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GENERAL ARTICLES

Halothane Stimulates a Na+H+ Antiporter Involved in the Regulation of Intracellular pH in Alveolar Epithelial Cells

Philippe Juvin, MD^*, Christine Clerici, MD, PhD, Alain Loiseau, PhD, Jean Mantz, MD, PhD^*, Michel Aubier, MD, Gérard Friedlander, MD, PhD, and Jean-Marie Desmonts, MD^*

*Departement d’Anesthésie, Hôpital Bichat-Claude Bernard, Paris; Laboratoire de Physiologie, Hôpital Avicenne, Bobigny; and Faculté de Médecine Bichat, Paris, France

Address correspondence and reprint requests to Philippe Juvin, MD, Departement d’Anesthésie, Hôpital Bichat-Claude Bernard. 46, Rue Henri Huchard, 75877 Paris Cedex 18, France. Address e-mail to philippe.juvin{at}bch.ap-hop-paris.fr

	Abstract

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Changes in intracellular pH (pHi) of alveolar type II (ATII) cells have been involved in the pathophysiology of pulmonary edema. ATII cells have evolved several ions transporters to regulate their pHi, including a Na⁺H⁺ antiporter. Because halothane alters the activity of ion transporters in various cells types, it may also affect the activity of this Na⁺H⁺ antiporter. This study was performed 1) to characterize a Na⁺H⁺ antiporter in a model of ATII cells and 2) to investigate the effect of halothane on the activity of this antiporter. ATII cells were obtained from primary rat ATII cells transfected with a mutant of simian virus SV40 large T antigen (SV40-T2), and their pHi was monitored using the pH-sensitive fluorescent probe 2'-7' (bis carboxyethyl)-5(6')-carboxyfluorescein. We demonstrated in vitro that 1) a Na⁺H⁺ antiporter (apparent Km 6.8 ± 3.4 mM, Vmax 0.0105 ± 0.0013 UpHi/s) regulates the pHi of SV40-T2 cells and 2) at clinically relevant concentrations (10^-3 to 10^-5 M) and for a short exposure duration (60 min), halothane enhances the activity of this antiporter. Because ATII cell acidification has been associated with alterations in the alveolar epithelial barrier, halothane-induced intracellular alkalinization might exhibit some protective effect in clinical situations, such as aspiration pneumonia.

Implications: In vitro, halothane induces an intracellular alkalinization of pneumocytes II via the activation of a Na⁺H⁺ antiporter. Because acidification of these cells has been associated with alterations in the alveolar epithelial barrier, halothane might exhibit some protective effect in clinical situations, such as aspiration pneumonia.

	Introduction

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Volatile anesthetics affect lung function at both physiologic and cellular levels (1). In animals, they increase the protein permeability of the alveolar barrier during oxidant-stress injury (2) and decrease the normal alveolar epithelial fluid clearance (3). Because these two variables play a critical role in the resolution of alveolar edema, the administration of volatile anesthetics has become controversial in patients with impaired alveolar-capillary exchanges (3).

Changes in intracellular pH (pHi) of alveolar type II (ATII) cells are also involved in the pathophysiology of pulmonary edema. Intracellular acidosis of ATII cells may lead to alterations in the integrity of the epithelial alveolar barrier (4). ATII cells have evolved several ions transporters to regulate pHi, especially a Na⁺H⁺ antiporter (4). As halothane alters the activity of ion transporters in various cells types (5–7), it may also affect the activity of this Na⁺H⁺ antiporter. The aim of this study was 1) to characterize a Na⁺H⁺ antiporter in a model of ATII cells and 2) to investigate the effect of halothane on the activity of this antiporter.

	Methods

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ATII cells were obtained from primary rat ATII cells transfected with a transformation-defective mutant of simian virus SV40 large T antigen (SV40-T2). Cells were used between passages 25 and 35. They were plated in culture on plastic flasks in a medium consisting of Ham’s F12 supplemented with 5% fetal bovine serum (FBS). Confluent cultures were passaged with Versene-EDTA every 6 days. For experiments, cells were mobilized with Versene-EDTA at 37°C and suspended in Ham’s F12 medium with 10% FBS over 1 min. Cells were then resuspended in medium A composed of FBS 0.1 g/L and (in mM): NaCl 135, KCl 3, MgSO₄ 1, KH₂PO₄ 0.2, K₂PO₄ 0.8, HEPES 10, CaCl₂ 1, glucose 5, and L-leucine 5. Medium A was bubbled with 100% O₂ and adjusted to pH 7.4 with Tris (hydroxymethyl) aminomethane base.

