INTRODUCTION

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THE CELLULAR INTEGRITY and epithelial structure of A6 epithelial cells are protected against extracellular osmotic perturbations by activation of volume regulatory mechanisms. Similar to many other tissues (22, 27, 30), this renal epithelial cell line exhibits a biphasic response to an abrupt reduction of the extracellular osmolality. During the fast initial phase, cell volume increases rapidly as a result of the equilibration of water across the basolateral membrane, which has a large hydraulic conductivity. During the following phase, termed regulatory volume decrease (RVD), efflux of intracellular solutes and osmotically obliged water causes the cells to shrink to their initial size.

Previous studies demonstrated that A6 epithelia regulate their volume by excreting predominantly inorganic ions, mainly K⁺ and Cl, and to a smaller extent by the loss of amino acids (8, 9). In addition, these studies showed that swelling-activated K⁺ and Cl excretion occurred through conductive pathways that are highly sensitive to quinine but rather resistant to Ba²⁺. In many different cell types, mechanical stretch of the membrane as a result of cell swelling was thought to trigger the activation of the volume-activated pathways. These mechanosensitive channels not only mediate inorganic ion efflux but also have a substantial permeability for organic anions and uncharged osmolytes (20, 31). Micromolar concentrations of La³⁺ or Gd³⁺ block this pathway from the intracellular side (6, 19, 41). Besides lanthanides, Ca²⁺ and Mg²⁺ were also found to inhibit stretch-activated channels (SACs) (40, 41). On the other hand, volume regulation in many cell types is mediated by Ca²⁺ influx through stretch-activated nonselective channels (7), whereas these Ca²⁺-permeable SACs were proposed to be the sensing elements of the hypotonic cell swelling-induced Ca²⁺ increase (6).

The present study aims to further investigate the properties of the volume-activated pathway for K⁺ efflux probed by the use of ⁸⁶Rb as a substitute for K⁺. Evidence is given for the activation of a poorly selective cation channel responsible for K⁺ excretion during RVD, which, as far as its sensitivity to lanthanides is concerned, has some resemblance to the SACs. Although the activation of the channel depends on cell swelling, the maximum ⁸⁶Rb efflux is noticeably delayed compared with the peak in cell volume, suggesting that membrane stretch is not directly involved in the activation of the volume regulatory mechanism.

	MATERIALS AND METHODS

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Cell preparation. As described previously (34), A6 cells obtained from Dr. J. P. Johnson (University of Pittsburgh, Pittsburgh, PA) were cultured on permeable filter supports (pore size = 0.2 µm; Nunc Intermed, Roskilde, Denmark). For this study we utilized passages 103-113. The cells were seeded at a density of 10⁵ cells/cm² and used for our experiments after 10-20 days of growth.

Cell thickness measurements. Before the cells were seeded, the filters were coated with 1-µm-diameter fluorescent microspheres (catalog no. L-5081, Latex Fluospheres, sulfate, Molecular Probes, Eugene, OR). The apical surface of the epithelium was labeled with fluorescent avidin-coated microbeads (catalog no. F-8776, Molecular Probes). Cell thickness (T_c) was recorded as the vertical distance between the lower and the upper beads, as described previously (36). Taking into account the diameter of the microspheres, we corrected the T_c values by subtracting 1 µm from the measured thickness. An improved version of the software used in the present experiments enabled us to record simultaneously and continuously T_c of up to 20 cells. Only records where T_c was followed over the entire duration of the experiments were used for evaluation of cell volume. Cell volume is expressed as percentage of the value recorded just before the hyposmotic challenge is imposed. The different periods of the experiment were precisely timed to enable us to average the T_c records. T_c was measured in the absence of apical Na⁺. Transepithelial conductance (G_t) was recorded at 14-s intervals with 5-mV pulses under voltage clamp. Under these conditions, G_t mainly reflects the conductance of the paracellular pathway. Continuous monitoring of G_t enabled us to verify the integrity of the structure of the epithelium.

⁸⁶Rb efflux experiments. Cells were loaded by exposure of the basolateral surface to a K⁺-free isotonic solution that contained 50 µCi of ⁸⁶Rb. The loading solution had the same composition as the control Cl solution (Table 1), with KHCO₃ replaced by NaHCO₃. Depending on the specific activity of the isotope, the Rb⁺ concentration varied between 0.6 and 1.3 mM. Tissues were removed from the filter cups and mounted in a two-compartment chamber. The membrane area exposed to the perfusates was 1.54 cm². Both compartments were continuously perfused. Rapid basolateral washout was achieved by vigorous stirring with a motor-driven magnetic stirring bar. The apical and basolateral perfusates were collected in test tubes that were replaced at 1-min (Fig. 1) or 3-min intervals. ⁸⁶Rb was measured by gamma counting. The data are presented as release per unit time and expressed as radioactivity released in each collected sample as percentage of the total amount in the cells at that time. At the end of the experiment the cells were lysed with a 5% (wt/vol) TCA solution, and five additional samples of the chamber perfusate were collected to determine the total amount accumulated in the cells, which was on average 10⁵ counts.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Time course of 86Rb efflux and volume changes during a hypotonic shock. Experiments were done with HCO3-buffered solutions. 86Rb release (RRb) was sampled at 1-min time intervals. A: RRb increased from 0.17 ± 0.02 min1 in isosmotic conditions to a peak of 5.49 ± 0.23 min1 [number of experiments (N) = 4] 11 min after initiation of hypotonic challenge. Apical RRb (RapRb) was 0.12 ± 0.01 min1 in isosmotic conditions and was not noticeably affected by hypotonic shock. Cell thickness (Tc) increased to 164% and reached its peak 4 min after osmolality was reduced. Mean absolute value of Tc in isosmotic conditions is 6.31 ± 0.19 µm [N = 6, number of cells (n) = 73]. Tc data recorded during final isosmotic period are omitted for clarity. RblRb, basolateral RRb; bl, basolateral osmolality. B: relation between Tc and RRb values recorded during hypotonic period. Even after volume recovery, RblRb remained markedly above its control.

