Extracellular Ca2+ regulates the stimulation of Na+ transport in A6 renal epithelia

Danny Jans,1 Jeannine Simaels,2 Els Larivière,2 Paul Steels,1 and Willy Van Driessche2

1Laboratory of Physiology, Biomedical Research Institute, Limburgs Universitair Centrum, Universitaire Campus, B-3590 Diepenbeek; and 2Laboratory of Physiology, Katholieke Universiteit Leuven, Campus Gasthuisberg O/N, B-3000 Leuven, Belgium

Submitted 3 November 2003 ; accepted in final form 8 June 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We investigated the involvement of intracellular and extracellular Ca2+ in the stimulation of Na+ transport during hyposmotic treatment of A6 renal epithelia. A sudden osmotic decrease elicits a biphasic stimulation of Na+ transport, recorded as increase in amiloride-sensitive short-circuit current (Isc) from 3.4 ± 0.4 to 24.0 ± 1.3 µA/cm2 (n = 6). Changes in intracellular Ca2+ concentration ([Ca2+]i) were prevented by blocking basolateral Ca2+ entry with Mg2+ and emptying the intracellular Ca2+ stores before the hyposmotic challenge. This treatment did not noticeably affect the hypotonicity-induced stimulation of Isc. However, the absence of extracellular Ca2+ severely attenuated Na+ transport stimulation by the hyposmotic shock, and Isc merely increased from 2.2 ± 0.3 to 4.8 ± 0.7 µA/cm2. Interestingly, several agonists of the Ca2+-sensing receptor, Mg2+ (2 mM), Gd3+ (0.1 mM), neomycin (0.1 mM), and spermine (1 mM) were able to substitute for extracellular Ca2+. When added to the basolateral solution, these agents restored the stimulatory effect of the hyposmotic solutions on Isc in the absence of extracellular Ca2+ to levels that were comparable to control conditions. None of the above-mentioned agonists induced a change in [Ca2+]i. Quinacrine, an inhibitor of PLA2, overruled the effect of the agonists on Na+ transport. In conclusion, we suggest that a Ca2+-sensing receptor in A6 epithelia mediates the stimulation of Na+ transport without the interference of changes in [Ca2+]i.

intracellular calcium concentration; magnesium; phospholipase A2; osmolality


THE ACTIVATION MECHANISM THAT elevates Na+ transport in the renal epithelial A6 cell line in response to changes in osmolality has been the subject of many studies performed in different laboratories, including the team of Wills et al. (7, 19, 38), Marunaka et al. (20–23), and our own group (12). Although already a half-century ago the general concept of Na+ transport in tight epithelia was modeled by Ussing and co-workers (13, 35), the stimulation of Na+ transport by hypotonicity remains poorly understood.

According to the two-membrane hypothesis, tight epithelia, such as those found in the distal tubule of the kidney and in the distal colon, transport Na+ transcellularly in a two-step process (13, 35). Na+ reabsorption is enabled by the Na+-conductive apical membrane that provides passive entry of Na+ through the amiloride-sensitive epithelial Na+ channel (ENaC) driven by 1) the negative intracellular potential established by the K+-conductive basolateral membrane and 2) by the activity of the Na+-K+-ATPase in the basolateral membrane that is responsible for active extrusion of Na+ from the cell. It has been suggested that upregulation of Na+ pump activity is the initial trigger to boost Na+ transport in the A6 epithelium (20). Within this concept, cell swelling activates tyrosine kinases that upregulate a basolateral Cl conductance that stimulates the Na+ -K+-ATPase, a prerequisite to increase Na+ transport in the A6 epithelium (20). On the other hand, there is more consensus that apical Na+ entry constitutes the rate-limiting step for Na+ transport and, conceivably, for its regulation by hypotonicity as well. Consequently, many investigators have explored several factors that could regulate ENaC activity (32). One cellular component that is well known to modulate ion channel activity is intracellular Ca2+ concentration ([Ca2+]i). Recently, it has been reasoned that neural precursor cell-expressed, developmentally downregulated protein (Nedd4) reduces Na+ transport activity. The ubiquitin ligase lowers the number of functional Na+ channels in the apical membrane by catalyzing the conjugation of {beta}-ENaC to ubiquitin, thereby introducing its degradation by the proteasome. Such a concept was indirectly proven to be effective in A6 epithelia (18). Nedd4 translocates from the cytosol to the apical plasma membrane after binding Ca2+ to its C2 domain, allowing a connection of the C2 domain to annexin, thereby enabling interaction of the WW domain of Nedd4 with the PY motif in {beta}-ENaC (26). On the other hand, inactivation of Nedd4 is mediated by phosphorylation, catalyzed by activated serum- and glucocorticoid-inducible kinase isoform 1 (SGK1) (8). In recent years, it has become evident that SGK1 acts as a point of convergence in the stimulation of epithelial Na+ transport in response to hormones, such as aldosterone and insulin (25), but also to other factors, such as hypotonicity, as demonstrated recently in A6 epithelia (33). Nevertheless, insulin and aldosterone elevate [Ca2+]i, which is thought to augment the expression of ENaC at the apical membrane (30). Due to a number of conflicting findings, general agreement on whether [Ca2+]i actually enhances or diminishes Na+ transport activity is still lacking. Measurements of [Ca2+]i changes in fura 2-loaded A6 epithelia showed a biphasic increase in [Ca2+]i when polarized A6 cells were subjected to a sudden decrease in osmolality (11). We related the initial rapid phase of the [Ca2+]i increase to the release of ATP across the basolateral membrane that occurred as a response to the sudden decrease in osmolality (11). A linear relationship was observed among the degree of cell swelling, the maximal rate of ATP release, and the initial change in [Ca2+]i (11). However, suppressing the initial phase of the rise in [Ca2+]i by emptying intracellular Ca2+ stores did not change the stimulation of Na+ transport by the decrease in osmolality. A complete suppression of the changes in [Ca2+]i during hypotonicity obtained by including Mg2+ in the basolateral bath to block Ca2+ entry had no effect on the rise of Na+ transport in these conditions.

