Pflügers Arch - Eur J Physiol (2001) 442:297–303 DOI 10.1007/s004240100548

O R I G I N A L A RT I C L E

T. Grosse · I. Heid · J. Simaels · F. Beck · W. Nagel W. Van Driessche · A. Dörge

Changes in element composition of A6 cells following hypotonic stress

Received: 2 November 2000 / Received after revision: 29 January 2001 / Accepted: 30 January 2001 / Published online: 20 March 2001 © Springer-Verlag 2001

Abstract Cellular element concentrations and dry weight contents were determined in A6 epithelia using electron microprobe analysis. This was done to assess the quantitative contributions of Na, K and Cl to the regulatory volume decrease (RVD) and isovolumetric regulation (IVR) after decreasing the basolateral osmolality from 260 to 140 mosmol/kg in a stepwise or gradual way. Two minutes after inducing acute hypotonic stress the cells behaved almost like ideal osmometers, as indicated by a pronounced increase in cell height and decreases in the cellular dry weight and concentrations of all measured elements by about the same degree. Sixty minutes after inducing acute hypotonic stress the dry weight and concentrations of the impermeant elements P, Mg and Ca had returned approximately to control values, indicating normalized cell volume. Na, K and Cl concentrations, however, remained greatly reduced. The cellular amounts of Na, K and Cl diminished during RVD by approximately 31%, 24% and 46%, respectively. The dry weights and element concentrations measured 60 min after inducing acute hypotonic stress were similar to those obtained after a continuous reduction of basolateral osmolality. The cellular loss of Na and K following hypotonic stress exceeded that of Cl by about 40 mmol/kg wet wt., suggesting the exit of an other anion and/or the titration of fixed negative charges. The contribution of Na, K and Cl to total cellular osmolality increased from about 75% under control conditions to about 85% during RVD and IVR. Since only approximately 70% of the loss of cellular osmolytes necessary for the observed RVD A. Dörge (✉) Physiologisches Institut, Pettenkoferstr. 12, 80336 Munich, Germany Tel.: +49-089-5996504, Fax: +49-089-5996532 T. Grosse · I. Heid · F. Beck · W. Nagel · A. Dörge Physiologisches Institut der Universität München, Munich, Germany J. Simaels · W. Van Driessche Laboratory of Physiology, Katholieke Universität Leuven, Campus Gasthuisberg, Louvain, Belgium

and IVR is accounted for by the cellular exit of Na, K and Cl, other osmolytes, possibly amino acids, must leave the cells following hypotonic stress. Keywords Cell volume · Cellular dry weight content · Electron microprobe analysis · Hypotonic stress · Isovolumetric regulation · Regulatory volume decrease

Introduction A6 epithelia, composed of amphibian kidney cells of a continuous cell line, are treated as a model for studying transepithelial Na and Cl transport across tight epithelia [18, 22,30] and have also been used extensively to clarify volume regulatory processes [7, 14,27]. The cells of A6 epithelia are able to maintain their volume despite large decreases in basolateral osmolality. An acute reduction of basolateral osmolality leads to cell swelling and subsequent a regulatory volume decrease (RVD) [9,11]; however, no alteration in cell volume is observed if the basolateral osmolality is reduced continuously and slowly (isovolumetric regulation=IVR) [26]. Using different approaches various studies have shown that cellular losses of K and Cl contribute to cell volume regulation of A6 cells during hypotonic shock. Measurements of Cl and Rb fluxes have revealed that the cellular efflux of Cl and K from A6 cells increases during RVD and IVR [4,26]. Evidence that these ions leave through basolateral channels was provided by using various K and Cl channel blockers, which altered the characteristics of cellular K efflux following hypotonic stress, diminished hypotonicity-induced basolateral conductance and blocked RVD [5, 8,14]. The patch-clamp technique has revealed that various types of quiescent Cl channels are activated during the first few minutes of hypotonic stress, and that the activity of Cl channels already in use also increases at this time [1,5]. Although direct evidence for the hypotonicity-induced loss of K and Cl from A6 cells has been obtained by measuring cellular K content [26] and Cl activity [4],