Experimental protocols. For cell volume and isotope efflux measurements, cells were subjected to hypotonic conditions only after a stable baseline in isotonic conditions (260 mosmol/kgH₂O) was recorded. The last 25- or 30-min period of this phase is shown. During the subsequent 60-min period, we reduced the basolateral osmolality (_bl) to 140 mosmol/kgH₂O; _bl was restored to 260 mosmol/kgH₂O during the final 30-min period.

Solutions. The composition of the apical and basolateral solutions is depicted in Table 1. Cl was the principal anion in all solutions. Apical solutions were hyposmotic and Na⁺ free, with N-methyl-D-glucamine (NMDG⁺) as the major cation. In experiments with large concentrations of inorganic blockers, we kept the osmolality constant by reducing the NMDG⁺ concentration. The osmolality of the solutions was verified with a cryoscopic osmometer (Osmomat 030, Gonotec, Berlin, Germany). Osmolality of the iso- and hyposmotic basolateral solutions was 260 ± 2 and 140 ± 2 mosmol/kgH₂O, respectively. Basolateral Ca²⁺-free experiments as well as experiments with Gd³⁺ and La³⁺ were executed with HEPES-buffered solutions. Quinine (0.5 mM), lidocaine (0.2 mM), and verapamil (0.2 mM) were added to both sides of the epithelium. Ba²⁺ (30 mM) and Mg²⁺ (0.5 or 5 mM) were added as Cl salts. All experiments were performed at room temperature (22°C) in air-conditioned rooms. Values are means ± SE; N is the number of experiments, and n is the number of cells (or T_c records) used to calculate the averaged curves.

	RESULTS

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To avoid interference of Na⁺ uptake with the volume regulatory mechanisms (10), we used Na⁺-free apical solutions in all experiments. Because the water permeability of the apical barrier is negligible (10), we could keep the osmolality of the apical bath at 140 mosmol/kgH₂O during the entire experiment. With this procedure, no osmotic or ionic gradients are created during the hyposmotic phase of the experiment. Transepithelial anion gradients during the hypotonic phase were also avoided by matching the anion composition of the apical and basolateral hyposmotic solutions; in this way, possible interference of transepithelial anion currents with the volume regulatory processes was prevented. Cell swelling was invoked by lowering the _bl.

Figure 1A compares the time course of T_c and R_Rb recorded with NMDG-Cl and NaCl solutions applied to the apical and basolateral sides, respectively. On reduction of _bl, cells swell and regulate their volume completely back to their normal size. The peak T_c was achieved 4 min after the osmolality was reduced. The time constant of the T_c decay is 4.6 min (Table 2). The R_Rb data in Fig. 1A demonstrate that in isosmotic conditions the basolateral ⁸⁶Rb release (R^bl_Rb) is relatively small (0.17 ± 0.02 min¹). R^bl_Rb was drastically augmented by hypotonicity and reached a maximum (5.49 ± 0.23 min¹) 11 min after initiation of the osmotic perturbation. This relatively large delay in R^bl_Rb suggests that cell volume expansion does not directly activate the pathway for cation release. This is conspicuously illustrated in Fig. 1B, which depicts the relation between T_c and R^bl_Rb during the hypotonic phase of the experiment. Figure 1B shows that the maximal isotope efflux was recorded only after T_c declined to 15% above control. Furthermore, during the last phase of the hypotonic period, when cell volume had completely recovered, R^bl_Rb remained clearly above its control. On the other hand, R^bl_Rb only reached 0.92 min¹ (20% of its full response) at the moment when we recorded the maximal T_c increase. The data in Fig. 1A also show that the apical efflux of ⁸⁶Rb was extremely low (0.06 ± 0.01 min¹) during the isosmotic period. Moreover, during the hypotonic treatment of the tissue, apical efflux of ⁸⁶Rb did not increase (0.08 ± 0.01 min¹). Because of the negligible amount of ⁸⁶Rb release at the apical barrier, we confine this report to R^bl_Rb.

During the loading period of the ⁸⁶Rb release experiments, the tissues accumulated large amounts of cold Rb⁺. We calculated that, depending on the activity of ⁸⁶Rb, intracellular accumulated Rb⁺ concentration was as much as 50-85 mM. Therefore, we verified whether volume regulation was affected by this marked change in cell K⁺ and loaded cells with cold Rb⁺ to obtain concentrations similar to those reached in the tracer experiments. Figure 2 compares volume recovery in control with that in Rb⁺-loaded tissues. It is clear that the replacement of large amounts of cellular K⁺ by Rb⁺ did not affect the RVD. This finding justifies the use of Rb⁺ as a tracer for studying K⁺ release during RVD.

