Molecular Signaling Group and Departments of Medicine and Biochemistry and Molecular Biology, The University of Louisville, Louisville 40202 and Veterans Affairs Medical Center, Louisville, Kentucky 40206
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ABSTRACT |
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Calcium transport across a monolayer of Madin-Darby canine kidney (MDCK) cells was measured in response to stimulation of the basal surface with calcium-sensing receptor (CaR) agonists. Stimulation of the CaR resulted in a time- and concentration-dependent inhibition of calcium transport but did not change transepithelial voltage or resistance. Inhibition of transport was not altered by pretreatment of cells with pertussis toxin but was blocked by the phospholipase C (PLC) inhibitor U-73122. To determine a potential mechanism by which the CaR could inhibit calcium transport, we measured activity of the plasma membrane calcium ATPase (PMCA). Stimulation of the CaR on the basal surface resulted in an inhibition of the PMCA in a concentration- and PLC-dependent manner. Thus stimulation of the CaR inhibits both calcium transport and PMCA activity through a PLC-dependent pathway. These studies provide the first direct evidence that calcium can inhibit its own transcellular absorption in a model of the distal tubule. In addition, they provide a potential mechanism for the CaR to inhibit calcium transport, inhibition of PMCA.
kidney tubules; distal; calcium-transporting adenosine 5'-triphosphatase; receptors; cell surface; kidney; ion transport; Madin-Darby canine kidney; plasma membrane calcium adenosine 5'-triphosphatase
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INTRODUCTION |
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CALCIUM, AN IMPORTANT INTRA- and extracellular cation, plays an essential role in diverse functions ranging from forming structural elements in bone matrix to intracellular signaling. Calcium homeostasis is maintained by changes in the function of the small intestine, where absorption of calcium is modulated; the skeleton, where both reabsorption and deposition into bone are regulated; and the kidney, where renal tubular reabsorption of filtered calcium is controlled on the basis of the body's overall need for calcium retention or loss (14). Because the kidney is the only one of these organs primarily involved in excretion, it has a dominant role in regulation of calcium metabolism (16). The renal tubule has the ability to vary its reabsorption of filtered calcium to adjust for changes in intake or increased calcium requirements. Under normal conditions, <1% of filtered calcium is excreted in the urine (16).
Calcium is reabsorbed throughout the nephron (26); however, most of the regulation of reabsorption occurs in the distal nephron. In the thick ascending limb of the loop of Henle (TAL), calcium reabsorption occurs through a primarily paracellular pathway, but active, transcellular reabsorption may occur as well during stimulation with parathyroid hormone (PTH) (16). In the distal convoluted tubule (DCT), calcium reabsorption occurs through an active transcellular process against an electrochemical gradient. Calcium enters DCT cells at the apical surface through a calcium channel, traverses the intracellular space, and exits the cell at the basolateral surface either through a sodium-calcium exchange mechanism or by a plasma membrane calcium ATPase (PMCA). About 10% of the filtered calcium is reabsorbed in the DCT. The amount of calcium reabsorbed in the DCT, however, represents a key portion for regulation of reabsorption (36). Because only a small percentage of filtered calcium is excreted in the urine, and the DCT is the last major sight for reabsorption of calcium, relatively small changes in the fraction reabsorbed in the DCT result in large changes in the total amount of calcium lost in the urine. Reabsorption in the DCT is regulated by PTH (14, 38). When serum calcium concentration decreases, PTH release from the parathyroid glands increases. This effect is mediated by the calcium-sensing receptor (CaR) in the parathyroid gland. A direct mechanism by which calcium could inhibit its own transcellular reabsorption in the distal tubule (either TAL or DCT) has not been described previously. Several lines of evidence support the hypothesis that the CaR is present on the basolateral surface of DCT where it could inhibit calcium transport. CaR mRNA has been identified in distal tubule (33, 43) and cultured distal tubular cells (4), and the receptor has been localized by immunocytochemistry to the basolateral surface of the distal tubule in rat kidney (32). Activation of the CaR at the basal surface of dissected rat distal tubules causes release of calcium from intracellular stores (30).