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it remains unclear precisely how much these ions contribute to cell volume regulation. Electron microprobe analysis of cellular element concentrations has provided detailed information about the participation of small electrolytes in RVD, as demonstrated for rabbit kidney cells [23]. In the present study this technique was applied to assess quantitatively the changes in element concentrations and contents of A6 cells following hypotonic stress. Measurements were performed 2 min and 60 min after inducing acute hypotonic stress or after continuously reducing basolateral osmolality from 260 to 140 mosmol/kg by decreasing the NaCl concentration.

Materials and methods Cell culture Renal A6 cells (Xenopus laevis) were obtained from the American Type Culture Collection (Rockville, Md., USA) and cultured in flasks (Nunc, Wiesbaden, Germany, area 75 cm2). Cells at passages 100–116 were incubated in a special culture medium and maintained in a humidified incubator (MCO-17 AI, CO2, Sanyo, Japan) gassed with 2% CO2 at 27°C. About 1 l (1043 ml) of culture medium contained: 350 ml Leibovitz’s F-15 medium (Gibco, Karlsruhe, Germany; 11415–049), 350 ml Nutrient Mixture Ham’s F-12 medium (Gibco; 21765–029), 200 ml (pyrogen-free) water, 100 ml fetal calf serum (Seromed, Berlin, Germany; S0213), 20 ml of 200 mmol/l L-glutamine solution (Gibco; 25030–024), 20 ml penicillin-streptomycin solution (Gibco; 15070–022) and 3 ml of 7.5% NaHCO3 solution (Gibco; 25080–060). The osmolality of the culture medium was about 260 mosmol/kg and the pH 7.4. For subculture the cells were detached weekly from the culture flask bottom by incubating the cells with a trypsin-EDTA solution (Gibco; 45300–019) for 20 min. To stop enzyme activity, relatively large volumes of culture medium were added to the cell suspension. The isolated A6 cells were then seeded either again in culture flasks at a density of 5×105 cells/cm2 or on collagen-coated, permeable large (5.47 cm2) or small (0.7 cm2) Millicell-CM filters (Millipore, Eschborn, Germany) at a density of 2×105 cells/cm2. Coating solution was obtained by dissolving 10 mg rat tail collagen Type I (Sigma, Heidelberg, Germany, C7661) in 4 ml of 0.2% acetic acid. The cells in the culture flasks and those on the filter membranes were fed twice weekly by renewing the culture medium. Confluent monolayers obtained 10–12 days after seeding were used for the experiments. Incubation Epithelia cultured on the large filters were exposed to either a stepwise decrease in basolateral hypotonicity for 60 min (longterm hypotonicity) or to a continuous decrease in basolateral osmolality (gradual hypotonicity). On epithelia grown on the small filters the basolateral side was made hypotonic for only 2 min (short-term hypotonicity). Basolateral osmolality was always reduced from 260 to 140 mosmol/kg by omitting NaCl. Media used for incubation were normal Ringer (260 mosmol/kg), apical Nafree hypotonic (140 mosmol/kg) and basolateral Na-containing hypotonic (140 mosmol/kg) solution. All solutions contained 1 mmol/l CaCl2 and 2.5 KHCO3 and were adjusted to pH 8 by adding KOH. In addition, normal Ringer contained 140 mmol/l NaCl, Na-free hypotonic solution 70 mmol/l N-methyl-D-glucamine chloride (NMDG-Cl) and Na-containing hypotonic solutions 70 or 50 mmol/l NaCl. For long-term and gradual hypotonicity a central part of the filter membrane with the epithelium on top was glued onto a Lucite ring (OD 12, ID 8 mm), cut out at the outer circumference of