View larger version (11K): [in this window] [in a new window]   Fig. 2.   Volume regulation in cells loaded with Rb+. Cells were loaded with cold Rb+ as in 86Rb release experiments. Before and during experiment, apical and basolateral sides of epithelium were exposed to solutions in which Rb+ (2.5 mM) replaced K+. Incubation time before hypotonic challenge was 60 min. From measurements of total 86Rb accumulated in cells and concentration of cold Rb+ in loading solution, we estimated a cell Rb+ concentration of 65 mM. Tc values in isosmotic conditions are 6.31 ± 0.19 (N = 6, n = 73) and 6.81 ± 0.23 µm (N = 6, n = 54) for control (Ctrl) and Rb+-loaded cells, respectively.

Previously, we demonstrated that submillimolar concentrations of quinine abolish the RVD completely (9, 26), whereas with high concentrations of Ba²⁺, cells remained swollen during the 25-min hyposmotic period (9). In this study (Fig. 3) we show that Ba²⁺ is not able to prevent RVD and that volume recovery can take place over an extended period of 60 min. With Ba²⁺ as well as quinine, the initial volume increase was markedly larger than in control (Table 2). The increase of T_c above the expected ideal osmometric swelling could be due to an increased osmotic activity of the cell as a result of a change in cell composition and/or complex formation of the intracellular solutes. Alternatively, it is conceivable that Ba²⁺ and quinine block an osmolyte release that might take place during the rising phase of T_c. Ba²⁺ markedly retarded the volume recovery (time constant = 20.0 min¹), and at the end of the hypotonic phase, T_c remained clearly above control. R^bl_Rb was noticeably inhibited by Ba²⁺ and almost completely abolished by quinine. The peak value of R^bl_Rb of 4.69 ± 0.25 min¹ recorded in control was reduced to 2.27 ± 0.08 min¹ in the presence of Ba²⁺. This reduced R^bl_Rb still enables an almost complete, but markedly delayed, RVD. On the other hand, quinine almost completely blocked R^bl_Rb and totally abolished the RVD. This result supports the idea that quinine is an efficient blocker of volume-activated K⁺ channels, whereas Ba²⁺ is a poor inhibitor of this channel type. Volume-activated K⁺ channels studied in epithelia by permeabilization of apical membranes are inhibited not only by quinine (14) but also by verapamil (21) and even more effectively by lidocaine (33). In these studies the apical membranes were treated with nystatin in the presence of high K⁺ concentrations, which were assumed to increase cell K⁺ and cell volume. Figure 4 illustrates the effect of these agents on volume recovery. It is clear that lidocaine only slightly affected the RVD, whereas verapamil exerted a marked effect on volume recovery. These supposedly contradictory effects are most likely due to the fact that volume increase induced by osmotic cell swelling gives rise to the activation of pathways that differ from those stimulated by permeabilization of the apical membranes.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Effect of quinine and Ba2+ on volume regulation and 86Rb efflux. BaCl2 (30 mM, Ba2+) and 0.5 mM quinine (QNE) were administered to both compartments. Quinine was added to control solution, and Ba2+ was used in 30 Ba2+ solution (Table 1). A: Tc records. In control and Ba2+-treated tissues, Tc reached a peak and returned toward control. Peak values were 164 and 200% for control and Ba2+-treated cells, respectively. At end of hypotonic phase, Tc reached plateau values of 98 and 118% in control and during Ba2+ inhibition, respectively. In presence of quinine, Tc initially increased to 205% and gradually increased further to 220% at end of hyposmotic shock. Initial absolute Tc values are 6.31 ± 0.19 (N = 6, n = 73), 4.91 ± 0.20 (N = 6, n = 83), and 6.33 ± 0.74 µm (N = 6, n = 78) for control and Ba2+- and quinine-treated cells, respectively. B: RblRb sampled at 3-min intervals. RapRb was negligible and not affected by osmotic perturbation (see Fig. 1). For clarity, RapRb is omitted. In isosmotic conditions, quinine and Ba2+ reduced basal RblRb from 0.46 min1 (control) to 0.36 and 0.16 min1, respectively. In control, RblRb reached a peak of 4.69 min1. Ba2+ reduced peak in RblRb to 2.27 min1, whereas quinine almost completely abolished 86Rb release.

View larger version (12K): [in this window] [in a new window]   Fig. 4.   Effect of lidocaine and verapamil on volume recovery. Lidocaine (0.2 mM) and verapamil (0.2 mM) were added to both sides of epithelium. Mean absolute Tc values in isosmotic conditions are 6.31 ± 0.19 (N = 6, n = 73), 5.57 ± 0.22 (N = 6, n = 86), and 5.26 ± 0.25 µm (N = 5, n = 36) for control and verapamil- and lidocaine-treated cells, respectively.

