INTRODUCTION

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REGULATION OF CELLULAR VOLUME is important for cells to function properly (12). Cells that undergo regulatory volume decrease (RVD) after exposure to hyposmotic medium adjust their volume through the release of intracellular electrolytes and osmolytes (organic solutes such as amino acids, polyols, and trimethylamines) accompanied by a net efflux of water (3, 14). Osmolyte release is thought to occur via osmolyte channels located in the membranes of the cells. In many cells, the osmolyte channels show the properties of a swelling-activated anion channel (10, 18, 20). In fish erythrocytes, however, the osmolyte channel is proposed to be the anion exchanger band 3 or an associated channel (4, 6, 8, 9, 11, 15).

Previous studies have examined the role that ionic strength plays in cell volume regulation (2, 5, 11, 13, 15, 17). With the use of trout red blood cells (RBC), Motais and collaborators (11, 15) tested the idea that ionic strength regulates the activation of osmolyte channels by measuring changes in electrolyte levels in the erythrocytes and correlating these changes with those of osmolyte channel activity following cell swelling in different media. Changes in levels of electrolytes were taken to reflect changes in ionic strength. The authors concluded that the change in cellular ionic strength induced by swelling is the stimulus for hyposmotic volume regulation; explaining that when ionic strength drops, the cells adopt hyposmotic swelling patterns (allowing release of taurine and electrolytes), but when swelling is accomplished by an increase in ionic strength, the cells follow isotonic swelling patterns resulting in less activation of the taurine channel and activation of ion channels (15).

The results obtained by Cannon et al. (2) in studies with mammalian C6 glioma and Chinese hamster ovary (CHO) cells were consistent with Motais' findings regarding the role of ionic strength in hyposmotic cell volume regulation. The former showed that hyposmotic cell swelling activates an anion channel, volume-sensitive organic osmolyte/anion channel (VSOAC), and that this channel's activity is inhibited by elevated intracellular electrolytes in the C6 glioma cells. In addition, an increase of intracellular electrolyte (CsCl) concentration in CHO cells resulted in a concentration-dependent decrease in channel activation. They concluded that both the volume set point and rate of swelling-induced activation of VSOAC are specifically modulated by cytoplasmic ionic strength (2).

Nilius et al. (17) have studied the effect of intracellular ionic strength on a volume-activated Cl current in cultured calf pulmonary endothelial cells. They compared the anion currents measured in whole cell patch clamp of cells dialysed with a pipette solution of normal ionic strength with anion currents measured in cells dialysed with a pipette solution of decreased ionic strength but equal Cl concentration and found that a large current developed in the cells exposed to the solution of reduced ionic strength. Therefore, current changes could be attributed solely to changes in ionic strength. Further experiments showed that an increase in the intracellular ionic strength reduced the swelling-activated current and that extracellular hypertonicity inhibited the effect of reduced intracellular ionic strength. On the basis of these results, the investigators concluded that a decrease in intracellular ionic strength activates an anion current identical to that activated by cell swelling in calf pulmonary endothelial cells (17).

The purpose of the present study was to extend earlier findings on the role of intracellular electrolyte concentration and ionic strength in volume regulation, using the skate RBC as the test model. Through measurements of intracellular conductivity and electrolytes in skate RBC swollen in different media, we addressed the question of whether the opening of the taurine channel is related to conductivity and electrolyte levels in the cytoplasm of the skate RBC.

	METHODS

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Animals. Little skates (Raja erinacea) were obtained from either the coast of Mount Desert Island, ME or Cape Cod, MA. They were kept in seawater tanks at 10-20°C for no longer than 10 days.

Incubation media. Blood was drawn from a skate caudal vessel using a heparinized syringe with a 21-gauge needle. The blood was spun for 2 min at top speed in a DYNAC centrifuge (Clay Adams). The plasma and buffy coat were removed from the cells, and the cells were washed in isosmotic (940 mosmol/kgH₂O) elasmobranch incubation medium [EIM: (in mM) 300 NaCl, 5.2 KCl, 2.7 MgSO₄, 5.0 CaCl₂, 15.0 Tris, and 370 urea, pH = 7.5]. The tube was again centrifuged for 2 min, and the supernatant was removed.

pH measurements. Intracellular pH was determined in lysed RBC as described previously by Davis-Amaral et al. (4).