the ring and inserted into an Passing type chamber (exposed area 0.5 cm2). During the preincubation period the epithelia were incubated for the first 30 min with normal Ringer on both sides and then for a further 30 min with normal Ringer on the basolateral and Na-free hypotonic solution on the apical side. Thereafter the incubation was either continued under the same conditions for a further 60 min (control) or the basolateral side was also made hypotonic (140 mosmol/kg), either by 60 min of perfusion with Nacontaining hypotonic solution (long-term hypotonicity) or by decreasing the NaCl concentration and thereby the osmolality in the basolateral perfusion solution continuously using a gradient mixer (gradual hypotonicity) as described by Van Driessche et al. [26]. The epithelia were kept short-circuited throughout the chamber incubation by an automatic clamping device (Frankenberger, Germering, Germany). Voltage pulses (1 mV, 1 s duration, 10 s interval) were applied to calculate, by Ohm’s law, transepithelial conductances from the current deviations. Each half-chamber (volume 1.5 ml) was perfused with a peristaltic pump at about 5 ml/min. For short-term hypotonicity the small filter inserts (cups) with the epithelia upwards (apical side) were placed in Petri dishes (basolateral side). For the first 30 min both apical and basolateral sides contained normal Ringer solution. Thereafter, the apical side was exposed for 32 min to the Na-free hypotonic solution. For the last 2 min of incubation the Ringer solution on the basolateral side was replaced by the Na-containing hypotonic solution. A pipette was used to fill the filter inserts and the Petri dishes with incubation solution and also to remove it. To avoid hydrostatic pressure differences detaching the epithelia from the permeable supports, the Petri dishes were only filled and emptied if incubation fluid was in the filter cups. Tissue preparation The preparation of freeze-dried cryosections for electron microprobe analysis is described in detail elsewhere [13,24]. In short, at the end of the incubation the A6 epithelia were removed quickly from the chambers or the Petri dishes, freed from adherent incubation fluid, covered on both sides with a thin layer of appropriate albumin standard solutions and shock frozen in a mixture of liquid isopentane/propane (20/80%) cooled to liquid N2 temperature. Adequate albumin standard solutions were prepared by dissolving 1 g bovine albumin (Behring, Marburg, Germany) in 4 ml of the apical and basolateral solutions used at the end of incubation. Less than 10 s elapsed between the end of incubation and shock freezing. It was necessary to cover both sides of the epithelium with albumin solution to provide sufficient mechanical stability to prevent the epithelium from detaching from the filter membrane when the tissue was frozen and cut. Cryosections about 1 µm thick were cut perpendicular to the epithelial surface in a modified cryoultramicrotome (Reichert, OmU3, Vienna, Austria) at –90°C. The sections were sandwiched between thin formvar and collodium films and freeze-dried at –80°C and 10–4 Pa (10–6 mbar). Electron microprobe analysis As described earlier in more detail [13,24], electron microprobe analysis of freeze-dried cryosections was performed using a scanning electron microscope (Stereoscan S150, Cambridge Instruments, Cambridge, UK) with an energy dispersive X-ray detecting system (LINK System, High Wighcombe, UK). The acceleration voltage was 20 kV, the probe current 0.3 nA and the analysis time 100 s. Areas of about 0.5 µm2 were scanned during analysis within A6 cells and in the albumin standard layer adherent to the apical surface. The emitted X-rays were assessed in the energy range 0–20 keV. This range encompasses the K-lines of the biologically relevant elements Na, Mg, P, Cl, K and Ca. Discrimination between the elements’ characteristic radiation and the background of the X-ray spectrum was performed with a specially designed computer program [2]. Quantification of cellular element concentra-

299 tions (in mmol/kg wet weight) and dry weight contents (in g/100 g wet weight) was performed by comparing the intensities of the element characteristic radiation and those of the background radiation of cell and standard spectra, respectively. Statistics The data were obtained under each osmotic situation from six to ten experiments. The element concentrations in about 20 cells were determined for each experiment. The data are given as means ±SEM. The significance of differences between means was calculated using the Student’s t-test (for paired samples). A value of P<0.05 (two tailed) was regarded as significant.