Our data show that only quinine is able to block completely the RVD and the related ⁸⁶Rb efflux, whereas Ba²⁺, a potent K⁺ channel blocker, was unable to prevent volume recovery. Therefore, we assayed the effect of Cs⁺, which is also known to inhibit certain types of K⁺ channels (11). Figure 5A illustrates the effect of 10, 30, and 60 mM Cs⁺ on volume regulation. It is clear that high concentrations (30 mM) of Cs⁺ are required to block volume regulation. On the other hand, after replacement of 60 mM Na⁺ in the basolateral solution by Cs⁺, hypotonicity leads to a massive increase of T_c. As early as 25 min after the beginning of the osmotic challenge, this augmentation of cell volume increased T_c to 305 ± 14%. This extreme increase in cell volume could be caused by an additional cellular accumulation of osmolytes during hypotonicity, which under these conditions is most likely caused by the entry of Cs⁺ across the basolateral membrane. The replacement of 60 mM basolateral Na⁺ by Cs⁺ did not affect cell volume in isosmotic conditions, indicating that the basolateral membranes are impermeable for Cs⁺ in isosmotic conditions. However, hyposmotic swelling seems to open a pathway with poor selectivity that enables Cs⁺ to enter the cell. Comparison of ⁸⁶Rb release data and T_c measurements in Fig. 5B confirms this idea. Here 40 mM Cs⁺ blocked the RVD almost completely. On the other hand, R^bl_Rb increased to a level approaching the peak release in control and remained relatively high during the entire osmotic challenge. If Cs⁺ acted as a pure blocker of K⁺ efflux, R^bl_Rb would have been reduced, as in the experiment with quinine (Fig. 3). Thus Cs⁺ seems to permeate the cell membrane, thereby keeping intracellular cation content high and, in this way, preventing net osmolyte loss. The lack of cellular osmolyte decrease keeps cells swollen, as demonstrated in the T_c record, which maintains the ⁸⁶Rb release pathway activated. Also, in the initial phase of the hyposmotic shock, the increase of R^bl_Rb is noticeably delayed compared with control. This observation suggests a direct inhibitory effect of Cs⁺ on the ⁸⁶Rb efflux.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Influence of Cs+ on regulatory volume decrease (RVD) and 86Rb efflux during a hypotonic shock. Solution is described in Table 1. To avoid a transepithelial gradient, both sides of epithelium were exposed to Cs+. A: inhibitory effect of 10, 30, and 60 mM Cs+ on RVD. Initial absolute Tc values are 6.31 ± 0.19 (N = 6, n = 73), 5.42 ± 0.30 (N = 3, n = 47), 7.76 ± 0.19 (N = 4, n = 73), and 7.45 ± 0.40 µm (N = 4, n = 46) for 0 (control), 10, 30, and 60 mM Cs+, respectively. B: RblRb and Tc traces in control and in presence of 40 mM Cs+. At end of hyposmotic pulse, RblRb was 0.46 ± 0.02 (N = 7) and 1.65 ± 0.08 min1 (N = 4) in control and Cs+-treated cells, respectively. Initial absolute Tc with 40 mM Cs+ is 5.76 ± 0.22 µm (N = 5, n = 57).

Inasmuch as classical K⁺ channel blockers (Ba²⁺ and Cs⁺) were not able to abolish volume regulation and because the Cs⁺ experiments indicated the rather poorly selective nature of the regulatory pathway, we decided to test the effect of lanthanides, which are known to block poorly selective and stretch-activated ion channels in other tissues (7, 40, 41). Figure 6 demonstrates that chronic exposure of the basolateral side to 0.5 mM Gd³⁺ completely abolished volume regulation and R^bl_Rb. On the other hand, the peak in the R^bl_Rb response was only slightly reduced if Gd³⁺ was acutely added to the bath when the osmolality was reduced. However, acute application also blocked the R^bl_Rb fully toward the end of the osmotic pulse, indicating a marked delay in the inhibitory action of the lanthanide. This delay also appears from the T_c record (Fig. 6A), showing a clear onset of volume regulation, which, however, was interrupted after T_c had declined from 171 to 122%. This striking difference between the two modes of application suggests an intracellular site of action. Whether Gd³⁺ exerts its action on the signaling pathway or directly on the cation channels remains unresolved. Effects similar to those shown with Gd³⁺ were obtained with La³⁺. Figure 6C compares the chronic and acute effects on RVD of addition of the lanthanide at 0.5 mM to the basolateral side. Here also chronic preincubation with 0.5 mM lanthanide fully blocked RVD, whereas acute administration did not affect the initial phase of volume recovery. This result confirms an intracellular site of action of the lanthanides.

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Chronic and acute effect of 0.5 mM basolateral Gd3+ and La3+. In chronic experiment, lanthanide was present in iso- and hyposmotic solution and added 60 min before hypotonic challenge. Acute effects of Gd3+ and La3+ were tested by adding lanthanide when osmolality was reduced. Experiments were performed in HEPES-buffered solutions (Table 1). A: effect of Gd3+ on volume regulation. Initial absolute Tc values are 7.46 ± 0.25 (N = 6, n = 59), 6.27 ± 0.35 (N = 6, n = 51), and 6.06 ± 0.22 µm (N = 5, n = 59) for control and acutely and chronically administered Gd3+, respectively. B: effect of Gd3+ on RblRb. Peak RblRb was 5.17 (N = 4), 3.65 (N = 6), and 0.47 min1 (N = 4) for control and acutely and chronically administered Gd3+, respectively. C: effect of La3+ on volume regulation. Initial absolute Tc values are 7.46 ± 0.25 (N = 6, n = 59), 7.07 ± 0.22 (N = 7, n = 70), and 5.33 ± 0.25 µm (N = 6, n = 68) for control and acutely and chronically administered La3+, respectively.