The Madin-Darby canine kidney (MDCK) cell line was originally derived
from dog kidney and retains a number of characteristics of distal
tubule, including the ability to form high-resistance monolayers that
polarize, express antigens of the distal tubule, and transport calcium
and other ions (20, 21, 28, 35). MDCK cells have also been
shown to be capable of transepithelial calcium transport that is
stimulated by PTH (23). Thus MDCK cells are a good model
for the transcellular calcium absorption that occurs in DCT. We have
grown MDCK cells on permeable supports and measured ion transport
across cell monolayers. This laboratory has demonstrated previously
that MDCK cells possess both mRNA (2) and protein
(3) for CaR and that the CaR couples to Gi
and Gq
heterotrimeric G proteins (2). We
hypothesized that the CaR in the MDCK cell model of distal tubule
inhibits transcellular calcium transport. In the present study, we
demonstrate that the CaR inhibits calcium transport across an MDCK cell
monolayer through a pathway that requires phospholipase C (PLC) but not Gi
. Inhibition of calcium transport is potentially
mediated through a basal calcium pump because the CaR inhibits PMCA
activity through a pathway that is also PLC dependent.
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METHODS |
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Materials. Trypsin-EDTA, MEM media containing Earle's salts and L-glutamine, and Ham's F-12 nutrient mixture media containing L-glutamine were obtained from GIBCO-BRL Life Technologies (Rockville, MD). [45Ca2+]CaCl2 and [32P]NAD were obtained from NEN (Boston, MA). Pertussis toxin was obtained from two sources, List Biologicals (Campbell, CA) and Biomol (Plymouth Meeting, PA). U-73122 and U-73343 were obtained from Biomol. Gadolinium (III) chloride hexahydrate was obtained from Aldrich (Milwaukee, WI). Falcon 3091 polyethylene terephthalate tissue culture inserts were obtained from Fisher Scientific (Pittsburgh, PA). Monoclonal antibody against PMCA was obtained from Affinity Bioreagents (Golden, CO). All other reagents were obtained from Sigma (St. Louis, MO). MDCK cells (line CCL-34) were obtained from American Type Culture Collection (Rockville, MD). Studies were done in either MDCK cells or a clonal line of MDCK cells that showed similar transport characteristics and inhibition of calcium transport by calcium (1).
Unidirectional calcium transport assays.
MDCK cells were grown in MEM media containing 10% fetal bovine serum
(FBS) without antibiotics. Cells were dispersed with trypsin-EDTA and
plated at 200,000 cells/well onto Falcon 3091 tissue culture inserts
containing 3.0-µm pores. The inserts were placed in six-well tissue
culture plates under sterile conditions and grown to confluence (~7
days). Confluence and generation of tight junctions were confirmed by
measurement of resistance across the membrane. We have previously shown
that calcium movement is more than six-fold higher in the apical to
basal direction than for basal to apical movement by using this model
(1). Cells were placed in Ham's F-12 medium (0.3 mM
Ca2+) with 10% FBS for 24 h before the study. Cells
were washed with 37°C transport buffer that contained (in mM) 136 NaCl, 16.5 HEPES, 5.4 KCl, 0.1 CaCl2, and 0.1 KH2PO4, pH 7.4. Unless otherwise indicated, calcium concentration in apical and basal compartments was 0.1 mM. The
cell culture inserts were transferred to a six-well tray containing
transport buffer, CaR agonists, and appropriate inhibitors in the lower
chamber. One milliliter of transport buffer containing calcium-45 (1 µCi/ml) was added to the cell culture insert above the cell
monolayer, and the assay was incubated at 37°C. At the end of the
designated time, the transport was stopped by removing the insert from
the tray. Calcium-45 transport was measured over 20 min unless
otherwise indicated. Aliquots (200 µl) from the lower chamber were
transferred to vials containing 5 ml of scintillation fluid and counted
on a Packard 2200CA liquid scintillation analyzer. Unidirectional
calcium transport was determined from the amount of calcium-45
appearing in the lower chamber. Unidirectional calcium transport was
expressed as picomoles per square centimeter determined by the
following formula
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Electrical measurements. Transepithelial voltage and resistance were measured by using a Millicell-ERS electrical resistance system (Millipore, Bedford, MA). Voltage (mV, apical surface relative to basal surface) and resistance were measured in five sets of inserts. Resistance was corrected for the contribution of the fluid and the insert itself and expressed as Ohms times square centimeters. MDCK cell monolayers were compared after 20 min of 0.1 or 5 mM calcium transport buffer on the basal surface. Both sets had transport buffer containing 0.1 mM calcium at the apical surface.