Besides its blocking effect on Ca2+ influx, basolateral Mg2+ also enhances the stimulation of Na+ transport by hyposmotic treatment. We reported this stimulatory effect of Mg2+ on Na+ transport in a previous paper and suggested the presence of a Ca2+-sensing receptor at the basolateral border (11). In this report, we further explore the putative Ca2+-sensing receptor at the basolateral membrane of the A6 epithelium. A proper evaluation of the effect of extracellular Ca2+ requires removal of the divalent cation from the bath, a maneuver that causes the loss of epithelial integrity (15). This problem can be at least partially prevented by treating the tissues with the PKC inhibitor H-7 (6, 14, 16, 17). Removal of extracellular Ca2+ in the presence of H-7 severely attenuated the activation of Na+ transport during a hyposmotic shock. Interestingly, several agonists of the Ca2+-sensing receptor (4), i.e., Mg2+, Gd3+, neomycin, and spermine, were able to replace Ca2+ and stimulate Na+ transport during hypotonic conditions in the absence of extracellular Ca2+. The present observations thus confirm our previous suggestions and point to the existence of a Ca2+-sensing receptor in the basolateral membrane of A6 cells that is involved in the stimulation of Na+ transport.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. The epithelial A6 cell line is derived from the distal nephron of the African clawed toad Xenopus laevis. The cells were a kind gift of Dr. J. P. Johnson (University of Pittsburgh, Pittsburgh, PA). All experiments were carried out in A6 epithelial monolayers that were allowed to polarize on permeable supports (pore size 0.2 µm; Anopore, Nunc Intermed, Roskilde, Denmark). Cell cultures were kept at 28°C in a humidified incubator inflated with 1% CO2. The growth medium was renewed twice weekly and consisted of a 1:1 mixture of Leibovitz's L-15 and Ham's F-12 media, supplemented with 10% fetal bovine serum (Sigma, St. Louis, MO), 3.8 mM L-glutamine, 2.6 mM NaHCO3, 95 IU/ml penicillin, and 95 µg/ml streptomycin. Cells were seeded at a density of 2.5 x 105/cm2. The monolayers for the [Ca2+]i measurements were used between days 5 and 9 of culturing, whereas those for electrophysiological recordings remained in culture for 8–15 days.

Solutions and chemicals. Hyposmotic solutions (140 mosmol/kgH2O) contained (in mM) 70 Na+, 2.5 K+, 2.5 HCO3, 1 Ca2+, and 72 Cl (pH 8.0). Hyposmotic Ca2+-free solutions contained (in mM) 71 Na+, 2.5 K+, 5 HEPES, 2 EGTA, and 68.5 Cl (pH 7.4). Isosmotic solutions (260 mosmol/kgH2O) were prepared by adding 65 mM NaCl. Solutions at 200 mosmol/kgH2O contained (in mM) 102 Na+, 2.5 K+, 2.5 HCO3, 1 Ca2+, and 104 Cl. A hypotonic shock was elicited by a sudden decrease in basolateral osmolality, whereas the osmolality of the apical perfusate was lowered to 140 mosmol/kgH2O at least 30 min in advance. The water impermeability of the apical membrane of the A6 cells allows this procedure. Experiments with solutions at 200 mosmol/kgH2O were bilaterally equiosmolal all the time.

Mg2+ was added as a chloride salt and, in case its concentration exceeded 2 mM, equiosmolal NaCl was omitted. Similar steps were taken when 10 mM CaCl2 was included in the medium. Neomycin, H-7, spermine, and quinacrine were purchased from Sigma.

Measurements of [Ca2+]i. A6 cells, grown on permeable supports for at least 5 days, were incubated with an apical solution containing 10 µM fura 2-AM (Sigma) and 0.2 g/l pluronic acid (F-127, Molecular Probes, Eugene, OR) for 120 min at 28°C in 1% CO2. The excess dye was washed off gently before the monolayer was placed in an Ussing-type chamber and mounted on the stage of an inverted fluorescence microscope equipped with a x40 objective (Zeiss LD, Achroplan, Carl Zeiss, Jena, Germany). The apical surface of the epithelium faced toward the objective of the microscope. Both the apical and basolateral surfaces of the monolayer were perfused independently at room temperature. Tissues were excited with light that alternated between 340 and 380 nm. The fluorescence emission at each wavelength was filtered through a band-pass filter centered at 510 nm (6 nm) and detected by photon counting using a photomultiplier tube (Hamamatsu H3460–04, Hamamatsu Photonics, Shizuoka, Japan). Photon counts of the emission at each of the excitation wavelengths were corrected for autofluorescence, using signals from monolayers (n = 10) not exposed to the dye. The ratio of corrected fluorescence excited at 340 nm to that excited at 380 nm (i.e., R = I340/I380) was then used to estimate [Ca2+]i (9)

where Rmax and Rmin are the corrected fluorescence ratios in the presence of ionomycin (5 µM) under saturating and Ca2+-free conditions, respectively; Kd is the dissociation constant of fura 2 for Ca2+ and was taken as 224 nM (9); and Rc is the ratio of the corrected fluorescence intensities at 380-nm excitation in zero and saturating Ca2+. In a previous paper from our laboratory, we included a table with the electrophysiological characteristics of A6 epithelia that have been cultured between days 5 and 9 (11).

