1Department of Biophysics, 2Department of Cell Biology, Institute of Cell Physiology, National University of Mexico, Mexico City, Mexico
Submitted 14 May 2003 ; accepted in final form 15 January 2004
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ABSTRACT |
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volume regulation; taurine; hyposmolarity; isovolumetric regulation; regulatory volume decrease
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MATERIALS AND METHODS |
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Solutions and gradually diluted solutions. The isosmotic medium contained (in mM) 135 NaCl, 1.0 CaCl2, 1.17 MgSO4, 4.7 KCl, 5 dextrose, and 10 HEPES (300 mosM, pH 7.4). The gradually diluted solutions (1.8 mosM/min) were obtained by the procedure described by Lohr and Grantham (7) and by Van Driessche et al. (22). Briefly, the system consisted of two identical glass cylinders interconnected at their bases by a tube with an interrupting valve. The first container was filled with isosmotic medium and the second with the same volume of hyposmotic medium (50%). Media were kept at 39°C by placing the cylinders on a temperature-controlled hot plate and stirring. Superfusion medium was pulled from the first container with a polystaltic pump, allowing the hyposmotic medium to begin to enter this cylinder, mixing gradually and continuously with the isosmotic medium. In this way, a solution with linear dilution of 1.8 mosM/min was produced, which at the end of the experiment (82 min later) reached 150 mosM (50% hyposmotic). The linearity of the diluted solutions and the osmolarity of all solutions were verified in a freezing-point osmometer (Osmete A, Precision Systems). Reductions in osmolarity are indicated throughout the manuscript as hyposmolarity of negative percent change in each case. For instance, a 3% reduction is expressed as H-3%.
Estimation of changes in cell volume. Cell volume measurements were performed by estimating the changes in relative cell volume with a large-angle light-scattering system (12, 16). C6 cells were cultured on rectangular cover glasses (10 x 50 mm) at 90% confluence at the time of experiments. Cover glasses were placed at a 50° angle relative to the excitation light in a cuvette filled with isosmotic medium (300 mosM) in an Aminco-Bowman Series 2 luminescence spectrometer. Cells were excited at 585 nm with an argon arc lamp, and emission was detected at the same wavelength. Data are expressed as the inverse of the emission signal, because light intensity inversely correlates with cell volume. Cell volume changes were calculated according to the equation lo/lt, where lo is isosmotic emission signal average and lt is emission signal at time t.
Electrophysiological recordings.
Currents were monitored with an Axopatch 200 patch-clamp amplifier (Axon Instruments, Foster City, CA). All recordings were performed at 35°C with a diluted solution-generating system. Whole cell membrane currents were measured by using ruptured patches. The time course of whole cell currents was obtained by following voltage protocols of holding potential of 70 mV to potentials ranging from 120 to +100 mV in 20-mV increments for 350 ms. Electrophysiological recording was carried out on cells seeded on 35-mm petri dishes, as described above. Once the whole cell configuration was obtained, cells were perfused for 5 min with isosmotic solution before the gradual dilution in osmolarity was initiated. The voltage protocol was carried out every 5 min for the duration of the experiment while the dilution gradient continued. This manipulation did not interfere with the whole cell recordings, because the entire whole cell voltage protocol lasted only 12 s. The voltage protocol was carried out fast enough to be concluded while the osmolarity reduction was only 0.36 mosM (H-0.2%) during the 12-s period. This protocol was repeated every 5 min for the duration of the experiments, as indicated. Patch electrodes were prepared from 1.5-mm OD, 1.5-mm ID borosilicate glass (World Precision Instruments), and the resistance was between 3 and 5 M when filled with the pipette solution. The recorded signal was filtered at 10 kHz with a low-pass Bessel filter and transferred to a computer with the Digidata 1200 interface (Axon Instruments). Whole cell currents were analyzed with pCLAMP6 software (Axon Instruments).
The standard pipette solution contained (in mM) 110 K+ aspartate, 30 KCl, 1 MgCl2, 10 HEPES, 5 EGTA, and 5 Mg ATP, pH 7.4 adjusted with KOH (300 ± 3 mosM). In the anion-substitution experiments, K-aspartate and KCl were fully replaced by 140 mM CsCl. In experiments to isolate K+ currents, aspartate was the major anion in the pipette and only 10 mM Cl was present in the intracellular solution.
Efflux of amino acids. Cells were incubated for 60 min in culture medium containing the labeled amino acids D-[3H]aspartate (as a tracer for glutamate) and [3H]taurine (0.5 µCi/ml). After the loading period, cultured dishes were superfused for 20 min with isosmotic medium, and the superfusion continued either with isosmotic medium or with the gradually diluted solutions (1.8 mosM/min) during the time indicated. Samples were collected every minute, and radioactivity in samples and in cells after the experiment was quantified by scintillation. Labeled amino acid fluxes were calculated as efflux rate constants, i.e., the amount of radioactivity released in any given fraction divided by the total amount of label present in cells at that moment.
