat ventricular myocytes and Hopf et al. (21) with dystrophic mdx muscle cells. This increase resulted from Ca2+ influx via specific Ca2+ leak channels. The physiological significance of these channels was not defined, but it was suggested that their activity could account for the resting Ca2+ conductance. In newborn CCD cells, the nifedipine-induced Ca2+ influx may take place through Ca2+ leak channels. Indeed, our preparation exhibited some of the characteristics reported by Wang et al. (44) for this kind of channel. First, the results from both studies show similar profiles in relation to the activation of Ca2+ entry after exposure to nifedipine and BAY K 8644. Second, Wang et al. noted that Ca2+ leak via K+ channels was unlikely because effective K+ channel blockers such as Ba2+ (5 mM) did not block Ca2+ entry. Third, the activity of Ca2+ leak channels was augmented by H2O2. However, it must be pointed out that, in our case, polycationic compounds such as protamine, which inhibits Ca2+ leak channels (7, 44), only inhibited the H2O2-dependent Ca2+ increase and not the nifedipine-dependent effect.

To date, it is clear that at least two different families of Ca2+ channels coexist in the apical membrane of DCT cells. First, isoforms of the L-type Ca2+ channels, extensively studied in excitable cells, are present in DCT cells as confirmed by the molecular characterizations of the alpha - and beta -subunits (2, 46, 47). The second type of channel appears to be more specific to epithelial cells. In DCT cells, the presence of one of these channel types, i.e., the recently cloned epithelial Ca2+ channel (ECaC), the molecular structure and electrophysiological properties of which have been described in extensive studies carried out by Bindels' group (17, 18, 42), is now well established. This channel presents some of the molecular structural characteristics of the transient receptor proteins (TRP) and the capsaicin receptor VR1 (17). The TRP channels mediate capacitive Ca2+ currents (35), as does the Ca2+ leak channel (20). However, the entry of Ca2+ via TRP follows the depletion of Ca2+ stores and is activated by either IP3 or a decrease of luminal [Ca2+] (4). The similar kinetics and pharmacological characteristics of the Ca2+ permeability presented in our study support the hypothesis that the Ca2+ permeability of rat newborn CCD cells in culture could correspond to a Ca2+ leak channel-like mechanism. At worst, we can postulate that the protein in charge of the DHP-sensitive Ca2+ entry in CCD cells belongs to the large cationic channel family composed of six transmembrane-domain proteins, which includes ECaC, CaT1 (Ca2+ transporter 1) (32), SIC (stretch-inhibitable nonselective cation channel) (38), and TRP.

In conclusion, this study presents the characterization of a newly identified Ca2+ permeability in the membrane of cultured CCD cells isolated from newborn rat. This DHP-activated Ca2+ channel, which has a heightened expression in the newborn kidney compared with the adult kidney, might be a specific Ca2+ influx pathway in the newborn. This permeability is located in the distal segments of the nephron and could represent the main apical pathway for transepithelial Ca2+ transport occurring in this segment. Together, the high sensitivity of the channel to DHP and the strong Ca2+ influx induced by such compounds fit well with the activity of Ca2+ channels in CCD cells. Our results support the hypothesis of a large Ca2+ reabsorption in the distal part of the newborn rat renal tubule, regulated by vasopressin.

Finally, these results indicate that an important and atypical Ca2+ transport pathway exists in the apical membrane of CCD cells of newborn rats. This Ca2+ transport is enhanced in the newborn compared with the adult rat. This characteristic reflects the fact that Ca2+ reabsorption by renal cells (hence, apical Ca2+ channel activity) may be important during growth, although it remains to be shown whether these channels also exist in vivo. It is also possible that these channels be a feature of immature cells, and, as such, they might exist in other cell types. The type of Ca2+ channel involved in CCD cells now remains to be identified with electrophysiological and molecular approaches.


    ACKNOWLEDGEMENTS

This work was supported by the Program of Scientific Collaboration Mexico-France, ANUIES-ECOS (M96-B02), with the participation of Consejo Nacional de Ciencia y Tecnología, Mexico. L. Valencia, E. Melendez, and E. Sanchez received fellowships from Secretaria de Educacíon Pública, Mexico.


    FOOTNOTES

Address for reprint requests and other correspondence: P. Poujeol, UMR CNRS 6548, Bâtiment Sciences Naturelles Université de Nice-Sophia Antipolis, Parc Valrose, O6108 Nice Cedex 2, France (E-mail: poujeol{at}unice.fr).

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. Section 1734 solely to indicate this fact.

Received 29 June 2000; accepted in final form 6 December 2000.


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