Pertussis toxin studies. To determine whether the effect of activation of the CaR was mediated through a pertussis toxin-sensitive G protein, monolayers of MDCK cells were treated with 200 ng/ml pertussis toxin for 16 h before transport assays. Calcium transport was measured as described above. Pertussis toxin from two different sources was used. To confirm that pertussis toxin completely ADP-ribosylated pertussis toxin-sensitive G proteins, in vitro ribosylation of membranes from cells that were treated with pertussis toxin, as described above, was compared with ribosylation seen in membranes from cells that were not pretreated with pertussis toxin. After treatment of monolayers with pertussis toxin, the media was aspirated and the cells were washed twice with cold PBS, then 400 µl of lysis buffer (5 mM Tris-2 mM EGTA-10 µl/ml Sigma protease inhibitor cocktail) were added and the cells were immediately scraped into a Dounce homogenizer. The insert was washed with an additional 400 µl lysis buffer that were transferred to the Dounce and homogenized. Homogenate was centrifuged for 30 min at 13,000 rpm. The pellet was resuspended in 150 µl of 50 mM Tris (pH 7.4), washed twice with the same buffer, and resuspended in a volume of 100 µl of ribosylation buffer [(in mM) 25 Tris (pH 8.0), 100 NaCl, 1 dithiothreitol (DTT), 2.5 MgCl, 0.5 EDTA, and 10 thymidine, as well as 10 µM NAD, 100 µM ATP, 10 µM GTP, 5 µg preactivated pertussis toxin (per condition)] (24) and 5 µCi [32P]NAD at 37°C. At the end of 1 h, the sample was centrifuged at 14,000 g for 15 min, separated on a 12% SDS-PAGE gel, and bands were visualized by autoradiography.
PMCA activity. MDCK cells were grown on permeable tissue culture inserts as described above. On day 5, the basolateral medium was changed to Ham's F-12 nutrient mixture with 10% FBS. On day 6, cells were treated for 20 min with buffer containing 130 mM NaCl, 16.5 µM HEPES, 5.4 µM KCl, 0.1 mM K3PO4, and the appropriate concentration of CaCl2. Immediately following treatments, inserts were transferred to six-well trays on ice containing chilled phosphate-buffered saline. Subsequent steps were performed on ice. Cells were harvested with cell scrapers into chilled lysis buffer containing 5 mM Tris, 2 mM EGTA, 20 µM DTT, and Sigma protease inhibitor cocktail (100 µl/107 cells). Cells were homogenized in a Dounce homogenizer and sonicated at the minimum setting with three short bursts (Braun-Sonic 2000). Cell debris was removed by centrifugation at 700 g for 3 min. Membranes were pelleted by centrifugation at 15,000 g for 30 min and resuspended in 50 mM Tris · HCl (pH 7.4), 0.3 M sucrose, 0.1 M KCl, 2 mM DTT, and Sigma protease inhibitor cocktail (100 µl/107 cells). PMCA activity of 10-20 µg of membranes was assayed by using a coupled enzyme system in which ATP hydrolysis is coupled to NADH oxidation with pyruvate kinase (PK), lactate dehydrogenase (LDH), and phosphoenolpyruvate (PEP), as previously described (12). The assay was performed in a 200-µl volume in a Costar UV-compatible 96-well plate at 37°C with a Bio-Tek Powerwave X plate reader. The assay substrate included 0.01 M TES, 0.1 M KCl, 420 µM PEP, 5 mM ATP, 5 mM MgCl2, 400 µM NADH, 7.5 U/ml PK, 18 U/ml LDH, 2 mM DTT, 50 nM calmodulin, 200 nM thapsigargin, and either 10 µM CaCl2 or 5 mM EGTA. Absorbance at 340 nm was monitored continuously for 3 min before and 13 min after addition of membranes. The difference in the rate of ATP hydrolysis in the presence of calcium and EGTA was used to calculate the calcium-ATPase activity. The use of thapsigargin eliminated contributions from internal membrane calcium ATPases.