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RESULTS |
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Whole cell currents activated during GOR in nominally Ca2+-free intracellular solution. We first explored the currents activated by GOR in the patch-clamp whole cell configuration with 5 mM EGTA in the patch pipette. Figure 3A illustrates the activation and progressive increase with time of whole cell currents in this condition. As indicated in Fig. 3A, the outward current activated by changes in osmolarity was more prominent than the inward current, even though the time course was similar in both cases. No whole currents were activated when cells were maintained in isosmotic medium for the same time as those exposed to GOR (Fig. 3A). The membrane potential progressively depolarized within the first 30 min of the osmolarity decrease, then reached a steady-state level of about 54 mV at H-18% (Fig. 3B). Figure 3C illustrates the current-voltage (I-V) relationship for control (isosmotic) and 25, 50, and 75 min after GOR onset (H-15%, H-30%, and H-45%, respectively).
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All of these results show that the initial increments of whole cell currents induced by GOR in the intracellular Ca2+-free condition result from the activation of a Cl conductance, followed minutes later by the activation of a K+-selective conductance. The maximal conductance values obtained were 1.6 ± 0.18 nS (n = 7) for Cl and 4.1 ± 0.47 nS (n = 6) for K+.
Whole cell currents activated by GOR in presence of intracellular Ca2+. All of the above described recordings were performed in nominally intracellular Ca2+-free conditions, and therefore all observed currents occurred through Ca2+-independent channels. To investigate whether Ca2+-dependent currents are elicited during the first minutes of osmolarity reduction and can contribute to cell volume control in cells showing small volume increase, whole cell currents were recorded in the presence of 200 nM Ca2+ in the patch pipette. As indicated in Fig. 6A, the change in osmolarity evoked inward and outward currents with similar time courses, the outward current being more prominent than the inward current at all osmolarities. Activation of these currents occurs at changes in osmolarity as low as H-3%. Figure 6B illustrates the change in membrane potential during osmolarity reductions. The potential remained hyperpolarized during the first 10 min (H-6%). After H-6%, the membrane potential depolarized to reach approximately 63 mV at H-9% and remained constant for the duration of the experiment. Figure 6C shows the I-V relationship for control (isosmotic) and at H-1%, H-3%, H-6%, H-9%, and H-21%. From H-6%, the maximal amount of current was reached. It is noteworthy that the total current at all points explored was lower in the Ca2+-free condition than in the presence of Ca2+ (Figs. 3 and 6), indicating the superposition of Ca2+-activated conductances.
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DISCUSSION |
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In both the sudden and the gradual model, at small reductions in osmolarity and less swelling cell volume control was more efficient in the presence of Ca2+, suggesting a Ca2+-dependent element in the volume-regulatory mechanism in this condition. The swelling-activated Cl channels, as well as the osmosensitive amino acid fluxes, are largely Ca2+ independent (15), thus making the K+ efflux pathway the likely candidate to be influenced by Ca2+. Volume-activated K+ channels show marked differences with respect to Ca2+ dependence, depending on the cell type (reviewed in Ref. 15). In epithelial cells hyposmolarity activates Ca2+-dependent K+ channels, whereas in most nonepithelial cells the volume-sensitive K+ channels are Ca2+ independent (15). Interestingly, in the present work we found the operation of both Ca2+-dependent and Ca2+-independent K+ channels in the same cell type, which activate at different times and by different signals. For the Ca2+-dependent channel, the activation signal may not primarily be the change in osmolarity but the increase in intracellular Ca2+ concurrent with swelling, known to occur in most cells (11), whereas the Ca2+-independent channel may respond to the osmolarity reduction or the magnitude of the volume change. The two types of K+ channels also differ in their pharmacological profile. The Ca2+-dependent channel is sensitive to charybdotoxin and insensitive to clofilium, whereas the opposite is found for the Ca2+-independent channel. This coincidence of two different types of K+ channels activated by swelling in the same cell has not been previously reported, to our knowledge, but was first suggested by an interesting study in jejunal villus epithelial cells showing that volume regulation at small volume increases is Ca2+- and charybdotoxin sensitive, although it turns to be Ca2+ independent and charybdotoxin insensitive at larger cell volume increases (10).
