Print version ISSN 0716-9760
Biol. Res. vol.35 no.2 Santiago 2002
Non-selective cation channels and oxidative stress-
induced cell swelling
FELIPE SIMON, DIEGO VARELA, ANA RIVEROS, ANA LUISA EGUIGUREN, ANDRES STUTZIN
Laboratorio de Fisiopatología Molecular, Programa de Fisiopatología, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile. Independencia 1027, Santiago, Chile.
Necrosis is considered as a non-specific form of cell death that induces tissue inflammation and is preceded by cell swelling. This increase in cell volume has been ascribed mainly to defective outward pumping of Na+ caused by metabolic depletion and/or to increased Na+ influx via membrane transporters. A specific mechanism of swelling and necrosis driven by the influx of Na+ through nonselective cation channels has been recently proposed (Barros et al., 2001a). We have characterized further the properties of the nonselective cation channel (NSCC) in HTC cells. The NSCC shows a conductance of ~18 pS, is equally permeable to Na+ and K+, impermeant to Ca2+, requires high intracellular Ca2+ as well as low intracellular ATP for activation and is inhibited by flufenamic acid. Hydrogen peroxide induced a significant increase in cell volume that was dependent on external Na+. We propose that the NSCC, which is ubiquitous though largely inactive in healthy cells, becomes activated under severe oxidative stress. The ensuing Na+ influx initiates via positive feedback a series of metabolic and electrolytic disturbances, resulting in cell death by necrosis.
Key terms: Cell death, Cell Volume, Nonselective cation channels, Flufenamic acid.
Necrotic cell death differs fundamentally from apoptosis and is generally considered to arise as a non-specific phenomenon after an exogenous traumatic insult. Accordingly, necrosis does not appear to be a programmed (normal) physiological process. Nonetheless, despite its accidental nature, necrotic cell death should be considered an active process, that can be triggered by relatively short and/or mild insults, which do not lead to complete and permanent inhibition of energy metabolism. Instead, such insults initiate acute ionic and chemical changes that trigger numerous, highly interactive processes and eventually cell death (see also discussion in Barros et al., 2002).
Cell death is commonly observed under pathological conditions such as hypoxia and oxidative stress. The necrotic form of cell death is initiated by changes observed as a consequence of inhibition of oxidative phosphorylation. These changes include, among others, decreased ATP and pHi, free radical overload, increased cell Na+ (Hermoso et al., 2001) and Ca2+ content, membrane depolarization and triggering of the inflammatory response (Lipton, 1999). However, the precise sequence of events leading to necrosis after initial inhibition of oxidative phosphorylation and ATP depletion, remains unknown (Rosser & Gores, 1995). Interestingly, recent work indicates that apoptosis requires maintenance of intracellular ATP levels, whilst in necrosis ATP significantly decreases (Halestrap, 2000). From a functional point of view, another distinguishing characteristic of necrotic cell death is the net gain of intracellular Na+ and water. This sodium and water overload, which cannot be controlled under oxidative stress conditions, leads to a marked increase in cell-volume and subsequent cell lysis (Majno & Joris, 1995; Leist & Nicotera, 1997; Barros et al., 2001b; 2002). Such behavior stands in striking contrast to events observed during apoptosis, which is preceded by a reduction in cell-volume (shrinkage) and net loss of K+ ions via the delayed rectifier K+ channel, as reported, for instance, in neurons (Yu et al., 1997) and other cell types (see also Razik and Cidlowski, 2002). It appears, therefore, that the observed, highly distinct volume changes and intracellular ion composition are not simply associated, secondary features of cell death process but essential prerequisites for both, necrotic and apoptotic cell death.
Due to the fact that Na+ is the most abundant extracellular osmolite, necrotic cell-volume increase should imply an intracellular Na+ overload. In most cell types, this Na+ accumulation has been regarded as passive, i.e. due to the progressive inability of the cell to extrude the cation under conditions of ATP shortage. However, some neuronal cell types swell and undergo necrotic changes as a result of an active process (excitotoxicity) that involves the specific activation of cation channels, such as the AMPA sub-type of the glutamate receptor family (Lee et al., 1999). In this respect, it is worth mentioning that in a chicken retina model, blockage of NMDA receptor-mediated current by fenamates under ischemic or excitotoxic conditions, was sufficient to protect the cells from excitotoxic damage (Chen et al., 1998).