The fact that Gd³⁺ acts on an intracellular site implies that the lanthanide could enter the cell and that the cell membrane has a finite permeability for this cation. The permeability of the basolateral membrane for Gd³⁺ also appears in experiments where the effect of Gd³⁺ on the ⁸⁶Rb release is measured in isotonic conditions (Fig. 7). The addition of 0.5 mM Gd³⁺ to the basolateral side transiently increased R^bl_Rb, which reached a maximum 15-18 min after addition of the lanthanide. The apical release of ⁸⁶Rb remained unaffected by this treatment. We also tested the effect of 10 µM nifedipine on the basal and Gd³⁺-induced R^bl_Rb. The data in Fig. 7 show that, in the absence of Gd³⁺, nifedipine only slightly decreased R^bl_Rb from 0.19 ± 0.01 to 0.15 ± 0.01 min¹. On the other hand, the peak value reached during Gd³⁺ treatment was reduced from 0.81 ± 0.08 to 0.45 ± 0.07 min¹ (N = 4). This result is in agreement with the idea that Gd³⁺ entry proceeds at least partly through L-type Ca²⁺ channels. Addition of Gd³⁺ to the basolateral solution in hypotonic conditions did not noticeably increase R^bl_Rb (data not shown). This result suggests that R^bl_Rb after acute administration should not be overestimated by an effect of Gd³⁺.

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Effect of basolaterally applied Gd3+ on Rb+ efflux in isosmotic conditions. Gd3+ (0.5 mM) was present during period indicated by open bar. Experiments were performed in HEPES-buffered solutions. Effect of nifedipine was tested by exposing basolateral membrane to 10 µM nifedipine during entire experiment. Only effect of the Ca2+ channel blocker on RblRb is shown, because RapRb was negligible.

Experiments with reduced amounts of Gd³⁺ (Fig. 8) demonstrated that 0.5 mM was needed to block the RVD completely. The chronic addition of 0.2 mM Gd³⁺ to the basolateral surface reduced the RVD markedly. During the hypotonic shock, T_c increased to 177% and remained markedly above control, reaching 136% at the end of the recovery phase. Gd³⁺ (10 µM) had only a small effect on RVD, which returned T_c to 108%.

View larger version (14K): [in this window] [in a new window]   Fig. 8.   Dose dependence of basolaterally applied Gd3+. Tc was recorded with addition of 0.01, 0.2, and 0.5 mM Gd3+ to basolateral side. Experiments were performed in HEPES-buffered Cl solutions. Numerical data are shown in Table 2. Mean absolute Tc values in isosmotic conditions are 7.46 ± 0.25 (N = 6, n = 59), 8.67 ± 0.25 (N = 5, n = 59), 7.34 ± 0.60 (N = 2, n = 18), and 6.27 ± 0.35 µm (N = 6, n = 51) for control and 0.01, 0.2, and 0.5 mM Gd3+, respectively.

Inasmuch as the comparison of effects of an acute and chronic addition of Gd³⁺ suggested an intracellular effect, we also tested whether Gd³⁺ could reach this site when added to the apical solution. Figure 9 illustrates effects of chronic application of Gd³⁺ to the apical surface of the epithelium. The T_c records show that the lanthanide at 0.5 mM hardly affected volume recovery. Also R^bl_Rb was not influenced by this amount of Gd³⁺. A 10-fold higher dose of this trivalent cation was needed to reduce R^bl_Rb markedly and to exert a pronounced effect on volume recovery. The fact that this rather high dose of apical Gd³⁺ could exert this inhibitory effect demonstrates that the apical border has a finite permeability for the lanthanide, which enables the cation to reach its intracellularly located inhibitory site.

View larger version (19K): [in this window] [in a new window]   Fig. 9.   Chronic application of Gd3+ to apical surface. Experiments were performed in HEPES-buffered solutions. A: Tc in control and with 0.5 and 5 mM Gd3+ added to apical perfusate. Initial absolute Tc values are 7.46 ± 0.25 (N = 6, n = 59), 7.20 ± 0.22 (N = 8, n = 114), and 7.17 ± 0.33 µm (N = 5, n = 60) for control and 0.5 and 5 mM Gd3+, respectively. B: RblRb with 0.5 and 5 mM Gd3+ added to perfusate. Peak RblRb values were 5.17 ± 0.21 (N = 4) and 2.70 ± 0.09 min1 (N = 4) for 0.5 and 5 mM apical Gd3+, respectively.