Measurements of electrophysiological parameters. Electrophysiological measurements were performed in Ussing-type chambers that allowed separate and continuous perfusion of the apical and basolateral surfaces of the epithelium. Voltage and current were recorded with Ag-AgCl electrodes in 1 M KCl that were in contact with the perfusion solutions through agar bridges. Transepithelial voltage was controlled with a high-speed voltage clamp. Computer hardware for the recordings is based on two digital signal processing (DSP) boards (model 310B, Dalanco Spry, Rochester, NY) equipped with two high-speed (300 kHz) analog-to-digital converters (14 bit) and two digital-to-analog converters (12 bit). This method has been extensively described in a previous paper from our laboratory (36).


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Effect of the hypotonic shock on short-circuit current, transepithelial conductance, and [Ca2+]i. The hypotonic shock was introduced by a sudden decrease in basolateral osmolality from 260 to 140 mosmol/kgH2O through the removal of 65 mM NaCl. Figure 1A illustrates the biphasic increases in short-circuit current (Isc) and transepithelial conductance (GT). Similar results are obtained when extracellular osmolality is reduced by removing sucrose from the solutions. The exposure time required to obtain a stable response of Isc and GT was ~60 min. During this time period, Isc increased from 3.4 ± 0.4 to 24.0 ± 1.3 µA/cm2, whereas GT was augmented from 0.19 ± 0.05 to 0.33 ± 0.04 mS/cm2. We measured the amiloride-sensitive part of Isc in both isosmotic and hyposmotic conditions by adding 100 µM amiloride to the apical bath. The amiloride-insensitive part of Isc was barely affected by the treatment and amounted to 0.98 ± 0.37 and 0.73 ± 0.19 µA/cm2 in isosmotic and hyposmotic conditions, respectively. Similarly, hypotonicity did not change GT values in the presence of amiloride, which were 0.18 ± 0.06 and 0.13 ± 0.03 mS/cm2 in iso- and hypotonic conditions, respectively. The ability of amiloride to completely block Isc together with the marked reduction of GT indicates that amiloride-sensitive Na+ channels are involved in the transport of Na+ that accounts for the Isc increase during hyposmotic conditions.



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Fig. 1. Effects of lowering basolateral osmolality on short-circuit current (Isc), transepithelial conductance (GT), and intracellular Ca2+ concentration ([Ca2+]i). A: responses of Isc and GT (n = 6). [Ami], amiloride concentration; ap, apical. B: response of [Ca2+]i (n = 10). bl, Basolateral. Monolayers were perfused with NaCl-Ringer solutions on the apical and basolateral sides. During the indicated time period, basolateral osmolality was reduced from 260 to 140 mosmol/kgH2O by removing 65 mM NaCl. During hypotonicity, apical and basolateral solutions had an identical composition. Solid lines represent the mean values recorded during n experiments. Dashed lines are means ± SE.

 
We also monitored the effect of the hypotonic shock on [Ca2+]i. During isosmotic conditions, the mean value for [Ca2+]i was 245 ± 12 nM (n = 10). Lowering the osmolality of the apical perfusate to 140 mosmol/kgH2O did not result in noticeable changes in [Ca2+]i for up to 30 min (data not shown). The sudden change in hyposmotic solutions at the basolateral border elicited a transient biphasic increase in [Ca2+]i, as illustrated in Fig. 1B. The underlying mechanisms involved in the [Ca2+]i changes during hypotonicity were explored in a previous study (11). In summary, the first phase of the [Ca2+]i changes is caused by the release of Ca2+ from intracellular stores, whereas the second phase depends on Ca2+ entry from the extracellular solution across the basolateral membrane. Both phases are independent of each other, and blocking one of them does not affect the other. Emptying the intracellular Ca2+ stores through the application of three successive basolateral treatments with ATP (0.5 mM), before the hyposmotic shock, suppresses the first phase of the [Ca2+]i changes (11). The second phase of the [Ca2+]i changes is abolished by including 2 mM Mg2+ in the basolateral solution (11). Figure 2B illustrates that the combination of emptying intracellular Ca2+ stores and blocking basolateral Ca2+ entry suppresses the hypotonicity-induced [Ca2+]i changes completely. We used this approach to verify the relationship between the [Ca2+]i changes and the stimulation of Na+ transport during hypotonicity. Figure 2A demonstrates that the magnitude of the responses of Isc and GT to hypotonicity is not influenced by the inhibition of [Ca2+]i changes. On the contrary, hypotonicity increased Isc from 1.7 ± 0.2 to 27.0 ± 2.1 µA/cm2 and GT from 0.10 ± 0.02 to 0.34 ± 0.03 mS/cm2 (n = 3). It is noteworthy that the increase in Isc is larger in this series of experiments, i.e., 14.9 times the control value, than the augmentation recorded in control conditions, i.e., 6.1 times the control value. Importantly, the above observations indicate that the [Ca2+]i changes during the lowered osmotic conditions are not related to the stimulation of Na+ transport.



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Fig. 2. Complete inhibition of [Ca2+]i changes during hyposmotic shock. A: effects on Isc and GT (n = 4). B: [Ca2+]i response (n = 4). Epithelia were pretreated with 3 successive basolateral ATP (0.5 mM) pulses and subsequently subjected to a hyposmotic shock in the presence of ATP. All basolateral solutions contained 2 mM Mg2+. Solid lines represent the mean values recorded during n experiments. Dashed lines are means ± SE.