Results The conditions during chamber incubation were the same as those used by Van Driessche and coworkers [26] to provoke an RVD and IVR. Before applying hypotonic stress by decreasing the basolateral osmolality, the apical Ringer solution was replaced with a Na-free hypotonic solution to eliminate the influences of transepithelial Na transport on volume regulatory systems and to avoid possible apical-to-basolateral osmotic gradients, which could open the paracellular pathway. Since the permeability of the basolateral membrane of A6 cells to water is considerably higher than that of the apical membrane [11], this procedure had no influence on cellular volume. Table 1 shows cellular element concentrations and the dry weight content of A6 cells obtained by microprobe analysis under control conditions. During chamber incubation, the apical side of the epithelia was perfused with the Na-free, hypotonic solution and the basolateral side with normal Ringer. Unlike the concentrations of Na, Mg, Cl and Ca, those of K and P were relatively high. The dry weight content accounted for about one-fifth of the total cellular mass. Incubation of the apical side with normal Ringer had no measurable effect upon cellular element concentrations and dry weight contents (data not shown). Within the first few minutes, A6 cells react to acute hypotonic stress by swelling. In the present study this was indicated by an increase in cell height as could be seen from the freeze-dried cryosections obtained under control conditions and 2 min after the acute hypotonic stress had been induced. The long-term effect of hypotonic stress upon the cellular element composition of A6 cells is demonstrated by two X-ray spectra shown in Fig. 1. The spectra were obtained under control conditions and 60 min after an acute reduction of basolateral osmolality from 260 to 140 mos-

Fig. 1 X-ray spectra obtained from A6 cells under control conditions and 60 min after an acute reduction of basolateral osmolality from 260 to 140 mosmol/kg (Hypo 60)

Fig. 2 Dry weight contents (dw) in g/100 g wet wt. and the concentrations of P, K, Cl, Na and Mg of A6 cells in mmol/kg wet wt. under control conditions and 2 and 60 min after reducing the basolateral osmolality from 260 to 140 mosmol/kg. The deviations in SEM are not larger than the symbols for the mean values

mol/kg. The P and Mg peaks are almost identical in both spectra whereas the Na, Cl and K peaks of the spectrum obtained from a cell exposed to hypotonicity are significantly smaller than in the control spectrum. Figure 2 shows the time course of the cellular dry weight content and the element concentrations after an acute reduction of basolateral osmolality. Two minutes after inducing hypotonic stress the dry weight content and the concentrations of all measured elements are drastically reduced by about 40–60%. The dry weight content and the concentrations of Mg and P decreased to

Table 1 Cellular element concentrations (mmol/kg wet) and dry weight (g/100 g wet) of A6 cells under control conditions

Control

Na (mmol/kg wet wt.)

Mg (mmol/kg wet wt.)

P (mmol/kg wet wt.)

Cl (mmol/kg wet wt.)

K (mmol/kg wet wt.)

Ca (mmol/kg wet wt.)

Dry weight (g/100 g wet wt.)

20.4±0.2

6.2±0.4

138.9±0.1

28.2±1.5

127.9±1.5

1.5±0.1

19.2±0.2

300 Table 2 Cellular element concentrations (mmol/kg dry weight) under control conditions and 60 min after acute (Hypo 60 min) and immediately after gradual reduction (Ramp; 1 mosmol·l–1·min–1) of the basolateral osmolality from 260 to 140 mosmol/l

Control Hypo 60min Ramp

Na (mmol/kg wet wt.)

Mg (mmol/kg wet wt.)

P (mmol/kg wet wt.)

Cl (mmol/kg wet wt.)

K (mmol/kg wet wt.)

Ca (mmol/kg wet wt.)