Electrolyte assays (Na+, K⁺, Cl). In these experiments, 0.3 ml cell suspension was added to experimental flasks containing 3.0 ml solution and sampled at specified time points. The time points were 0 and 30 min except for those with ammonium salts, which were 0 and 40 min. At each time point, duplicate 1.25-ml samples were transferred to preweighed tubes and immediately spun for 5 s in a microcentrifuge (Fisher). The supernatant was removed from the tubes, and the RBC pellet in each tube was washed once with 1.0 ml isosmotic mannitol (in mM: 550 mannitol, 370 urea, and 15 Tris, osmolality = 940 mosmol/kgH₂O, pH = 7.5) to remove extracellular electrolytes. Cells treated hyposmotically were washed instead with 1.0 ml hyposmotic mannitol (in mM: 193 mannitol, 250 urea, and 15 Tris, osmolality = 460 mosmol/kgH₂O, pH = 7.5). Separate experiments showed that extracellular electrolytes trapped in the RBC pellet were negligible after one wash. The tubes were spun again for 5 s, the supernatant was removed, the sides of the tubes blotted dry, and the weights of the pellets were recorded. The pellets were then extracted with eight volumes of distilled water and placed at 4°C overnight. The following day, one volume of 70% perchloric acid was added to nine volumes of lysed RBC, and the mixture was placed on ice for 15 min. The tubes were spun in the microcentrifuge for 5 min, and the supernatant was removed. Na⁺ and K⁺ concentrations were assayed on the supernatant using a flame photometer (Instrumentation Laboratory), and the assay for Cl concentration was performed using an Aminco-Cotlove chloride titrator (American Instruments, Silver Spring, MD). Electrolytes were expressed as cellular concentrations (µeq/g RBC).

Conductivity. For conductivity experiments, 0.6 ml cell suspension at 50% hematocrit was added to experimental flasks containing 3.0 ml solution, and the mixture was sampled at specified time points. In 1,120/940/460 EIM, ethylene glycol, propylene glycol, dimethylurea, and diethylurea experiments, the time points were 0 and 30 min; in ammonium chloride and ammonium nitrate experiments, the time points were 0 and 40 min. At each time point, duplicate 1.5-ml samples were transferred to preweighed tubes and immediately spun for 5 s in a microcentrifuge (Fisher). The supernatant was removed from the tubes, and the RBC pellet in each tube was washed with 1.0 ml 940 mosmol mannitol. The tubes were spun again for 5 s, the supernatant was aspirated, and the samples were snap-frozen in a dry ice/ethanol bath. The samples were stored in a 80°C freezer for 1-5 days. To measure the conductivity of the intracellular fluid, the blood cells were lysed by thawing samples at room temperature. Then, 0.075 ml of lysed cells were added to 0.5 ml of 0.01 M KCl. Separate experiments showed that, under these conditions, conductivity was linear for blood volumes between 0.05 and 0.1 ml. The blood cell-KCl mixture was brought to 15°C using a constant-temperature circulating water bath (Lauda), and the conductivity was measured with a conductivity electrode (Microelectrodes, model MI-905) attached to a conductivity meter (Orion, model 115). The conductivity of the cells was determined by subtracting the conductivity of the KCl solution from that of the blood cell-KCl mixture.

Hematocrits. For hematocrit experiments, RBCs were resuspended to a 50% hematocrit, and 0.2 ml of the suspension was added at staggered time points to vials containing 0.8 ml of test solution in the 15°C shaking water bath. Samples were drawn into hematocrit tubes (at the same time points as for the electrolyte experiments) and sealed at one end with Critoseal. The tubes were then spun in a hematocrit centrifuge (Clay Adams) for 3 min. Finally, they were removed, and the ratio of RBC to total volume (hematocrit) was determined using a finely graded ruler and magnifying glass. The ratio of packed RBC to total volume at the experimental time points was divided by the ratio obtained at the zero time point and expressed as relative cell volume.