 
Effects of removing extracellular Ca2+ on Isc and GT. The more pronounced stimulation of Isc in the experiments in which Ca2+ stores were depleted and Mg2+ blocked basolateral Ca2+ entry could be related to the basolateral effect of Mg2+ on Na+ transport previously reported (12). In this study, we hypothesized that Mg2+ exerted its effect on a Ca2+-sensing receptor in the basolateral membrane. To further explore this idea, we intended to evaluate the effect of extracellular Ca2+ on the stimulation of INa by reducing osmolality. Because the presence of Ca2+ in the basolateral solution is crucial for epithelial integrity (14), we included the nonspecific protein kinase inhibitor H-7 (50 µM) in the perfusion solutions. At the concentration used in our experiments, H-7 has been described as blocking PKC (6) and preventing excessive increases in GT on removal of extracellular Ca2+ (14). Because PKC has been demonstrated to modulate INa (1, 3), we first evaluated the effects of the blocker on Isc and GT. Figure 3 shows the effects of the hyposmotic shock on Isc and GT in four different conditions. In the presence of extracellular Ca2+, but in the absence of extracellular Mg2+ and H-7 (solid lines), hypotonicity increased Isc from 3.4 ± 0.3 to 24.9 ± 0.5 µA/cm2 and GT from 0.38 ± 0.02 to 0.77 ± 0.04 mS/cm2 (n = 4). The responses of Isc and GT were not markedly altered by applying H-7 to Ca2+-containing solutions (dashed lines); their values increased from 2.7 ± 0.3 to 19.1 ± 0.5 µA/cm2 and from 0.27 ± 0.02 to 0.35 ± 0.03 mS/cm2, respectively (n = 4). However, a conspicuous inhibition of the activation of Na+ transport was observed in conditions of zero Ca2+: the hyposmotic shock increased Isc merely from 2.2 ± 0.3 to 4.8 ± 0.7 µA/cm2. The record of GT in Fig. 3 shows that the opening of the paracellular pathway that is observed after removal of Ca2+ from the bath was markedly retarded by the H-7 treatment. The GT values remained within limits that still enabled reliable recording of Isc. In the absence of H-7, removing Ca2+ from the extracellular solutions dramatically increases GT to levels that do not allow for trustworthy electrophysiological recordings (dashed-dotted line in GT).



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Fig. 3. Effects of extracellular Ca2+ removal on Isc and GT during hyposmotic shock. The hyposmotic shock was induced through the removal of 65 mM NaCl from the basolateral bath. Solid line, control experiments performed in Ca2+-containing bathing media; dashed line, in the presence of extracellular Ca2+ and protein kinase inhibitor H-7; dotted line, bilateral extracellular Ca2+-free conditions and with H-7; short dashed-dotted line in GT, bilateral extracellular Ca2+-free conditions and without H-7. H-7 (50 µM) was applied 30 min before the hyposmotic shock was induced. Ca2+ was bilaterally removed at the time when hypotonicity was induced (n = 6).

 
Effect of Ca2+-sensing receptor agonists in the absence of extracellular Ca2+. The above experiments demonstrate the requirement of extracellular Ca2+ for Isc stimulation by hyposmotic shock. Because of the stimulatory effect of Mg2+ on Isc, we considered that Mg2+ could function as a substitute for Ca2+ in stimulating Na+ transport in response to hypotonicity. Figure 4A illustrates the effects of 2 mM Mg2+ on Isc and GT during hyposmotic shock in the absence of extracellular Ca2+ and with H-7 in the perfusion solutions. The lack of a stimulatory effect of hyposmotic solutions on Isc in Ca2+-free solutions is overruled by addition of Mg2+ to the basolateral bath. With Mg2+ the rise in Isc was from 3.4 ± 0.2 to 25.7 ± 0.4 µA/cm2, whereas GT was augmented from 0.15 ± 0.01 to 0.51 ± 0.03 mS/cm2 (n = 4). This augmentation in GT only partly covers an increase in apical conductance but also reflects an increase in paracellular conductance. It should be noted that in Ca2+-free solutions the integrity of the epithelium was better preserved in the presence of Mg2+. Thus far, we observe a stimulation of Isc in hyposmotic solutions only in the presence of one of the divalent cations, Ca2+ and Mg2+. This kind of behavior is reminiscent of a receptor-like mechanism to be involved in the stimulation of Na+ transport during hyposmotic conditions. The sensitivity to both divalent cations has led to the molecular identification of the Ca2+-sensing receptor that was originally cloned from the bovine parathyroid gland (4). This finding established Ca2+ as a first messenger in these cells involved in the secretion of parathyroid hormone (4). The Ca2+-sensing receptor lacks specificity and is also sensitive to the divalent cation Mg2+, to the trivalent cationic lanthanides Gd3+ and La3+, and to polyvalent compounds, such as neomycin and spermine. We tested a number of these cations to create an agonist profile for the putative Ca2+-sensing receptor in A6 cells involved in the stimulation of Na+ transport elicited by hypotonicity in Ca2+-free conditions. As illustrated in Fig. 4B, the presence of either neomycin or spermine in the basolateral bath is sufficient to stimulate Na+ transport during hypotonicity in the absence of extracellular Ca2+. In the presence of 1 mM spermine, hypotonicity stimulated Isc and GT from 3.3 ± 0.1 to 19.2 ± 0.4 µA/cm2 and 0.15 ± 0.02 to 0.55 ± 0.05 mS/cm2 (n = 4), respectively. With 0.1 mM neomycin in the basolateral perfusate during hyposmotic shock, Isc and GT increased from 4.9 ± 0.1 to 18.7 ± 0.5 µA/cm2 and 0.16 ± 0.03 to 0.52 ± 0.06 mS/cm2 (n = 4), respectively. Pilot experiments with Gd3+ (0.1 mM) also showed that the lanthanide was able to take over the role of Ca2+. Other divalent cations, such as Ni2+, Cd2+, or Zn2+, were unable to replace Ca2+ in the stimulation of Isc in response to hypotonic shock.



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Fig. 4. Effect of agonists of the Ca2+-sensing receptor on hyposmotic stimulation of Isc and GT in the absence of extracellular Ca2+. A: 2 mM Mg2+ (dashed line). B: 1 mM spermine (dotted-dashed line) and 0.1 mM neomycin (dashed line). H-7 (50 µM) was added 30 min before the hyposmotic shock was induced. Extracellular Ca2+ was removed when the hyposmotic shock was applied. Agonists were present in the basolateral bath for the entire duration of the experiment.