Several reports (1, 6, 7, 40) demonstrate that lanthanides exert an inhibitory effect on Ca²⁺ entry through SACs. Therefore, it is conceivable that the effects of Gd³⁺ and La³⁺ added to the basolateral bath are related to the blockage of Ca²⁺ entry. To verify this hypothesis, we performed experiments with basolateral Ca²⁺-free solutions prepared by removing Ca²⁺ and chelating the remaining Ca²⁺ with 0.5 mM EGTA. In one type of experiment we added 0.5 mM Gd³⁺ to the apical solution. This treatment kept the transepithelial resistance above 300  · cm², indicating that the epithelial structure was maintained. Similarly, an inhibition by La³⁺ of the increase in G_t after Ca²⁺ removal has recently been reported for the epithelium of frog urinary bladder (25). However, the nature of the protective effect of Gd³⁺ remains unexplained. In the absence of the lanthanide in the apical bath, we limited the duration of the hyposmotic shock to 30 min because of an extremely large drop in transepithelial resistance. The records in Fig. 10 demonstrate a considerable increase of the peak value of T_c, indicating that Ca²⁺ removal markedly augments the osmotic activity of the cell. During the hypotonic period, cell volume almost completely recovered, with a time constant of 8.2 min (Table 2). In the experiment with Gd³⁺ added to the apical surface, where we could extend the osmotic perturbation to 60 min, volume recovery was noticeably delayed but still almost complete. These results demonstrate that the development of the RVD does not require basolateral Ca²⁺ entry. Moreover, the inhibitory effect of the lanthanides does not seem to occur through the blockage of Ca²⁺ entry.

View larger version (12K): [in this window] [in a new window]   Fig. 10.   Effect of Ca2+ removal from basolateral bathing solution. Control trace is from Fig. 6. Basolateral Ca2+-free solutions were prepared by removal of Ca2+ and addition of 0.5 mM EGTA. In one type of experiment, we added 0.5 mM Gd3+ to apical solution. This treatment kept transepithelial resistance high (>300  · cm2) during entire hyposmotic shock of 60 min. In experiment performed without Gd3+, we limited hypotonic exposure time to 30 min because of a marked drop of transepithelial resistance to 270  · cm2. Mean absolute Tc values in isosmotic conditions are 7.46 ± 0.25 (N = 6, n = 59), 5.76 ± 0.29 (N = 2, n = 27), and 4.78 ± 0.32 µm (N = 3, n = 30) for control, 0 Ca2+, and 0 Ca2+ + apical Gd3+, respectively.

Data reported by Basavappa and Strange (2) demonstrate that volume-activated channels are sensitive to Mg²⁺. Effects of the acutely and chronically administered Mg²⁺ on volume recovery and R^bl_Rb are illustrated in Fig. 11. In contrast to the lanthanides, the action of Mg²⁺ did not depend on the preincubation time of the divalent cation. Indeed, acute as well as chronic administration delayed volume recovery, which is reflected in the time constant of the T_c decay (Table 2). Moreover, the volume recovery was incomplete in both types of experiments, keeping T_c 17% above control. The effects on volume recovery are reflected in the ⁸⁶Rb release, which demonstrates an almost identical reduction of R^bl_Rb for both types of treatment (Table 2). Inasmuch as we did not find any difference between the acute and chronic administration, we cannot exclude an extracellular effect of the divalent cation. Therefore, we tested a 10-fold higher dose. If Mg²⁺ exerted its effect directly on an extracellular site of the pathway for osmolyte release, the increased dose would exert a more pronounced effect on RVD as well as R^bl_Rb. However, increasing the dose of Mg²⁺ from 0.5 to 5 mM did not improve the inhibitory effect of Mg²⁺ (Table 2).

View larger version (21K): [in this window] [in a new window]   Fig. 11.   Chronic and acute effects of 0.5 mM Mg2+. Divalent cation was added to both sides of epithelium. In acute experiment, Mg2+ was added when basolateral osmolality was reduced. In chronic experiment, tissue was preincubated with Mg2+ 30 min before hyposmotic shock. A: Tc. Mean absolute Tc values in isosmotic conditions are 6.31 ± 0.19 (N = 6, n = 73), 7.48 ± 0.43 (N = 3, n = 23), and 7.14 ± 0.32 µm (N = 4, n = 29) for control and acutely and chronically administered Mg2+, respectively. B: effect of Mg2+ on RblRb. Peak RblRb was 2.40 (N = 4) and 2.03 min1 (N = 4) for acutely and chronically administered Mg2+, respectively.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

We have shown that cell swelling caused by a basolateral hyposmotic challenge results in a substantial increase of the unidirectional efflux of K⁺ (⁸⁶Rb), which takes place during the volume regulatory phase. A striking agreement between the ⁸⁶Rb efflux data and the T_c records was observed: Except for the Cs⁺ experiments, the inhibition of volume recovery was reflected in all cases in a reduction of the ⁸⁶Rb release. Quinine completely blocks the RVD and ⁸⁶Rb efflux, whereas lidocaine does not noticeably affect volume regulation. Also Ba²⁺, even at high concentrations, only displays a weak inhibitory effect. Furthermore, chronic administration of lanthanides (La³⁺ and Gd³⁺) blocks R^bl_Rb, resulting in a complete inhibition of the RVD. No difference was observed between chronic and acute administration of Mg²⁺, which significantly affected RVD and R^bl_Rb. The inhibition of RVD observed with high Cs⁺ concentrations without blocking the ⁸⁶Rb release suggests that hypotonicity opens a pathway for the entrance of this monovalent cation into the cell. These results suggest that hypotonicity activates a poorly selective cation channel that is highly sensitive to lanthanides and quinine and markedly inhibited by Mg²⁺.