Sodium overload and cell swelling are not exclusive findings for metabolically stressed excitable cells, like neurons. In fact, it has been reported that in liver cells exposed to oxidative stress or hypoxic conditions, significant cytoprotection was achieved by replacing extracellular Na+ with a non-permeant cation, such as choline (Carini et al., 1995a). Others authors have shown that similar protection can be also obtained in liver cells by inhibiting the Na+-K+-2Cl- cotransporter, the Na+-H+ exchanger and the Na+-HCO3- cotransporter (Carini et al., 1995b; Fiegen et al., 1997). Taken together, these results suggest that under oxidative stress conditions, hepatocytes become progressively less competent in their ability to regulate cell volume, a step that appears central to the onset of necrosis.
Defective extrusion of Na+ secondary to the failure of energy-dependent transporters, as discussed above, is reasonably well documented. In contrast, however, possible mechanisms of augmented Na+ influx during oxidative stress are less well characterized.
Cation-selective channels are widely distributed among different tissues but vary considerably with respect to conductance, selectivity sequence and modulation (Thorn & Petersen, 1992; Eguiguren et al., 1996). In general, however, they exhibit some common features, such as a very low open probability when studied in situ (cell-attached) in non-stimulated cells, a conductance of 15-30 pS under symmetrical Na+ conditions, and relatively voltage independent gating. In addition, when studied in the inside-out configuration, the channel is activated by Ca2+ and blocked by ATP. Interestingly, a channel with identical characteristics found in colonic cells has been reported to be inhibited by fenamates (Gögelein et al., 1990). On the other hand, oxidative stress-mediated cation channel activation is not without precedent (Koliwad & Elliot, 1996; Barros et al., 2001b). Taken together, these observations suggest that activation of such a conductance may be coupled to the metabolic state of the cell.
Recently, a functional interaction between oxidative stress, membrane Na+ permeability and cell volume in hepatoma cells has been proposed (Schlenker et al., 2000). Indeed, stressed cells tend to have high levels of intracellular Ca2+ and low levels of ATP. This metabolic combination, in addition to free radicals and/or still unknown specific signaling components, are thought to activate the NSCC. Due to the highly favorable gradient, Na+ enters the cell followed by water, and cell swelling occurs. As a consequence of the metabolic disorder, cells will not be able to handle this overload and lysis will eventually ensue.
In the present work, we have characterized further the basic properties of a 18 pS- NSCC present in HTC cells in particular under conditions of oxidative stress.
HTC (rat liver cell line derived from Morris hepatoma) were grown at 37oC in a 5% / 95% : CO2/air atmosphere in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum, 80,000 I.U/l penicillin and 50 µg/l streptomycin. In addition, cells were pretreated with BSO (DL-buthionine-S,R-sulfoximine, SIGMA) for 18 hours before subjecting them to the experimental conditions. This treatment depletes cellular glutathione stores (Kinnula et al., 1992) rendering the cells more sensitive to oxidative stress.
Single-channel currents were measured from isolated HTC cells at room temperature using the patch-clamp technique with an Axon 200B and/or an EPC-7 amplifier (Axon Instruments, Inc, USA; List Medical, Germany) as described elsewhere (Hamill et al., 1981; Eguiguren et al., 1996). The bath and pipette solutions used are described in the legends to the figures. The signal was low-pass filtered at 1 kHz (-3 dB) and digitized at 5 kHz. Acquisition, analysis and fitting were done with the pClamp software suite (Axon Instruments, Inc., USA). For analysis of PO, patches were held at the desired potential for at least 3 min and the records were analyzed off-line using the QuB software suite (Qin et al., 1996; 1997).