 
Additive effects of Ca2+ and Mg2+ on Isc at constant osmolality. So far, our experiments underlined the requirement of at least one of the agonists of the Ca2+-sensing receptor for the activation of INa during hyposmotic shock. Activation of the Ca2+-sensing receptor by extracellular Ca2+ shows positive cooperativity (29). Although the molecular mechanism is undefined, it has been suggested to result from multiple Ca2+ binding sites in the extracellular domain of the receptor. To test positive cooperativity between the agonists of the putative Ca2+-sensing receptor in A6 cells, we monitored Na+ transport at constant lowered osmolality. Figure 5 illustrates an experiment in which cells were perfused with 200 mosmol/kgH2O solutions that contained 1 mM Ca2+. The addition of 10 mM Ca2+ to the basolateral bath increased Isc from 5.5 ± 0.2 to 10.2 ± 0.3 µA/cm2 (n = 4), corresponding to a 19% increase in GT. The effect of 10 mM Mg2+ was more pronounced and resulted in an increase in Isc from 7.1 ± 0.2 to 15.8 ± 0.3 µA/cm2 (n = 4), matching a rise in GT of 26%. These data suggest that the receptor is more sensitive to Mg2+ than to Ca2+. This also becomes apparent in experiments in which 1 mM Mg2+ was added to solutions that contained 10 mM Ca2+. The additional Mg2+ elevated Isc from 6.1 ± 0.1 to 13.6 ± 0.3 µA/cm2 (n = 4), parallel to a 33% increase in GT. This demonstrates the positive cooperativity between Ca2+ and Mg2+ in stimulating Na+ transport. A similar behavior could be observed for neomycin and spermine in relation to Ca2+ (data not shown).



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Fig. 5. Effect of basolateral Ca2+ and Mg2+ on Isc and GT at constant osmolality. Cells were incubated in Ca2+-containing solutions with an osmolality of 200 mosmol/kgH2O at both sides. At the indicated time, agonists of the Ca2+-sensing receptor were added: 10 mM Ca2+ (solid lines), 10 mM Mg2+ (dotted lines), and 10 mM Ca2++1 mM Mg2+ (dashed lines) (n = 4).

 
Signaling pathway. Because Na+ transport can be stimulated in the absence of changes in [Ca2+]i (Fig. 2B), it is quite likely that the activation of the putative Ca2+-sensing receptor in A6 cells does not augment [Ca2+]i. To verify this hypothesis, we recorded the effect of the agonists on [Ca2+]i. Figure 6A shows that the addition of 10 mM Ca2+ to 200 mosmol/kgH2O solutions increased [Ca2+]i from 370 ± 9 to 580 ± 12 nM (n = 4). However, Mg2+, neomycin, and spermine were all unable to increase [Ca2+]i, but rather induced a slow decrease in [Ca2+]i (Fig. 6B). Interestingly, the [Ca2+]i rise observed on increasing Ca2+ in the basolateral bath declined rapidly by adding 1 mM Mg2+ to the solution. This demonstrates that the increase in [Ca2+]i observed in response to elevating Ca2+ in the basolateral perfusion solution is due to Ca2+ entry through the Mg2+-blockable basolateral Ca2+ entry channels, which are gated to the open state by the reduction in osmolality in solutions of 200 mosmol/kgH2O (11), and not via the activation of the Ca2+-sensing receptor. Indeed, [Ca2+]i was not increased when 10 mM Ca2+ was added to isosmotic solutions.



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Fig. 6. Effect of basolateral Ca2+ and Mg2+ on [Ca2+]i at constant osmolality. A: cells were perfused with Ca2+-containing solutions having an osmolality of 200 mosmol/kgH2O at both sides. At the indicated time period, 10 mM Ca2+ was added to the basolateral bath with respect to constant osmolality by removal of equiosmolal NaCl. Next, 1 mM Mg2+ was supplied to the basolateral border at the indicated time. B: A6 epithelia were challenged with solutions at 200 mosmol/kgH2O. After 5 min of reduced osmolality, either spermine (1 mM, solid line) or neomycin (0.1 mM, dotted line) was added to the basolateral bath.

 
The absence of a rise in [Ca2+]i on addition of the receptor agonists suggests that PLC is not activated downstream of the receptor, as is the case for the receptor cloned from bovine parathyroid (4). To test whether the activation of PLA2 was involved, we evaluated the activity of the agonists in the presence of quinacrine, a common inhibitor of this enzyme. Application of quinacrine (100 µM) at the basolateral border abolished the increase in Isc completely in response to the hyposmotic shock (Fig. 7). A similar lack of activity of the agonists in stimulating Na+ transport at constant osmolality could be observed in the presence of quinacrine (data not shown). These results suggest that the stimulation of Na+ transport by activating the Ca2+-sensing receptor in A6 epithelia requires the activity of PLA2.