Taurine effluxes. Cells were preloaded with [³H]taurine by resuspending them to 20% hematocrit in 940 EIM and then preincubating them with 2.0 µCi/ml [³H]taurine for 3 h in an open-air 15°C shaking water bath. Extracellular radioactivity was removed by washing the cells three times with control (940) EIM containing decreasing concentrations of cold taurine: 10 mM, 1 mM, and 0.1 mM. Cells were then resuspended to 20% in control EIM, and 0.15 ml were added to flasks containing 940 EIM, 460 EIM, or the other appropriate EIM solutions so that the final hematocrit in the flask was 5%. Aliquots of 0.5 ml of blood were taken from each flask at 0 and 30 min (0 and 40 min for the ammonium compounds), spun in a microcentrifuge for 2 min, and 0.4 ml of supernatant were removed from each tube and added to 4 ml liquid scintillation fluid. The cells from the 0-min control flask were lysed with 1 ml of 70% perchloric acid (PCA), left on ice for 15 min, and spun in a microcentrifuge for 2 min. Supernatant (0.4 ml) was removed and added to 4 ml liquid scintillation fluid. Taurine efflux was determined by measuring radioactive counts from each sample and dividing the dpm/g RBC of the supernatant by dpm/µmol taurine in the pellet extracts (from the control 0 time point) using a value of 75 µmol/g RBC taurine in skate RBC.

Kinase activities. The activities of various protein kinases were measured in control and volume-expanded skate RBC using an immune complex assay protocol as described previously by Musch et al. (16). Briefly, cells were volume expanded for 5 min in each of the following EIM: 460 (hyposmotic); 940 diethylurea, 940 dimethylurea, 940 ethylene glycol, 940 propylene glycol (isosmotic nonelectrolyte media); 940 ammonium chloride or 940 ammonium nitrate (isosmotic electrolyte media). After 5 min, cells were rapidly pelleted and snap-frozen. Immediately before assay, cells were thawed on ice and lysed in nine volumes (450 µl) of Nonidet P-40 (NP-40) lysis buffer [immunoprecipitate (IP); 25 mM HEPES, pH 7.4, 225 mM NaCl, 1% vol/vol NP-40, 5 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 2 mM benzamidine with 10 µg/ml each leupeptin, aprotinin, pepstatin A, and antipain]. Lysates were solubilized for 10 min and then cleared by centrifugation (14,000 g for 10 min at 4°C). Supernatants were incubated with various antibodies specifically directed to the kinases, prebound to protein A Sepharose. Kinases were allowed to bind for 120 min, washed two times with IP wash (25 mM HEPES, pH 7.4, 150 mM NaCl, and 0.1% vol/vol NP-40 with protease inhibitors as above), and then washed once with assay buffer (50 mM HEPES, pH 7.4, 10 mM MnCl₂, with protease inhibitors). The activity of the kinases attached to the beads was measured at 37°C for 30 min in 50 µl assay buffer with 5 mM p-nitrophenylphosphate and 1 µM -[³²P]ATP. In the case of protein kinase C (PKC), 1 mM CaCl₂ and 10 µg/ml sonicated phosphatidyl serine were added. Reactions were terminated by the addition of 25 µl 3× SDS-PAGE stop solution, and samples were heated at 65°C for 10 min to elute the kinases. Beads were pelleted, and the sample was loaded onto a 10% SDS-PAGE, transferred to a polyvinylene difluoride membrane, and alkali treated before autoradiography. Images were quantitated using Image 1.54 software from National Institute of Health.