The relative contribution of the Ca2+-dependent and the Ca2+-independent K+ channel to the total currents evoked by GOR can be estimated by the current magnitude as well as by the changes in membrane potential observed in the presence or absence of intracellular Ca2+. In the Ca2+-free condition, the first event elicited by GOR is an early activation (H-5%) of a Cl current, which increases as the external osmolarity drops. In the absence of a significant accompanying K+ current, cells markedly depolarize from the resting potential of 79 ± 2 mV to 54 mV. Activation of a K+ current at H-19% prevents further depolarization and stabilizes the membrane potential. A different response is observed in the presence of Ca2+. During the first 10 min (up to H-6%), no significant change in membrane potential is observed, suggesting that Cl and K+ currents already activated at that time stabilize the membrane potential. After this time, cells slightly depolarize, reaching 63 mV at H-9%, and this value remains unchanged for the duration of the experiment, suggestive of a predominant Cl current. The maximal value of total currents is consistently higher in the presence of Ca2+ than in the Ca2+-free condition at the same osmolarities, most likely reflecting the contribution of the Ca2+-activated K+ current. However, larger differences are observed at small osmolarity reductions, suggesting a more important contribution of the Ca2+-dependent channel in this condition, in line with the observation in jejunal villus cells on Ca2+-dependent RVD at small cell volume changes (10). The influence of the two types of K+ channels with respect to Ca2+ dependence is also reflected in the cell volume regulation. At H-15%, volume regulation after SOR is less efficient and swelling is higher after GOR in the Ca2+-free condition, whereas Ca2+ has no significant influence at H-30%. These results, similar to those reported in jejunal epithelium cells (10), stress the fact that volume regulatory mechanisms in conditions of physiological cell volume changes, such as those elicited by nutrient uptake or ionic gradients, differ from those observed at large cell volume changes, which may not occur even in pathological conditions. The type of K+ channel activated appears to be determinant in the mechanisms operating in the two different situations. The fact that the same differences in the type of K+ channel involved in volume regulation at small or large cell volume changes were observed in conditions of hyposmolarity (Ref. 10 and present results) or isosmolarity-evoked swelling (9) supports the notion that it is the change in cell volume rather than in osmolarity that determines the type of K+ channel activation and the consequent mechanism of cell volume adjustment.
The Cl and K+ efflux pathways during GOR may or may not be identical to those operating for the RVD after SOR. In A6 cells, the anion selectivity for the Cl efflux pathway in GOR and SOR is different, suggesting different mechanisms for Cl efflux (22). In C6 cells, the electrophysiological and kinetic properties of the Cl current activated by GOR, including an outwardly rectifying I-V relationship and current inactivation at hyperpolarizing voltages, as well as its pharmacological sensitivity, are similar to those of the Cl conductance activated in SOR described by Jackson and Strange (3). As to the K+ channels, the sensitivity to charybdotoxin of RVD in jejunal epithelium cells (10) and of the K+ current in C6 cells (present results) suggests that the Ca2+-dependent, swelling-activated K+ channel is a maxi-K+ channel, which is distinctively sensitive to this blocker. This type of channel appears similar to that involved in RVD in most epithelial cells (15). The Ca2+-independent channel, which is apparently also voltage independent, activates only by osmolarity and shows a pharmacological profile characterized by insensitivity to typical K+ channel blockers such as TEA, quinidine, 4-AP, and Ba2+ but sensitivity to clofilium. This feature relates this channel to the volume-activated K+ channel recently described in Ehrlich ascites cells (2, 13). Putative candidates for this channel include members of the 4M2P family of channels (13).
The gradual decrease in osmolarity elicited the release of taurine and glutamate in C6 cells. However, the amino acid efflux was a delayed cell response, the fluxes being activated at very low osmolarities. This is in marked contrast with the early activation of a Cl current. This result strongly supports the notion of different permeability pathways for the two osmolytes (4, 20) and provides further evidence against a common pathway for amino acids and Cl efflux, which was based mainly on similarities in their pharmacological profile (17, 19). The sensitivity to Cl channel blockers, however, is suggestive of some interdependence of the two osmolyte pathways.
The contribution of amino acids, and possibly other organic osmolytes permeating through the same pathway, may be crucial for the efficacy of cell volume regulation elicited by small cell volume changes. According to their behavior in the face of GOR, three types of cell responses have been observed so far. In cells responding by IVR such as tubule renal cells, A6 cells, a subset of hippocampal neurons, and cerebellar granule neurons (7, 21, 22, 24), amino acid efflux activates early after the osmotic stimulus, as documented in brain cells (21). Other cells such as cultured myocytes (18) and C6 cells in the present study in conditions of GOR do not exhibit IVR but show a higher efficiency for volume regulation (evidenced as less swelling) in conditions of large cell volume increase, concomitant with a delayed activation of amino acid fluxes. Finally, in trout erythrocytes (1), cell swelling is the same in both SOR and GOR, with no efflux of taurine observed until very late. The importance of taurine, amino acids, and other organic osmolytes in preventing cell swelling in brain cells may operate also in vivo. The long-term swelling prevention in brain during chronic hyponatremia relies not on electrolytes but on the sustained decrease in the brain content of organic osmolytes, which may be as large as 90% in the case of taurine (23). The superior ability of neurons compared with C6 cells to resist to changes in external osmolarity, which seems to be based primarily on the contribution of organic osmolytes, may represent a protective mechanism to spare neurons from the deleterious consequences of swelling.
In summary, a main conclusion of the present study is that the mechanisms operating in response to small or large changes in cell volume differ essentially in the type of K+ channel involved as well as in the sensitivity of amino acid efflux pathways.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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