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Fig. 7. Use of the PLA2 inhibitor quinacrine during hyposmotic shock. Hyposmotic shock was introduced during the indicated time period. The PLA2 inhibitor quinacrine (100 µM) was present in the basolateral bath from the beginning of the experiment. CTRL, control.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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The data presented in this paper relate to the initial trigger for the stimulation of Na+ transport in the renal epithelial cell line A6 in response to a hyposmotic shock. We observed that extracellular Ca2+ functions as an essential component in the basolateral perfusion solution for increasing the rate of Na+ transport during the hyposmotic challenge. Our findings point to the presence of a Ca2+-sensing receptor in the basolateral membrane of the A6 epithelium. This suggestion is supported by 1) the requirement for extracellular Ca2+ to stimulate Na+ transport by hypotonicity and 2) the ability of agonists of the Ca2+-sensing receptor, i.e., Mg2+, Gd3+, neomycin, and spermine, to replace extracellular Ca2+ at the basolateral membrane and to restore the stimulatory effect of the hyposmotic solutions on Na+ transport in Ca2+-free conditions. The activation process has the following properties: 1) the receptor is more sensitive to Mg2+ than it is to Ca2+; 2) receptor activation with the agonists also stimulates Na+ transport at constant osmolality; and 3) downstream signaling does not require a change in [Ca2+]i, but rather activates PLA2. Unfortunately, molecular identity for the suggested Ca2+-sensing receptor is at this moment still lacking. So far, attempts that we made with RT-PCR screening of total mRNA were not successful. Failure to achieve a positive outcome in the RT-PCR screening can be attributed to the fact that primer design was based on the Ca2+-sensing receptor cloned from the gastric mucosa of the amphibian Necturus maculosus (5). The Necturus Ca2+-sensing receptor is closely homologous to that of the rat (31), and this receptor signals through increases in [Ca2+]i. Activation of the putative Ca2+-sensing receptor in A6 epithelia does not cause an increase in [Ca2+]i. The elevation in [Ca2+]i that is observed during hypotonicity in the A6 cells is related to the stimulation of P2 purinergic receptors in the basolateral membrane, because of ATP release across this border during cell swelling (11). We observed a rise in [Ca2+]i that takes place on increasing the extracellular Ca2+ concentration when A6 epithelia are perfused with solutions of 200 mosmol/kgH2O. However, this [Ca2+]i increase is caused by Ca2+ entry across the basolateral membrane and is blocked by Mg2+ in the basolateral bath. Thus the rise in [Ca2+]i on increasing the Ca2+ concentration in the basolateral bath is not related to the activation of the Ca2+-sensing receptor but to the opening of basolateral Mg2+-blockable Ca2+ entry channels in solutions of lowered osmolality. Addition of agonists of the Ca2+-sensing receptor had no effect on [Ca2+]i in A6 epithelia in isosmotic conditions. Palmer and Frindt (24) studied Na+ channels in the cortical collecting tubule of the rat and did not observe a direct effect of Ca2+ on the channel, but they noticed a decrease in ENaC activity on increasing cytosolic Ca2+ concentration by the use of the Ca2+-ionophore ionomycin. Ishikawa et al. (10) observed a similar downregulation of the activity of rat ENaC, expressed in Madin-Darby canine kidney cells, when ionomycin was used to elevate [Ca2+]i. Recently, Awayda et al. (2) reported a lack of ENaC activity when using BAPTA to chelate intracellular Ca2+ and mentioned a possible role of changes in [Ca2+]i for stimulating Na+ transport in A6 epithelia via either the cellular trafficking machinery or a second messenger, such as PLC. Our observations do not support the idea that activation of PLC regulates ENaC activity through changes in [Ca2+]i. Nevertheless, our observations do not exclude a role for Ca2+ in regulating the activity of the Na+ channel. It has been observed that reloading of intracellular Ca2+ stores can take place without an apparent change in [Ca2+]i (27). It is reasonable that local changes in [Ca2+]i exert important regulatory effects on protein conformation and operation.

The Ca2+-sensing receptor was originally cloned from chief cells of the bovine parathyroid gland, where increases in extracellular Ca2+ and Mg2+ concentration diminish the secretion of parathyroid hormone by the cell via a PLC-mediated increase in [Ca2+]i (4). Meanwhile, a Ca2+-sensing receptor has been cloned from various cells sharing some of the properties with the original. Although Ca2+ is the physiological regulator of this receptor, it can also sense and respond to other multivalent cations. The latter include the polyamine spermine and the aminoglycosidic antibiotic neomycin, which at physiological pH possess four and five positive charges, respectively, due to the presence of primary and secondary amine moieties. Agonist binding occurs within the large extracellular domain rather than in a pocket defined by the seven transmembrane helixes, as is characteristic of most G protein-coupled receptors. Interestingly, the extracellular domain of the Ca2+-sensing receptor and the NMDA receptor channel share limited homology, allowing all Ca2+-sensing receptor agonists mentioned here to modulate NMDA channel function. Sensitivity to cationic agonists might involve surface charge-shielding effects. Within this concept, a reduction in ionic strength was described to be a potent activator of the receptor (28). It should be noted that stimulation of Na+ transport can take place by decreasing the osmolality of the basolateral solution without a change in ionic strength of the solution, for instance, by taking out sucrose. In addition, keeping the osmolality constant by replacing the membrane-impermeant solute sucrose with the membrane-permeant solutes ureum or glycerol will stimulate Na+ transport to comparable levels in this epithelium. This is conceivable with the intracellular tonicity functioning as the actual trigger for activation of the receptor in A6 cells. The suggested Ca2+-sensing receptor that we describe shares the properties of the agonist profile with the original, despite the fact that we observed a higher sensitivity of the receptor for Mg2+ than for Ca2+.

Stimulation of the receptor by the agonists was inhibited in the presence of quinacrine, suggesting the involvement of PLA2, downstream of the receptor in A6 epithelia. This finding links our observations to the description of the involvement of PLA2 in the stimulation of Na+ transport (19, 39). Recently, it has been reported for A6 cells that activation of PLA2 at the basolateral border stimulated Na+ transport, whereas apical activation of the enzyme had the adverse effect (39). Interestingly, the authors suggested a tonic activity of the enzyme, an effect that may result from the continuous presence of Ca2+ at the basolateral membrane causing a constitutive activity of the receptor. A Ca2+-sensing receptor that signals via PLA2 without changing [Ca2+]i has also been reported in the thick ascending limb of the kidney (37). Because activation of Na+ transport in A6 epithelia by hypotonicity has been demonstrated to be mediated by SGK1 (33), a link between PLA2 and activation of SGK1 would be a next challenge. Possible pathways may involve K-Ras2A, which was recognized as an early aldosterone-induced protein in A6 cells, or the activation of phosphatidylinositol 3-kinase, which recently has been recognized to be involved in hypotonic stimulation of Na+ transport in A6 epithelia (33).