Cell volume measurements
Changes in cell water volume were assessed in single cells by measuring changes in concentration of an intracellularly trapped fluorescent dye (Alvarez-Leefmans et al., 1995). HTC cells were plated on coverslips, loaded with calcein-AM (5 µM, for 5 min) and then superfused with isoosmotic solution for 15 min before starting the experiment. The experiments were performed using a confocal laser imaging system (Carl Zeiss). Excitation light was 488 nm, and emitted light was measured at wavelengths longer than 515 nm. Pictures were obtained at 10-s intervals, and fluorescence of a ~10 µm2 area in the center of a cell was measured. The records were corrected for fluorescence decay independent of cell volume changes (primarily due to dye photobleaching). The data are presented as Vt/V0, where V0 = cell water volume in isoosmotic solution at t = 0, and Vt = cell water volume at time = t. This was calculated from F0/Ft (F = fluorescence intensity) as described previously (Alvarez-Leefmans et al., 1995; Stutzin et al., 1999).
BSO-treated HTC cells exposed to 1 mM H2O2, a free-radical donor, in the presence of external Na+, rapidly increasead their volume by ~ 20%, reaching a plateau in 15 min (Fig. 1A). To prove that H2O2-induced cell swelling depends on external Na+, the Na+-rich medium was replaced after initial swelling by a medium rich in D(-)-N-methylglucamine (NMDG), an impermeant cation. Upon Na+ replacement (Fig. 1B), cell swelling stopped and the cells recovered their original volume in the presence of H2O2. The ability of cells to display volume recovery was strongly dependent on the time of exposure to Na+ + H2O2. Thus, cells exposed to external sodium and hydrogen peroxide for more than 10 min were unable to recover their volume after switching to a zero-sodium condition and continued swelling (data not shown).
|Fig. 1. H2O2-induced cell swelling in HTC cells. A. Effect of hydrogen peroxide on cell volume. HTC cells were exposed to an isotonic solution [Iso NaCl; compositon in mM; 136 NaCl, 4.7 KCl, 1.25 MgSO4, 1.25 CaCl2, 20 HEPES and 5.6 glucose (pH 7.4, 298 mOsmol/l)]. During the time indicated by the bar, the same buffer solution plus 1 mM H2O2 was added. B. Hydrogen peroxide-induced cells swelling is dependent on external Na+. The same experimental protocol (as depicted in A.) was employed, except that after initial swelling, the external H2O2-Na+-rich medium was replaced by a medium rich in NMDG in the continuous presence of H2O2. Results shown are representative of 5-10 cells per coverslip and per experiment. Each experiment was repeated at least 3 times.|
The possible presence of the NSCC in HTC cells line was next examined. In the cell-attached configuration, channel activity was rarely observed (data not shown). Upon excision into a high-Ca2+ (2.6 mM) containing solution and in the absence of permeant anions, channels were rapidly activated and several conductances levels were usually observed with a slope conductance of ~18 pS and a linear current-voltage relationship, similar to that described previously (Gray & Argent, 1990; Eguiguren et al., 1996; Barros et al., 2001b). The calculated open probabilities, (PO) assuming channel independency and binomial distribution were ~ 0.5 and 0 at 2.6 mM and 0 mM Ca2+, respectively (Fig. 2). Relative cation permeability examined under biionic conditions (Na+/K+) gave a PNa+/PK+ of 1.11 and PCa2+/P Na+ < 0.01 (data not shown).
| Fig. 2. Calcium-induced activation of the 18 pS NSCC in HTC cells. A. Single-channel traces from an excised inside-out membrane patch recorded at room temperature and under symmetrical ionic conditions (bath-pipette solution containing (in mM: 140 NaGlutamate, 5 NaCl, 2.6 CaCl2, 1.3 MgCl2, 10 HEPES and 5.6 glucose; pH 7.4, 298 mOsmol/l) are shown. B. The bath solution was replaced by a Ca2+-free solution (in mM: 140 NaGlutamate, 5 NaCl, 1.3 MgCl2, 2 EGTA, 10 HEPES and 5.6 glucose; pH 7.4, 298 mOsmol/l). The membrane voltage (Vm) was kept at -56 mV throughout the experiment. (n=3).