106.3±2.2 73.1±2.6 79.4±2.9

32.3±0.4 31.9±0.6 34.9±0.6

723.4±6.8 718.8±8.0 733.1±8.0

146.9±1.9 80.0±1.8 66.9±2.3

666.1±8.1 507.5±7.6 511.9±7.0

7.8±0.3 7.5±0.5 6.8±0.4

Fig. 3 Dry weight contents (dw) in g/100 g wet wt. and the concentrations of P, K, Cl, Na and Mg of A6 cells in mmol/kg wet wt. under control conditions and after decreasing the basolateral osmolality continuously within 120 min from 260 to 140 mosmol/kg. The deviations in SEM are not larger than the symbols for the mean values

about 60% of control (60% for the dry weight, 63% for Mg and 59% for P). The concentrations of Na, K and Cl decreased to somewhat smaller values of 47%, 56% and 43%, respectively. Sixty minutes after the induction of hypotonic stress, the dry weight content and the concentrations of Mg and P had returned almost to control values, whereas the concentrations of Na, K and Cl remained significantly reduced. The dry weight content and the concentrations of Mg and P reached 83%, 82% and 82% of the control values; the concentrations of Na, K and Cl were still reduced to 57%, 64% and 45%, respectively. The influence of a continuous reduction of basolateral osmolality from 260 to 140 mosmol/kg within 120 min on the cellular composition is documented in Fig. 3. Similar to the results obtained after 60 min of acute hypotonic stress, the cellular dry weight content and the concentrations of Mg and P at the end of the osmolality ramp were only slightly diminished compared with control. In contrast, the concentrations of Na, K and Cl at the end of the osmolality ramp were substantially lower than under control conditions. Whereas the dry weight content and the concentrations of Mg and P were reduced during the gradual decrease in basolateral osmolality to only 83%, 95% and 85%, the concentrations of Na, K and Cl were diminished to 62%, 64% and 38%, respectively. The Ca concentrations, which because of their relatively small values are not shown in Figs. 2 and

Fig. 4 A Osmolalities of basolateral incubation solution (continuous line) and cellular osmolalities accounted for by K, Cl and Na under control conditions and 2 and 60 min after inducing acute hypotonic stress. Cellular osmolalities of the watery space were calculated from the ion concentrations in mmol/kg wet wt. and cell water (obtained from dry weight) assuming an osmotic coefficient for 0.93. B Cell volume as calculated from the dry weight contents 2 and 60 min after inducing acute hypotonic stress

3, behaved similarly to the dry weight contents and the concentrations of Mg and P. At 2 and 60 min after the acute induction of basolateral hypotonicity, the cellular Ca concentration had fallen from a control value of 1.5±0.3 to 0.9±0.2 and 1.2±0.3 mmol/kg wet wt., respectively. The continuous decrease of basolateral osmolality reduced the cellular Ca concentration to 1.1 mmol/kg wet wt. Table 2 shows the cellular element concentrations referred to the dry weight content under control conditions, 60 min after acute induction of hypotonicity and after the osmolality had been reduced continuously. Essentially, the concentrations of Mg, P and Ca were not altered compared with control after an acute or a continuous reduction of basolateral osmolality. In contrast, both manipulations reducing basolateral osmolality resulted in significant decreases of cellular Na, K and Cl concentrations.

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Figure 4 demonstrates the contribution of K, Cl and Na to total cellular osmolality under control conditions and 2 and 60 min after acutely reducing the basolateral osmolality; it also shows the changes in cell volume following hypotonic stress. The osmolalities provided by Na, K and Cl were calculated, assuming that these ions are present solely in the watery space of the cells and that the cellular osmotic coefficients are the same as in the extracellular solution [6, 15]. The cell volumes as a percentage of control were calculated from the dry weight contents. After acutely reducing the basolateral osmolality from 260 to 140 mosmol/l, as indicated in the upper part of the graph by the continuous line, the cell volume increased within 2 min to 167% of control. Sixty minutes later cell volume was again reduced to 120%. Similar changes in cell volume can also be calculated from the alterations in cellular Mg, P and Ca concentrations following the induction of basolateral hypotonicity. The osmolalities derived from cellular K, Cl and Na account for 75% of basolateral osmolality (continuous line) under control conditions and for 71% and 85% after 2 and 60 min of basolateral hypotonicity. The values observed after a gradual reduction of basolateral osmolality (data not shown) were similar to those measured 60 min after the induction of acute hypotonic stress.