In summary, we have substantial evidence for a Ca2+-sensing receptor in A6 epithelia that mediates stimulation of Na+ transport during hyposmotic shock and at steady osmotic conditions. The lack of a molecular relationship to the cloned Ca2+-sensing receptor impedes an effective functional evaluation, and hence its possible role in the collecting duct remains difficult to evaluate. Nevertheless, the presence of the putative Ca2+-sensing receptor that we describe in this paper may represent an important mechanism for the distal nephron to supplement the previous finding of an extracellular Ca2+-sensing receptor in the apical membranes of cells in the inner medullary collecting duct (34). During periods of diuresis when the renal tubules are filled with primary urine of low tonicity, water leaks toward the interstitial space even in the absence of antidiuretic hormone. Both systems mentioned above may cooperate to prevent urine concentration. Lowered intracellular tonicity of the principal cells of the cortical collecting duct, modeled by the A6 epithelium, may stimulate Ca2+-sensing receptor-mediated Na+ transport in this part of the nephron, whereas lowered tonicity in the lumen of the inner medullary sections activates the apical Ca2+-sensing receptor to diminish local water reabsorption.


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This work was supported through the "Fonds voor Wetenschappelijk Onderzoek Vlaanderen" (G.0277.03) and the "Foundation Alphonse en Jean Forton."


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Address for reprint requests and other correspondence: W. Van Driessche, Laboratory of Physiology, K. U. Leuven, Campus Gasthuisberg O/N, B-3000 Leuven, Belgium (E-mail: willy.vandriessche{at}med.kuleuven.ac.be)

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.