Inhibition of the NSCC by high (millimolar) intracellular adenine nucleotide concentrations is characteristic for many of these channels. Figure 3A-B shows a typical current record obtained in the presence of 0 and 1 mM ATP, respectively. Inhibition by ATP is summarized in the all-points amplitude histograms which reveal a drop of the P0 from 0.64 to 0. In addition to inhibition by adenine nucleotides, the NSCC is known to be blocked by flufenamic acid. Fig. 3C-E illustrates such an experiment. A representative single-channel record shows that channel activity is fully and reversibly blocked by 100 µM flufenamic acid.
|Fig. 3. Inhibition of the 18 pS NSCC in HTC cells by ATP. The same experimental conditions as described for Fig. 2 were used, except that Vm was kept at -59 mV. A. Control trace. B. Effect of the presence of 1 mM ATP in the bath solution. (n=3).|
From more recent data, it is becoming apparent that necrotic cell death involves specific, although largely unknown signaling events, including what has been proposed as a "cell death effector" (Barros et al., 2001b). Cell volume changes are known to play a crucial role in apoptosis (Bortner & Cidlowski, 1998) and necrosis (Carini et al., 1999; Barros et al., 2001b; Okada et al., 2001). Furthermore, the presence of external sodium ions is critical for the onset of cell swelling and subsequent necrotic death. Cells subjected to certain oxidative stress conditions swell and are likely to die by necrosis. In a previous study, we demonstrated that free-radical donors elicit cell swelling, NSCC activation and necrotic cell death in liver Clone-9 cells (Barros et al., 2001b). We now extend these findings to another epithelial cell line stressed by low concentrations of hydrogen peroxide.
Connecting free-radical production, Na+-dependent cell volume increase and NSCC channel activation is still a matter of speculation. A 28 pS NSCC has been shown to be directly activated by oxidized glutathione in endothelial cells exposed to free-radical donors (Koliwad & Elliot, 1996). Alternatively, a larger (70 pS) NSCC, which can also be activated during oxidative stress by high [Ca2+]i, low reduced glutathione and high NAD+, has been described in the insulin-secreting cell line CRI-G1 (Herson et al., 1999). Furthermore, a functional interaction between oxidative stress and increased membrane Na+ permeability has previously been reported in HTC cells (Schlenker et al., 2000). Thus, the 18 pS NSCC described above in HTC cells may in fact represent the molecular entity responsible for this previously reported increase in membrane Na+ permeability.
Fig. 4. Inhibition of the 18 pS NSCC in HTC cells by flufenamic acid. The same experimental conditions as described for Fig. 3 were used, except that Vm was kept at -47 mV. Control trace. B. Effect of the presence of 100 µM flufenamic acid in the bath solution. C. Wash-out of the fenamate. (n=5).
Known factors contributing to necrosis include intracellular ATP depletion and an increase in [Ca2+]. It is important to note that in our experiments ATP and Ca2+ have a profound impact on NSCC activity, suggesting that these may serve as linkers between oxidative stress, unbalanced cell swelling and necrosis. In addition, flufenamic acid, a NSCC channel blocker, reduces necrotic cell death (Chen et al., 1998; Barros et al., 2001b).
In conclusion, evidence is presented suggesting that external Na+ is a key factor in H2O2-induced cell swelling in HTC cells and that presumably sodium ions enter the cells via the Ca2+-dependent, ATP inhibited 18 pS NSCC. More work is required to determine the precise signaling events that link ATP decrease, calcium increase, and channel activation to necrotic cell death.
The authors wish to thank Marcela Hermoso for kindly helping with the cell volume experiments. This work was supported by Fondecyt 1010994 and FONDAP 15010006.
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Correspondence should be addressed to: Andrés Stutzin. Casilla 70058 Correo 7, Santiago, Chile. Phone 56 2 6786494. Fax 56 2 7376240. e-mail: email@example.com
Received: June 14, 2002. In revised form: June 24, 2002. Accepted: July 14, 2002