	RESULTS

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Swelling in skate RBCs. Table 1 shows examples of electrolyte concentrations and relative cell volumes in skate RBC volume expanded by hyposmotic and isosmotic electrolyte (ammonium chloride) and isosmotic nonelectrolyte (ethylene glycol) solutions. The cells swollen by these different treatments exhibited similar degrees of volume expansion, with the relative cell volumes ranging from 1.42 to 1.46 times the isosmotic control cell volume. However, the manner in which the cells were swollen varied. In the cells swollen by exposure to hyposmotic medium, a dilution of the intracellular fluid occurred with an influx of water. As a result, these cells showed a decrease in total electrolyte concentration following swelling. Cells swollen isosmotically by exposure to the nonelectrolyte ethylene glycol increased volume by the influx of ethylene glycol with accompanying water. Although the two methods of swelling (hyposmotic vs. isosmotic) differ, both resulted in significant decreases in electrolyte (Na⁺, K⁺, Cl) concentrations. In cells swollen isosmotically by exposure to an electrolyte (ammonium chloride), the volume expansion occurred as water followed the ammonium chloride into the RBCs. Under these conditions, swelling was accompanied by an increase in intracellular electrolyte concentration due to the influx of ammonium and chloride ions. Ammonium is thought to enter RBC as NH₃ and be converted to NH₄⁺ by reacting with H⁺ (21). Chloride accompanies the NH₃ by exchanging with intracellular OH (or HCO₃). The sum of Na⁺ + K⁺ + Cl concentrations varied from experiment to experiment even in cells maintained in isosmotic media (Table 1). However, intracellular conductivity was relatively constant (See Fig. 3).

Isosmotic swelling with electrolytes. In these experiments, skate RBCs were volume expanded with either isosmotic solution containing ammonium salts or with hyposmotic media, and the effects on electrolyte concentration, intracellular conductivity, and taurine efflux were compared. When cells were swollen with ammonium chloride, the final electrolyte concentrations of the cells were significantly higher than those of the control cells. The cells swollen in hyposmotic media, on the other hand, showed a drop in electrolyte concentrations. The data are shown in Fig. 1A. In addition, intracellular fluid conductivity was measured in cells treated with ammonium chloride, ammonium nitrate, and hyposmotic solutions. In cells treated with ammonium salts, the conductivity following swelling was significantly higher than in the control cells, whereas in hyposmotically treated cells, the conductivity was lower than that of the control cells (Fig. 1B).

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Intracellular electrolyte (Na+ + K+ + Cl) concentration (A) and conductivity (B) and taurine efflux (C) in skate red blood cells following swelling with isosmotic electrolytes. Values are means ± SE (average n = 7 for A, n = 8 for B, n = 8 for C). * Difference significant P < 0.01 vs. 940  >460 elasmobranch incubating medium (EIM);  difference significant at P < 0.05.

Hyperosmotic pretreatment. To test the effects of increased electrolyte concentration on the opening of the taurine channel in hyposmotically swollen cells, RBC were preincubated in hyperosmotic media. As shown in Fig. 2A, the cells preincubated in the hyperosmotic (1,120 mosmol EIM) solution had higher initial electrolyte concentrations than those in isosmotic control solution. In addition, although the control and hyperosmotically treated cells showed similar decreases in intracellular electrolyte concentration following cell swelling, the latter maintained higher total electrolyte concentrations. The intracellular conductivity showed the same trend as the electrolyte concentration in that the initial conductivity in the cells preincubated in hyperosmotic media was higher than for those in the isosmotic media, and this higher conductivity was maintained following swelling in the hyposmotic media (Fig. 2B). However, the increased initial electrolyte concentration in the hyperosmotically treated cells appeared to blunt stimulation of the osmolyte channel. Although cell swelling in both situations resulted in significant stimulation of the taurine channel, taurine efflux was lower in the cells that were preloaded with electrolytes by hyperosmotic treatment (Fig. 2C).

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Intracellular electrolyte concentration (A) and conductivity (B) and taurine efflux (C) in skate red blood cells following swelling in hyposmotic media (preincubation in isosmotic or hyperosmotic media). Values are means ± SE (average n = 7 for A, n = 7 for B, n = 12 for C). * Difference significant at P < 0.01 vs. 940 EIM;  difference significant at P < 0.05 (1,120  460 compared with 940  460 EIM);  difference significant at P < 0.05 vs. 940 EIM.