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  1. Awayda MS. Specific and nonspecific effects of protein kinase C on the epithelial Na+ channel. J Gen Physiol 115: 559–570, 2000.[Abstract/Free Full Text]
  2. Awayda MS, Boudreaux MJ, Reger RL, and Hamm LL. Regulation of the epithelial Na+ channel by extracellular acidification. Am J Physiol Cell Physiol 279: C1896–C1905, 2000.[Abstract/Free Full Text]
  3. Awayda MS, Platzer JD, Reger RL, and Bengrine A. Role of PKC{alpha} in feedback regulation of Na+ transport in an electrically tight epithelium. Am J Physiol Cell Physiol 283: C1122–C1132, 2002.[Abstract/Free Full Text]
  4. Brown EM, Gamba G, Riccardi D, Lombardi M, Butters R, Kifor O, Sun A, Hediger MA, Lytton J, and Hebert SC. Cloning and characterization of an extracellular Ca2+-sensing receptor from bovine parathyroid. Nature 366: 575–580, 1993.[CrossRef][ISI][Medline]
  5. Cima RR, Cheng I, Klingensmith ME, Chattopadhyay N, Kifor O, Hebert SC, Brown EM, and Soybel DI. Identification and functional assay of an extracellular calcium-sensing receptor in Necturus gastric mucosa. Am J Physiol Gastrointest Liver Physiol 273: G1051–G1060, 1997.[Abstract/Free Full Text]
  6. Citi S. Protein kinase inhibitors prevent junction dissociation induced by low extracellular calcium in MDCK epithelial cells. J Cell Biol 117: 169–178, 1992.[Abstract]
  7. Crowe WE, Ehrenfeld J, Brochiero E, and Wills NK. Apical membrane sodium and chloride entry during osmotic swelling of renal (A6) epithelial cells. J Membr Biol 144: 81–91, 1995.[ISI][Medline]
  8. Debonneville C, Flores SY, Kamynina E, Plant PJ, Tauxe C, Thomas MA, Munster C, Chraibi A, Pratt JH, Horisberger JD, Pearce D, Loffing J, and Staub O. Phosphorylation of Nedd4–2 by SGK1 regulates epithelial Na+ channel cell surface expression. EMBO J 20: 7052–7059, 2001.[Abstract/Free Full Text]
  9. Grynkiewicz G, Poenie M, and Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260: 3440–3450, 1985.[Abstract]
  10. Ishikawa T, Marunaka Y, and Rotin D. Electrophysiological characterization of the rat epithelial Na+ channel (rENaC) expressed in MDCK cells. Effects of Na+ and Ca2+. J Gen Physiol 111: 825–846, 1998.[Abstract/Free Full Text]
  11. Jans D, De Weer P, Srinivas SP, Lariviere E, Simaels J, and Van Driessche W. Mg2+-sensitive non-capacitative basolateral Ca2+ entry secondary to cell swelling in the polarized renal A6 epithelium. J Physiol 541: 91–101, 2002.[Abstract/Free Full Text]
  12. Jans D, Simaels J, Cucu D, Zeiske W, and Van Driessche W. Effects of extracellular Mg2+ on transepithelial capacitance and Na+ transport in A6 cells under different osmotic conditions. Pflügers Arch 439: 504–512, 2000.[CrossRef][ISI][Medline]
  13. Koefoed-Johnsen V and Ussing HH. The nature of the frog skin potential. Acta Physiol Scand 42: 298–308, 1958.[ISI]
  14. Kottra G. Protein kinase inhibitor H7 prevents the decrease of tight junction resistance induced by serosal Ca2+ removal in Necturus gallbladder epithelium. Cell Physiol Biochem 5: 211–221, 1995.[CrossRef][ISI]
  15. Lacaz-Vieira F. Calcium site specificity. Early Ca2+-related tight junction events. J Gen Physiol 110: 727–740, 1997.[Abstract/Free Full Text]
  16. Lacaz-Vieira F. Tight junction dynamics: oscillations and the role of protein kinase C. J Membr Biol 178: 151–161, 2000.[CrossRef][ISI][Medline]
  17. Lacaz-Vieira F and Jaeger MM. Protein kinase inhibitors and the dynamics of tight junction opening and closing in A6 cell monolayers. J Membr Biol 184: 185–196, 2001.[CrossRef][ISI][Medline]
  18. Malik B, Schlanger L, Al-Khalili O, Bao HF, Yue G, Price SR, Mitch WE, and Eaton DC. ENaC degradation in A6 cells by the ubiquitin-proteosome proteolytic pathway. J Biol Chem 276: 12903–12910, 2001.[Abstract/Free Full Text]
  19. Matsumoto PS, Mo L, and Wills NK. Osmotic regulation of Na+ transport across A6 epithelium: interactions with prostaglandin E2 and cyclic AMP. J Membr Biol 160: 27–38, 1997.[CrossRef][ISI][Medline]
  20. Niisato N and Marunaka Y. Activation of the Na+-K+ pump by hyposmolality through tyrosine kinase-dependent Cl conductance in Xenopus renal epithelial A6 cells. J Physiol 518: 417–432, 1999.[Abstract/Free Full Text]
  21. Niisato N and Marunaka Y. Hyposmolality-induced enhancement of ADH action on amiloride-sensitive Isc in renal epithelial A6 cells. Jpn J Physiol 47: 131–137, 1997.[ISI][Medline]
  22. Niisato N, Post M, Van Driessche W, and Marunaka Y. Cell swelling activates stress-activated protein kinases, p38 MAP kinase and JNK, in renal epithelial A6 cells. Biochem Biophys Res Commun 266: 547–550, 1999.[CrossRef][ISI][Medline]
  23. Niisato N, Van Driessche W, Liu M, and Marunaka Y. Involvement of protein tyrosine kinase in osmoregulation of Na+ transport and membrane capacitance in renal A6 cells. J Membr Biol 175: 63–77, 2000.[CrossRef][ISI][Medline]
  24. Palmer LG and Frindt G. Effects of cell Ca and pH on Na channels from rat cortical collecting tubule. Am J Physiol Renal Fluid Electrolyte Physiol 253: F333–F339, 1987.[Abstract/Free Full Text]
  25. Pearce D. SGK1 regulation of epithelial sodium transport. Cell Physiol Biochem 13: 13–20, 2003.[CrossRef][ISI][Medline]
  26. Plant PJ, Lafont F, Lecat S, Verkade P, Simons K, and Rotin D. Apical membrane targeting of Nedd4 is mediated by an association of its C2 domain with annexin XIIIb. J Cell Biol 149: 1473–1484, 2000.[Abstract/Free Full Text]
  27. Putney JW Jr. Capacitative calcium entry revisited. Cell Calcium 11: 611–624, 1990.[ISI][Medline]
  28. Quinn SJ, Kifor O, Trivedi S, Diaz R, Vassilev P, and Brown E. Sodium and ionic strength sensing by the calcium receptor. J Biol Chem 273: 19579–19586, 1998.[Abstract/Free Full Text]
  29. Quinn SJ, Ye CP, Diaz R, Kifor O, Bai M, Vassilev P, and Brown E. The Ca2+-sensing receptor: a target for polyamines. Am J Physiol Cell Physiol 273: C1315–C1323, 1997.[Abstract/Free Full Text]
  30. Rao US, Baker JM, Pluznick JL, and Balachandran P. Role of intracellular Ca2+ in the expression of the amiloride-sensitive epithelial sodium channel. Cell Calcium 35: 21–28, 2004.[CrossRef][ISI][Medline]
  31. Riccardi D, Park J, Lee WS, Gamba G, Brown EM, and Hebert SC. Cloning and functional expression of a rat kidney extracellular calcium/polyvalent cation-sensing receptor. Proc Natl Acad Sci USA 92: 131–135, 1995.[Abstract]
  32. Rotin D. Regulation of the epithelial sodium channel (ENaC) by accessory proteins. Curr Opin Nephrol Hypertens 9: 529–534, 2000.[CrossRef][ISI][Medline]
  33. Rozansky DJ, Wang J, Doan N, Purdy T, Faulk T, Bhargava A, Dawson K, and Pearce D. Hypotonic induction of SGK1 and Na+ transport in A6 cells. Am J Physiol Renal Physiol 283: F105–F113, 2002.[Abstract/Free Full Text]
  34. Sands JM, Naruse M, Baum M, Jo I, Hebert SC, Brown EM, and Harris HW. Apical extracellular calcium/polyvalent cation-sensing receptor regulates vasopressin-elicited water permeability in rat kidney inner medullary collecting duct. J Clin Invest 99: 1399–1405, 1997.[Abstract/Free Full Text]
  35. Ussing HH and Zerahn K. Active transport of sodium as the source of electric current in the short-circuited isolated frog skin. J Am Soc Nephrol 10: 2056–2065, 1999.[Medline]
  36. Van Driessche W, De Vos R, Jans D, Simaels J, De Smet P, and Raskin G. Transepithelial capacitance decrease reveals closure of lateral interspace in A6 epithelia. Pflügers Arch 437: 680–690, 1999.[CrossRef][ISI][Medline]
  37. Wang W, Lu M, Balazy M, and Hebert SC. Phospholipase A2 is involved in mediating the effect of extracellular Ca2+ on apical K+ channels in rat TAL. Am J Physiol Renal Physiol 273: F421–F429, 1997.[Abstract/Free Full Text]
  38. Wills NK, Millinoff LP, and Crowe WE. Na+ channel activity in cultured renal (A6) epithelium: regulation by solution osmolarity. J Membr Biol 121: 79–90, 1991.[ISI][Medline]
  39. Worrell RT, Bao HF, Denson DD, and Eaton DC. Contrasting effects of cPLA2 on epithelial Na+ transport. Am J Physiol Cell Physiol 281: C147–C156, 2001.[Abstract/Free Full Text]