Data analysis. Data were analyzed by using one-way repeated measures ANOVA followed by Tukey's test or by a paired t-test with Bonferroni correction for multiple comparisons.
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RESULTS |
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MDCK cells were grown to confluence on the tissue culture inserts
and developed electrically tight junctions. Calcium-45 transport was
measured across the monolayer and occurred in a time-dependent manner.
To determine whether stimulation of the CaR could inhibit calcium
transport across the monolayer, 5 mM CaCl2 was added to the
basal surface of the monolayer and calcium transport was measured over
a 20-min period (Fig. 1A).
Stimulation of the CaR resulted in an inhibition of unidirectional
calcium transport that was statistically significant after 10 min. To
determine whether the inhibition of calcium transport by the CaR
occurred within a physiologically relevant range, a
concentration-response curve with increasing concentrations of
CaCl2 was done (Fig. 1B). Stimulation of the CaR
on the basal surface of the MDCK cell monolayer inhibited calcium
transport over a range from 0.1 to 1.7 mM without a further decrease in
transport at concentrations as high as 10 mM CaCl2. To
confirm that changes in calcium concentration could inhibit calcium
transport in the physiological range, we stimulated the basal surface
of the monolayer with either 0.7 or 1.3 mM CaCl2 for 20 min. Stimulation with 1.3 mM calcium resulted in a 6% inhibition of
calcium transport relative to 0.7 mM (P < 0.01, n = 7). This indicates that the MDCK cell CaR inhibits
calcium transport over a physiologically relevant range.
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To confirm that calcium caused changes in transcellular calcium
movement and did not produce changes in the physical characteristics of
the membrane that would alter paracellular transport, we measured resistance and electrical potential across the MDCK cell monolayer. Changing basal calcium concentration from 0.1 to 5 mM did not alter
transmonolayer resistance (1,189 ± 197 vs. 1,581 ± 210 /cm2, n = 5) or transmonolayer potential
(
1.9 ± 0.3 vs.
1.6 ± 0.2 mV, n = 5).
These data demonstrate that changes in transepithelial calcium
transport were due to alterations in transcellular transport and not to
paracellular movement of calcium, and confirm the previous findings
that MDCK cells are capable of transcellular calcium transport.
To confirm that the inhibition of calcium transport produced by basal
administration of CaCl2 was mediated through the CaR and
not simply by a change in the concentration gradient for calcium across
the cell monolayer, we used two additional CaR agonists. Concentration-response curves were done with gadolinium and neomycin to
determine their effects on calcium transport after addition to the
basal surface. Both gadolinium (Fig.
2A) and neomycin (Fig. 2B) inhibited calcium transport in a concentration-dependent
manner. Inhibition by both agents occurred in the range that had been described previously for activation of the CaR.