Discussion Under control conditions the cellular electrolyte concentrations of A6 epithelia are similar to those measured by electron microprobe analysis in other epithelial cells. The sum of Na and K concentrations of A6 cells is 147 mmol/kg wet wt. (basolateral osmolality of 260 mosmol/kg). In the epithelia of amphibians [17] and mammals [12] usually exposed to lower (220 mosmol/kg) and higher external osmolalities (300 mosmol/kg), the sum of cellular Na and K concentrations is also lower and higher by about 20 mmol/kg wet wt., respectively. This positive correlation between cellular cations and basolateral osmolalities suggests that different epithelial cells have very similar compositions, and that Na and K contribute substantially to total cellular osmolality. Cell swelling as the first reaction of A6 cells to acute hypotonic stress is maximal at about 2 min after induction of the osmotic challenge [26]. If Na and/or Cl uptake across the apical membrane is inhibited, A6 cells behave in this early phase of hypotonic stress like ideal osmometers [7]. Evidence for such behavior can also be derived from the present cellular concentrations, which were determined in the absence of apical Na 2 min after the induction of hypotonic stress. Assuming that the cellular dry weight content under control conditions of about 20% represents the osmotic inactive volume of the cells and that osmotic perturbations of the basolateral incubation solution are balanced solely by water movement, the reduction in basolateral osmolality from 260 to 140 mosmol/kg should increase the cellular volume to

169% and decrease the cellular element concentrations and the dry weight content to 59%. Within the first 2 min of hypotonic shock, the cellular content of dry matter, whose main constituents are thought to be impermeant [23], and the concentrations of the essentially impermeant elements Mg, P and Ca were in fact reduced to around 60%. The finding that the concentrations of Na, K and Cl were decreased to somewhat lower values of 47%, 56% and 43%, respectively, might be accounted for if the RVD had already begun (see below). This interpretation compliments the observation that hypotonic stress applied to A6 cells leads to an immediate increase of cellular K and Cl efflux [14, 26]. Cell height measurements have shown that A6 cells respond to acute and gradual hypotonic stress with an RVD [9, 10, 11] and IVR [26], respectively. In the present study the RVD and IVR are indicated by the relatively small decreases in dry weight content and in the concentrations of Mg, P and Ca 60 min after the induction of an acute hypotonic stress and immediately after the induction of a gradual hypotonic stress. The involvement of Na, K and Cl in cell volume regulation (RVD, IVR) is documented by the decrease in the contents of these ions under both experimental conditions (Table 2). Despite the cellular loss of Na, K and Cl during RVD and IVR, the contribution of cellular osmolytes necessary to achieve osmotic equilibrium with the basolateral incubation solution increased from about 75% to 85%. Since basolateral H2O permeability is much higher than the apical [11], total cellular osmolality is represented by the basolateral osmolality. The reduced difference between total cellular osmolality and that accounted for by cellular Na, K and Cl indicates the loss of other, presumably organic, osmolytes during RVD and IVR. This behaviour of A6 cells is slightly different from that observed in rabbit kidney cells, in which the contribution of Na, K and Cl to total cellular osmolality was about 80% before and after the induction of hypotonic stress [23]. An explanation of this discrepancy might lie in differences in the incubation conditions. The less pronounced hypotonic stress applied to the kidney cells (osmolality decrease from 290 to 190 mosmol/l) could be accompanied by a smaller loss of cellular osmolytes other than Na, K and Cl. Furthermore, the high HCO3 concentration of 25 mmol/l in the solution incubating the rabbit kidney tubules should also result in high cellular HCO3 concentrations. Both processes should keep the cellular osmolality that is not accounted for by Na, K and Cl high. As indicated by the decreases in the cellular dry weight contents (Figs. 2 and 3) and demonstrated by Fig. 4 the volume regulation after an acute and after a gradual reduction of the basolateral osmolality was incomplete. From the decrease in basolateral osmolality and the reduced dry weight contents, the loss of cellular osmolytes necessary to establish the new cell volume can be calculated to account for about 370 mosmol/kg dry weight. The decreases in the Na, K and Cl concentrations referred to cellular dry weight (Table 2) explain about 70% of the expected fall in cellular osmolality af-