Swelling with isosmotic nonelectrolytes. Cells were swollen isosmotically with nonelectrolytes, and the effects on electrolyte concentration, conductivity, and taurine efflux were compared with those found in hyposmotically swollen cells. Figure 3A shows the decreases in electrolyte concentration in cells swollen under various conditions. Cells swollen hyposmotically or isosmotically with ethylene glycol had ~20% less measured electrolytes than 940 control cells, whereas isosmotic swelling with propylene glycol, dimethylurea, and diethylurea decreased measured electrolytes by 26-29%. The fall in intracellular conductivity in cells swollen in the nonelectrolyte media (Fig. 3B) ranged from 25-34% of control values. The somewhat greater fall in conductivity than in measured electrolyte concentrations suggests that other unmeasured intracellular inorganic and organic electrolytes contribute to the intracellular conductivity and its changes during volume expansion. Although the drop in electrolyte concentration and conductivity varied by a factor of <1.5, taurine effluxes (Fig. 3C) in cells swollen by different treatments showed an about fivefold range from 2.9 ± 0.3 µmol/g RBC in ethylene glycol to 14.0 ± 1.2 µmol/g RBC in 460 EIM. Furthermore, in every case, swelling with isomostic nonelectrolyte solutions was much less effective than hyposmotic swelling in stimulating taurine efflux. Thus some factor(s) other than, or in addition to, ionic strength must be contributing to the regulation of the osmolyte channel under these conditions.

View larger version (30K): [in this window] [in a new window]   Fig. 3.   Intracellular electrolyte concentration (A) and conductivity (B) and taurine efflux (C) in skate red blood cells following swelling with isosmotic nonelectrolytes [ethylene glycol (EG), propylene glycol (PG), dimethylurea (DMU), and diethylurea (DEU)]. Values are means ± SE (average n = 7 for A, n = 6 for B, n = 13 for C). * Difference significant at P < 0.01 vs. 940 EIM;  difference significant at P < 0.05 vs. 940 EIM.

Kinase activities. Musch et al. (16) previously observed an increase in the activities of the band 3 phosphorylating tyrosine kinases p72syk and p56lyn when skate RBC were volume expanded in hyposmotic media (460 mosmol/kg EIM), and the tyrosine kinase inhibitor piceatannol inhibited both the enzyme responses and taurine efflux. This suggested that these protein kinases could be involved in the volume regulatory pathway of hyposmotically volume expanded skate RBC. Therefore, in this study, the activities of the tyrosine kinases p72syk, p56lyn, and pp60src as well as PKC and MAP kinase/extracellular signal-regulated kinase kinase (MEKK-1) were measured after cells were volume expanded in hyposmotic media, isosmotic nonelectrolyte, or isosmotic electrolyte media.

	DISCUSSION

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After swelling, skate RBCs return to normal volume by releasing intracellular osmolytes (including taurine) and water (1, 4, 7-9, 16, 19). In this study, we examined the role of intracellular ionic strength in opening the osmolyte channel involved in the efflux of taurine and other osmolytes. It has been hypothesized that in trout RBCs and epithelial cells, a decrease in intracellular ionic strength triggers the opening of the osmolyte channel (11, 15, 18). In previous studies, changes in inorganic electrolytes were taken to represent changes in ionic strength. However, inorganic electrolyte measurements in cells only include the effects of certain ions (Na⁺, K⁺, Cl). Therefore, experiments to directly test the effects of intracellular ionic conductivity were performed as well. Conductivity detects all electrolytes, including phosphate and magnesium salts as well as organic salts such as basic and acidic amino acids. Because these compounds may have high intracellular concentrations, the aim of the conductivity experiments was to gain a more complete reading of the intracellular ionic strength.