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We had previously demonstrated that the CaR stimulates both
Gi and Gq
proteins in MDCK cells. To
determine whether Gi
proteins mediated the inhibition of
calcium transport by the CaR, MDCK cell monolayers were pretreated with
pertussis toxin (200 ng/ml, 16 h). Concentrations of CaR agonists
that gave a maximal response were used. Stimulation with the CaR
agonists calcium, gadolinium, and neomycin caused similar decreases in calcium transport. Pretreatment with pertussis toxin did not prevent the inhibition of calcium transport produced by basal administration of
the CaR agonists (Fig. 3,
A-C). To confirm that the treatment had ADP-ribosylated G
proteins, the ability of pertussis toxin to ADP-ribosylate G proteins
in membranes after overnight pertussis toxin treatment of intact cells
was evaluated. Membranes obtained from cells that were not pretreated
with pertussis toxin were able to be ADP-ribosylated by a 1-h treatment
with activated pertussis toxin in the presence of
[32P]NAD. Membranes from cells that were pretreated with
pertussis toxin did not show further ADP-ribosylation of G proteins
(Fig. 3D). These studies demonstrate that pertussis toxin
treatment of intact cells on tissue culture inserts fully
ADP-ribosylates the pertussis toxin-sensitive G proteins and that
Gi
proteins are not involved in the inhibition of
calcium transport by the CaR.
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To determine whether PLC was involved in the inhibition of calcium
transport by the CaR, the inhibitor U-73122 and its inactive analog
U-73343 were used (Fig. 4). U-73343 did
not effect the ability of the CaR to inhibit
JCa. U-73122 pretreatment increased basal
calcium transport across the cell monolayer compared with U-73343 and
blocked the ability of the CaR to inhibit calcium transport. Thus
whereas pertussis toxin-sensitive G proteins are not necessary for
the inhibition of calcium transport, PLC is required. In addition, PLC
may exert a tonic inhibition of calcium transport that is released by
treatment with U-73122.
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We next investigated a potential mechanism by which the CaR could
inhibit calcium transport, alteration of PMCA activity. PMCA activity
was measured in MDCK cells in response to stimulation of the CaR to
determine whether inhibition of PMCA was responsible for the decreased
calcium transport produced by the CaR. The basal surface CaR of the
intact MDCK cell monolayer was stimulated, as described, for
measurement of calcium transport. PMCA activity was compared in
purified membranes from cells exposed to low (0.1 mM) or high (5 mM)
calcium at the basal surface for 20 min before membrane preparation.
Basal PMCA activity in membranes from cells exposed to low calcium was
27 ± 3 nmol · min1 · mg
1.
Stimulation of the basal surface CaR with high calcium caused nearly
complete inhibition of PMCA activity (Fig.
5A). To determine whether CaR
activation inhibited PMCA activity in the relevant physiological range,
a concentration-response curve was done for PMCA activity after
stimulation of the basal surface of intact cell monolayers. Stimulation
of the basal surface CaR inhibited PMCA activity in a
concentration-dependent manner (Fig. 5B). Inhibition of PMCA
activity was nearly complete at a concentration of 1.4 mM calcium. In a
separate set of experiments, the effect of two calcium concentrations
on PMCA activity was compared. High calcium (1.3 mM) resulted in a 72%
reduction in PMCA activity compared with low calcium (0.7 mM,
P < 0.05, data not shown).
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Finally, to determine whether inhibition of PMCA by the CaR requires
PLC, MDCK cell monolayers were pretreated with the PLC inhibitor
U-73122 or its inactive analog U-73343 (5 µM, 1 h). After a
20-min treatment with or without calcium, cell membranes were prepared
and PMCA activity was determined and expressed as percent change
relative to the control (U-73343) without calcium (Fig.
6). Pretreatment with U-73122 increased
PMCA activity relative to treatment with U-73343 (P < 0.05). Membranes from cells pretreated with the control substance
showed the typical nearly complete inhibition of PMCA activity after
stimulation of the CaR for 20 min. In contrast, in cells pretreated
with U-73122, stimulation of the CaR did not cause a statistically
significant inhibition of PMCA activity. To determine the effect within
each treatment group, the effect of calcium on PMCA activity was
compared in U-73343- or U-73122-treated cells. CaR stimulation caused
an 84 ± 12% inhibition of PMCA in cells pretreated with U-73343.