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ter both an acute and a gradual decrease in basolateral osmolality. Since the loss of K causes the main effect, the generally accepted view that K plays a key role in cell volume regulation [19] is valid for A6 cells as well. Cellular electrolyte losses of A6 cells following hypotonic stress have been demonstrated by measuring the K content in non-polarized cells by chemical analysis [26] and the cellular Cl activity by a fluorescence dye [7]. Comparison of these data with the present values is difficult, since the K contents are given in µmol/mg protein and the Cl activities were obtained from preparations in which the initial external osmolality and Cl concentration were considerably lower than in the present experiments. The remaining loss of cellular osmolytes of about 30%, not explained by the exit of Na, K and Cl, is provided by substances that escape detection by electron microprobe analysis. Since it was shown by Van Driessche et al. [9] for A6 cells that the loss of amino acids covers about 17% of the expected total osmolyte loss during RVD, other osmolytes or a drop in the osmotic coefficient of non-permeant solutes [25] seems to play no or only a minor role in the volume regulation of A6 cells. Since the cellular release of Na (9 mmol/kg wet wt.) and K (46 mmol/kg wet wt.) occurring during RVD and IVR exceeds Cl release (15 mmol/kg wet wt.) considerably, a loss of other anions and/or titrations of negative, fixed charges has to accompany cellular cation exit. Besides the release of amino acids [9] that can contribute to the anion loss, an exit of HCO3 generated within the cells was proposed for kidney cells [23, 28]. The H thus formed is made osmotically inactive by binding to fixed negative charges of cellular buffers. The participation of cellular buffers in cell volume regulation as previously described for red blood cells [16, 20] appears feasible. As holds for other epithelial cells, the loss of K and Cl during hypotonic cell volume regulation is mediated by the increased efflux of these ions via activated K and Cl channels in the basolateral membrane [4, 5, 14]. A KCl cotransporter involved in the volume regulation of certain epithelial cells [29] does not seem to be responsible for the observed KCl efflux from A6 cells during RVD [26]. The observed fall in cellular Na concentration following hypotonic stress has not yet been described for A6 cells, but for several epithelial cell types of the rabbit kidney [23]. This decrease in cellular Na concentration could result from a reduced cellular Na influx and/or an enhanced efflux via the Na-K-pump. Although it cannot be excluded that the cellular Na concentration decrease during RVD and IVR is at least partially caused by inhibition of the basolateral Na-K-Cl-cotransporter [4], activation of the Na-K-pump seems more likely. This view is based on the observations that the Na permeability of the basolateral membrane of A6 cells is already very low under control conditions [3] and that the hypotonic stress caused a severalfold increase in Na-K-pump activity [21]. In summary, evidence is presented that RVD and IVR in A6 cells following hypotonic stress is mainly accom-

plished by the cellular release of Na, K and Cl (about 70%) and to a lesser extent by organic substances, possibly amino acids. The loss of Na and K exceeds that of Cl considerably, indicating the cellular exit of other anions. The contribution of Na, K and Cl to total cellular osmolality is about 75% under control conditions and about 85% after hypotonic stress. Acknowledgements We gratefully acknowledge the invaluable technical assistance of Ibrahim Öztürk. The work was supported by grants of the Deutsche Forschungsgemeinschaft.

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