In the present study, when swelling was achieved via hyposmolarity, our results were consistent with the previous hypothesis regarding the role of ionic strength in cell volume regulation; a fall in intracellular ionic strength activated the osmolyte channel. In addition, loading skate RBC with electrolytes by preincubating them in high NaCl medium blunted the taurine efflux response to hypotonicity. Furthermore, when skate RBC were swollen with NH₄⁺ salts so that the intracellular ionic strength was slightly elevated, only minimal taurine efflux occurred. Thus the effects of volume expansion achieved by hypotonicity, by treatment with hyperosmotic electrolyte solutions, and with NH₄⁺ salts are all consistent with the idea that intracellular ionic strength regulates the opening of the osmolyte channel in skate RBC (2, 5, 11, 15, 17).

It has been suggested that the electrolyte Cl is responsible for controlling the opening of the osmolyte channel in skate hepatocytes and C6 glioma cells (2). Therefore, we compared the effects of expansion of skate RBC by NH₄Cl versus NH₄NO₃ on taurine efflux to determine whether the osmolyte channel in skate RBC is specifically regulated by intracellular chloride. In contrast to skate hepatocytes (13) and C6 glioma cells (2), the osmolyte channel in skate RBC does not appear to be specifically regulated by intracellular chloride, because taurine efflux was similarly blunted when skate RBC were volume expanded by either ammonium chloride or ammonium nitrate.

In contrast to the results obtained with electrolyte loading and ammonium salts, the findings in experiments with isosmotic nonelectrolytes are not consistent with the idea that ionic strength alone regulates the opening of the osmolyte channel. Isosmotic cell swelling with nonelectrolytes resulted in decreases in electrolyte concentrations and conductivities similar to those found with hyposmotic cell swelling. However, taurine effluxes were found to be much lower with nonelectrolyte-induced swelling. Although the fall in electrolyte concentrations and intracellular conductivities were similar or greater in cells treated with isosmotic nonelectrolytes compared with hyposmotically treated cells, the rise in taurine efflux was much greater in hyposmotic than isosmotic nonelectrolyte media. Figure 4 shows a comparison between electrolyte concentration (Fig. 4A), conductivity (Fig. 4B), and taurine efflux in skate RBC incubated in hyposmotic and isosmotic nonelectrolyte media. It is clear that there is no correlation between a decrease in electrolyte concentration or conductivity and taurine efflux under these conditions. These results obtained with skate RBC differ from those of Motais and co-workers (11, 15), who found a direct correlation between a decrease in ionic strength and an increase in amino acid efflux in trout RBC under a variety of conditions.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Lack of relationship between taurine efflux vs. electrolyte concentration (A) and conductivity (B) in skate red blood cells incubated in isosmotic nonelectrolytes. Data taken from Fig. 3.

The results obtained in experiments with kinases are consistent with those with the taurine effluxes. As in the taurine efflux experiments, hyposmotic medium stimulated p72syk and p56lyn activities, whereas ammonium salts had no significant effects on kinase activities. In experiments with isosmotic nonelectrolytes, stimulation of kinase activities was much less than that found with hyposmotic stimulation, as was found for the taurine effluxes. In addition, there was a positive correlation between stimulation of kinase activities and stimulation of taurine efflux. For example, cells incubated in isosmotic nonelectrolytes showed similar stimulations of p72syk and p56lyn and taurine efflux. Thus whatever the reason is for the different effects of different isosmotic nonelectrolyte solutions on taurine effluxes, kinase activities are similarly affected.

Perspectives

Okada (18) has described several possible factors that may be involved in the activation of osmolyte channels. One is a volume-sensing, outwardly rectifying anion channel as a pathway for the release of organic solutes. Although the possible involvement of second messengers in modulation of the channel has been proposed in some cells (12), second messengers have not been clearly identified as responsible for channel activation in skate RBC (7). In many cells, the osmolyte channel is not osmosensing or stretch activated (18). However, it may be that in the skate RBC, the anion channel is osmosensing. Compared with trout RBCs and mammalian epithelial cells, skate RBCs have a much higher concentration of intracellular organic osmolytes (9). It is possible, therefore, that there are two sensors that regulate channel activation in skate RBCs. To stimulate the channel fully, a drop in both electrolytes and in organic osmolytes may be required.