In cells where PLC was inhibited, CaR stimulation resulted in a 35 ± 34% inhibition relative to U-73122 pretreatment alone. The response to CaR stimulation between the two groups was statistically significant (n = 6, P < 0.05, data not shown).
These results are consistent with the effect of inhibition of PLC on
calcium transport. The increase in calcium transport seen after
treatment with U-73122 may be mediated by an increase in activity of
the PMCA. Similarly, inhibition of PLC blocks the ability of the CaR to
inhibit both calcium transport and PMCA activity.
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DISCUSSION |
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The studies described herein provide evidence that the CaR
inhibits calcium absorption in a model of the renal distal tubule. Although inhibition of paracellular calcium reabsorption by the CaR in
the TAL has previously been postulated, this is the first evidence for
regulation of calcium transport by calcium via a transcellular pathway
in the distal tubule. Inhibition of the response by U-73122, but not
pertussis toxin, suggests that Gq, but not
Gi
, is involved. Furthermore, these studies demonstrate that inhibition of calcium transport by the CaR is caused, at least in
part, by inhibition of PMCA activity.
One possible mechanism by which addition of calcium to the basal surface of the monolayer could inhibit calcium transport is through a direct, nonreceptor-mediated effect based on the increased gradient against which the calcium is transported or an increase in calcium entry due to increased extracellular calcium concentration. The present study provides evidence that inhibition of calcium transport by calcium is mediated by the CaR. First, inhibition occurs across a range of calcium concentrations that includes physiological concentrations with little additional inhibition seen at higher calcium levels, as seen in Fig. 1. Second, the CaR agonists neomycin and gadolinium also inhibit calcium transport. The concentrations of gadolinium and neomycin used are consistent with its demonstrated potency at the cloned CaR (9, 17, 34). Third, the inhibition of the effect of the CaR by the PLC inhibitor U-73122 is most consistent with a receptor-mediated effect. Fourth, the effect was not due to a change in membrane resistance or electrical potential caused by higher concentrations of calcium, because these were measured and did not change. Finally, inhibition of PMCA activity by addition of calcium to the basal surface of the cell is most likely caused by a receptor, as the stimulation of the receptor occurred in the intact cell, whereas the activity was measured in isolated membranes that have been washed a number of times. In addition, the inhibition of PMCA by CaR is evidence that the effect on calcium transport is not mediated by a change in membrane potential but rather by a direct effect on the calcium pump.
The decrease in calcium appearing at the basal surface could also not be mediated by increased backflux of calcium-45. Initial concentration of "hot" calcium-45 in the apical compartment are 2.5 in 100 µM total ("hot" plus "cold") calcium. Thus, of the ~1,200 pmol/cm2 of calcium transport seen in Fig. 1, only 1/40 (30 pmol/cm2) is hot calcium-45. Corrected for the surface area of the membrane (4.2 cm2), this is 126 pmol of hot calcium-45 moving into the lower buffer containing 5 µM/ml cold calcium. Therefore, the specific activity of the apical solution is >100,000 times greater than that of the basal solution, even assuming that all of the apical-to-basal transport occurs immediately, which it does not. Because of the difference in specific activity, for increased backflux of calcium to account for the differences seen, the backflux would have to be 100,000-fold greater than apical-to-basal transport. We have shown that the backflux is more than sixfold less under resting conditions (1).
Hebert and colleagues (10) have proposed that activation of the basolateral CaR in the TAL inhibits calcium reabsorption from the TAL. This reabsorption occurs largely through a passive paracellular pathway driven by generation of a lumen positive gradient. Stimulation of the CaR in TAL inhibits apical potassium channels via a cytochrome P-450- (42) and phospholipase A2-dependent (41) pathway. Administration of calcium to the basal surface of intact TAL has also been shown to inhibit cAMP formation (39) and decrease chloride absorption in a phospholipase A2-independent manner (11). Inhibition of both the apical K channel and chloride reabsorption would be expected to decrease the lumen positive gradient and thus decrease calcium reabsorption. These data suggest, but do not prove, that calcium can regulate its own reabsorption in the TAL. In the absence of regulation of calcium reabsorption in the distal tubule, however, the effect of decreased TAL absorption would be diminished by increased reabsorption in the distal tubule, where up to 90% of delivered calcium can be absorbed (38).