In a variety of tissues, Ba²⁺ reduces the basolateral K⁺ conductance and affects volume regulation in a concentration-dependent manner (12, 39). We recorded a delayed and partially inhibited RVD with 30 mM Ba²⁺. The failure of Ba²⁺ to exert a distinct effect on RVD was shown in a previous study, where lower concentrations (1-5 mM) of the divalent cation were utilized (9). The low potency of Ba²⁺ in blocking volume regulation was also shown in HeLa cells (20), rabbit proximal straight tubules (38, 39), cultured astrocytes (29), and nonpolarized A6 cells (12). The failure of Ba²⁺ to inhibit volume regulation was also found in experiments where the basolateral osmolality was gradually reduced, which enabled the cell to adjust its volume without noticeable cell swelling (35). The low sensitivity to Ba²⁺ of the swelling-activated pathway compared with the effect of the divalent cation on the native K⁺ permeability of the basolateral membrane suggests that cell swelling opens a pathway for K⁺ excretion that has characteristics completely different from the native K⁺ channels. A similar conclusion was derived from experiments in which the basolateral K⁺ permeability was studied after the apical border was permeabilized with nystatin (13, 14). These studies mainly discriminated between the pathways on the basis of their degree of sensitivity to quinine.

Within this context, we compared pharmacological effects on volume regulation and on basolateral quinine-sensitive K⁺ channels studied in nystatin-treated tissues. Several reports demonstrated inhibitory effects of quinine as well as verapamil and lidocaine (13, 21, 33, 37). In these studies, K⁺ currents were generated by establishment of an electrochemical gradient for K⁺ and by permeabilization of the apical membrane with ionophores. The latter treatment enabled K⁺ to cross the apical barrier. K⁺ entering the cell in this way was assumed to cause cell swelling and activation of the quinine-sensitive pathway. Therefore, the quinine-sensitive pathway and volume regulation should display similar pharmacological properties. Our results, however, showed that lidocaine, which was the most potent inhibitor of the quinine-sensitive K⁺ channels in the studies mentioned above, failed to have a significant effect on RVD. Therefore, it appears that the basolateral K⁺ channels stimulated by permeabilization of the apical membrane and the subsequent cell swelling are different from channels activated by the hypotonic treatment. In this regard, besides its inhibitory effect exerted on volume-activated K⁺ channels (12, 29), quinine has been found to inhibit nonselective cation channels in rat distal colon cells (15). The pharmacological resemblance between the nonselective pathway in rat colon and the pathway for K⁺ excretion during volume recovery is quite striking and agrees with the above-stated hypothesis that volume recovery occurs through a poorly selective cation pathway. On the other hand, it appears that lidocaine-sensitive channels, activated by permeabilization of the apical membrane, are of a different nature. However, besides the direct blockage of ion channels, quinine might inhibit the volume-sensing mechanisms and/or transduction pathways.

The fact that the swelling-activated K⁺ release utilizes a pathway that differs from the native K⁺ channels appears also from the Cs⁺ data. Several types of K⁺-selective channels are capable of binding Cs⁺ (5, 11), resulting in a blockade of K⁺ current with millimolar concentrations of the inhibitor. Our data show that high concentrations of extracellular Cs⁺ were needed to reduce RVD significantly (Fig. 5). Although volume recovery was completely abolished at high Cs⁺ concentrations, the R^bl_Rb was not blocked. On the contrary, in the presence of 40 mM Cs⁺, ⁸⁶Rb efflux was activated to a degree similar to that in control and remained high during the entire hypotonic period. The apparently contradictory effects of Cs⁺ on RVD and R^bl_Rb seem to find their origin in the combination of two opposing phenomena. 1) In the initial phase of the hyposmotic shock, Cs⁺ clearly inhibits R^bl_Rb, most likely by occupying the binding site of Rb⁺ in the channel. 2) Then, when the pathway is fully activated, Cs⁺ permeates through the channel and substitutes for the intracellular K⁺ and Rb⁺, thereby keeping cell volume high and enhancing the isotope efflux. The entrance of Cs⁺ is favored by the electrochemical driving force for Cs⁺, which is inwardly directed. This results in a gradual increase of cellular volume in the presence of 60 mM Cs⁺. It is conceivable that the Cs⁺ uptake at least partly occurs through the Na⁺-K⁺-ATPase, as was reported for frog skeletal muscle (3). However, experiments not shown here demonstrated that 0.1 mM ouabain did not prevent the gradual increase of cell volume when the basolateral surface was exposed to 60 mM Cs⁺. This result supports the evidence for the uptake of Cs⁺ via the volume-activated pathway.

We found that the volume recovery was completely inhibited by lanthanides, especially Gd³⁺, which has been described as a potent blocker of stretch-activated K⁺ channels (SACs) (41). SACs act as Ca²⁺ entry pathways and thus contribute to the regulation of intracellular Ca²⁺ (1, 6). Therefore, an indirect effect of the lanthanides on the RVD via the inhibition of Ca²⁺ influx cannot be excluded. Indeed, in many cell types, cell volume regulation is mediated by Ca²⁺-activated K⁺ channels (6, 7). Within this context, the effect of Gd³⁺ on RVD via the inhibition of Ca²⁺ entry would presume the existence of SACs in the A6 epithelia. The experiments depicted in Fig. 10 demonstrate that an almost complete volume recovery could take place in the absence of extracellular Ca²⁺, suggesting that Ca²⁺ entry does not play a major role in volume regulation of the A6 epithelia. The initial volume increase was much larger than in control, indicating that Ca²⁺ removal markedly increases the intracellular osmotic activity. Volume recovery was noticeably delayed by using apical Gd³⁺ to maintain transepithelial resistance at a high level and, presumably, conserving in this way the integrity of the epithelial structure.