In the distal tubule, calcium reabsorption occurs through an active
transcellular transport process (16). Calcium enters at
the apical surface through a calcium channel and exits the cell at the
basal surface via a sodium-calcium exchanger (NCE) or PMCA
(14). In the dog, unlike most other species, NCE
is primarily localized in the connecting tubule and not in DCT
(8). In MDCK cells, NCE functional activity
(27) and Northern blot for NCE (31) are both
absent. Therefore, the most likely mechanism for extrusion from the
cell at the basal surface is by PMCA. Previous studies have localized
both PMCA (5) and the CaR (32) at the basal
surface of the distal tubule. In addition, we have demonstrated both
functional and immunological evidence for polarized localization of CaR
to the basal surface of MDCK cells (3). Because both PMCA
and CaR are located at the basal surface, inhibition of PMCA by the CaR
is a potential mechanism by which calcium could inhibit net calcium
transport across the tubule. PMCA is positively and negatively
regulated by a number of intracellular substances including calmodulin,
proteases, protein kinases, and acidic phospholipids (29).
In the distal tubule, PMCA activity is stimulated by PTH (40), which provides an indirect feedback loop to regulate
calcium absorption. The present study demonstrates that one mechanism by which the CaR inhibits calcium absorption is by inhibiting PMCA
activity. The effect of the CaR on both calcium transport and PMCA
activity was inhibited by U-73122. Because the CaR activates Gq, a likely sequence of intracellular events is CaR
activation followed by activation of Gq
and PLC. This
could lead to a change in the phosphorylation state of PMCA or a change
in other regulators of PMCA activity. These data do not rule out the
possibility that calcium uptake into the cell or NCE activity in intact
tubules could also be regulated by the CaR. Until recently, the pathway for calcium entry into renal epithelia cells was unclear. Bindels and
colleagues (22) have identified a novel calcium channel cloned from renal cells that may be responsible for calcium entry; however, its regulation has not been described.
The DCT is the primary segment for regulation of calcium absorption by PTH, calcitonin, and vitamin D3 (16). PTH increases absorption of calcium in the distal tubule (6, 25, 26, 37) and in cell culture models of distal tubule (13, 18, 23). Calcitonin increases calcium transport in distal tubule cells (19). Vitamin D increases calcium absorption in distal tubular membranes (7) and cells (15). In contrast, stimuli that decrease calcium reabsorption in the distal tubule have not been identified. Therefore, the role of serum calcium as a negative regulator of calcium reabsorption is important in overall calcium homeostasis. As serum calcium increases, the CaR on the basal surface of distal tubular cells is activated and results in decreased reabsorption of calcium and thus increased urinary losses. Acting in concert with this effect is the suppression of PTH release by increased serum calcium concentration and removal of the stimulus for reabsorption.
These studies have demonstrated for the first time that calcium can inhibit its own transcellular absorption in a model of distal tubule. This represents an important physiological mechanism for calcium homeostasis. In addition, we have shown that the regulation of calcium absorption is mediated through inhibition of the PMCA. The mechanism by which the CaR inhibits PMCA remains to be determined, but the MDCK cell model will prove useful in determining the signal transduction pathways involved.
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ACKNOWLEDGEMENTS |
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This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-54924, a Scientist Development Grant from the American Heart Association, and a National Kidney Foundation Young Investigator Grant. Portions of this paper have been previously presented in abstract form (FASEB J 14: A340, 2000).
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. Arthur, Kidney Disease Program, Baxter Biomedical Science Bldg., 570 S. Preston St., Louisville, KY 40202.
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 27 October 2000; accepted in final form 11 January 2001.
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