The marked differences between the effects of acute and chronic administration of lanthanides on the RVD and R^bl_Rb suggest an intracellular site of action. The intracellular concentration of Gd³⁺ reported to inhibit mechanosensitive ion channels in the yeast plasma membrane was 10 µM (18). It is conceivable that such an intracellular concentration is reached by addition of 500 µM Gd³⁺ to the basolateral bath. Evidence for a finite permeability of the cell membrane for Gd³⁺ and, thus, for an increase of the intracellular concentration was found in the marked transient stimulation of R^bl_Rb in isotonic conditions with an onset at ~12 min after administration of Gd³⁺ (Fig. 7). The noticeable delay in R^bl_Rb stimulation suggests that the lanthanide enters the cell and possibly activates the ⁸⁶Rb efflux by its action on Ca²⁺-activated K⁺ channels or by screening of negative surface charges (41). The latter concept assumes that the neutralization of negative surface charges reduces the local concentration of the permeant monovalent cation, which results in a transient increase of efflux of cations. The mechanism of action of Gd³⁺ and La³⁺ that causes the inhibition of RVD and R^bl_Rb during hypotonic conditions is unclear. At least two different mechanisms can be proposed. The first is a direct inhibitory effect of the lanthanides on the volume-activated channel. In this relation, Gd³⁺ inhibits SACs of very different conductances such as Xenopus (41), yeast (19), and bacteria (4). However, it seems unlikely that the inhibition of channels with such different sizes or structure occurs via a direct blocking mechanism. Alternatively, as proposed by Berrier et al. (4), the lanthanides could affect the stretch sensor that controls the gating of the cation pathway. Our data do not enable us to distinguish between these two possibilities.

Relatively small extracellular Mg²⁺ concentrations (0.5 mM) exhibit a significant inhibitory effect on volume recovery (Fig. 11). Increasing the amount of the divalent cation did not augment the effect on RVD and R^bl_Rb. Mg²⁺ acts much faster than Gd³⁺ and La³⁺. Indeed, in contrast to the lanthanides, with the divalent cation we found no difference in the responses to acute and chronic administration.

Our data show that the response of R^bl_Rb clearly lags behind the increase of T_c invoked by the hypotonic challenge (Fig. 1). This observation demonstrates that the ⁸⁶Rb efflux is not directly related to the size of the cell, which can increase by changing its shape or by increasing the membrane area. If cell swelling causes membrane stretch, this stimulus does not seem to directly activate the osmolyte release in A6 epithelia. Similarly, membrane bending and thus induced membrane stretch did not affect K⁺-Cl cotransport in rabbit red blood cells (23). Furthermore, Parker and co-workers (28) demonstrated that volume regulatory mechanisms are already activated in dog red blood cells at volumes 5-10% above normal. In this cell type the accommodation of this additional volume should be possible by relatively small changes in cell shape and thus without a noticeable increase in membrane stretch. Alternatively, it cannot be excluded that the initial phase of volume recovery is mediated by the excretion of organic osmolytes (31), whereas inorganic ion release accounts for the subsequent phase of the RVD. Within this context, membrane stretch cannot be excluded as the trigger for osmolyte excretion. Moreover, membrane stretch was suggested to trigger the increase of protein tyrosine kinase, which was demonstrated to regulate swelling-induced osmolyte excretion (32). Although the initial activation step of protein tyrosine kinase could take place at the moment cell volume reaches its maximum, different steps could delay the activation of the pathway for osmolyte excretion in the transduction system.

We found that, after an osmotic challenge of 120 mosmol/kgH₂O, cell volume returns to almost exactly its original size. The osmotic loss during such a complete RVD cannot be attributed solely to KCl but implies a major contribution of organic solutes (24, 31). The observation that Gd³⁺, La³⁺, and quinine are able to completely block the RVD thus implies that these inhibitors not only prevent the release of KCl but also abolish the organic osmolyte loss. This observation favors the hypothesis of an action of the lanthanide on the signal transduction pathway.

Two alternative mechanisms can be proposed for the mode of action of the inhibitors. 1) As suggested above, the lanthanides seem to exert their action on an intracellular site, and it is well documented that quinine can reach intracellular receptors. Therefore, it is conceivable that the inhibitors interfere with an intracellularly located volume sensor or with the signal transduction pathway, which activates the osmolyte release. 2) The inhibitors are known to be blockers of nonselective pathways (15, 16). This action would agree with the concept of a very unspecific large channel that enables the excretion of all osmotic substances during RVD (20). Hall et al. (20) suggested that this pore might be permeable for large anions and cations. The activation of such a pathway could explain the partial but pronounced volume recovery observed in Cl-depleted cells obtained by using gluconate or sulfate as the main anion in the bathing solutions (9).