As described previously, cells were loaded with the pH-sensitive fluorescence dye 2'-7' (bis carboxyethyl)-5(6')-carboxyfluorescein (BCECF) in medium A to monitor their pHi. This technique has been used in cells in suspension to study the epithelial transport of H⁺ ions (8). After one wash to remove the extracellular dye, the cells were prepared according to one of the two protocols described below. The first protocol was designed to characterize the Na⁺H⁺ antiporter in SV40-T2 cells. The second was designed to investigate the effect of halothane on the Na⁺H⁺ antiporter activity. After the appropriate protocol, the cells were washed again and diluted into glass cuvettes containing the experimental medium to reach a final cytocrit of approximately 1%. Cuvettes were placed in a Hitachi F2000 spectrofluorometer (Tokyo, Japan) to monitor BCECF fluorescence. Fluorescence intensity was recorded at one emission wavelength (525 nm), whereas the excitation wavelength was alternated automatically at 2-s intervals between 500 and 450 nm. The values of the fluorescence ratio F500 to F450 were converted into pHi values according to calibration curves performed by using Triton X-100 method (9). The rate of increase of extracellular fluorescence caused by extracellular leakage of BCECF was low (1.05% ± 0.13% per minute, n = 4) and neglected.

Protocol 1: Characterization of a Na⁺H⁺ Antiporter in SV40-T2 Cells
Na⁺H⁺ antiport activity was assessed by studying the sodium-dependent pHi recovery of sodium-depleted cells as described previously (10). Briefly, after incubation with BCECF, one aliquot of cells was taken to measure pHi in basal conditions. The rest of the cells were incubated and washed five times for 30 min in a sodium-free tetramethylammonium chloride (TMACl) medium (medium B, similar to medium A except for TMACl 135 mM, which was used instead of NaCl 135 mM) to produce intracellular sodium depletion by exchanging internal Na⁺ for external H⁺. Cells were then added into the spectrofluorometer cuvette containing medium B. Intracellular pH was measured over 20 s, and a TMACl (control) or a NaCl solution was suddenly added to the cuvette to reach various final concentrations up to 100 mM NaCl. The pHi was then measured over 180 s. Osmolarity of the final solutions was similar by replacing NaCl by TMACl isosmotically. The same experiments were repeated after 3 min incubation with amiloride 1 mM, a Na⁺H⁺ inhibitor. Kinetic values of changes in pHi after the addition of sodium were calculated by fitting the first 20 s of the pHi time course to a linear equation. The initial rate of change of pHi, which was a surrogate for the true proton flux rate, was plotted as a function of sodium external (Nae) concentration.

Protocol 2: Effect of Halothane on the Na⁺H⁺ Antiporter Activity
Dilutions of liquid halothane were obtained in dimethyl sulfoxide (DMSO). After incubation with BCECF, the cells were resuspended in medium A with either DMSO or liquid halothane 10^-3, 10^-4, or 10^-5 M over 30–120 min. After incubation with halothane or DMSO, cells were washed to remove the extracellular BCECF and added into the spectrofluorometer cuvette containing medium A. The pHi was then measured over 60 s. Experiments were repeated with amiloride 1 mM during the incubation course. Evaporation of halothane was minimized by covering the tubes with Parafilm (American National Can, Greenwich, CT). Final concentrations of halothane after dilution and equivalent periods were checked by chromatography as reported in our laboratory (11). The fluorescence was not affected by halothane regardless of the concentration or duration of incubation.

Data are expressed as mean ± SD or SEM of independent experiments run in triplicate. Statistical significance between experimental groups was assessed by using analysis of variance, followed by Student’s t-tests corrected for the number of comparisons. P < 0.05 was considered significant.

	Results

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Intracellular sodium depletion resulted in cell acidification (pHi 7.18 ± 0.05 and 6.83 ± 0.07 [P < 0.05] before and after sodium depletion, respectively). When NaCl was suddenly added to the cuvette, brisk intracellular alkalinization occurred. This was not observed after incubation with amiloride (Figure 1). The rate of cytoplasmic alkalinization increased with Nae concentrations. A hyperbolic curve, which obeyed Michaelis-Menten kinetics (Km 6.8 ± 3.4 mM, Vmax 0.0105 ± 0.0013 UpHi/s), was obtained when the initial rate of change of pHi was plotted as a function of Nae concentration (Figure 2).

View larger version (22K): [in this window] [in a new window]   Figure 1. Time course of intracellular pH (pHi) in sodium-depleted acidified alveolar type II cells after exposure to external sodium (Na). External sodium was added to the cell suspension at time 0, and the pHi was measured. Significant intracellular alkalinization (*P < 0.01) was caused by sodium alone (; final concentration of NaCl in the suspension 50 mmol/L, n = 8), compared with sodium plus 10-3 M amiloride (, n = 8) tetramethylammonium (TMA) chloride-treated (sodium-free) controls (•, n = 12). Data represent the means ± SD of experiments run in triplicate.

View larger version (11K): [in this window] [in a new window]   Figure 2. Dependence of the rate of the cytoplasmic alkalinization on external sodium concentration. Various external sodium concentrations were obtained in the suspension of the acidified cells. The initial rate of change of pHi (dpHi/dt) was measured as a function of external sodium ion concentration ([Nae]). Data represent the means ± SD of 6–12 experiments run in triplicate.

View larger version (17K): [in this window] [in a new window]   Figure 3. Time-dependent effect of exposure to halothane on intracellular pH (pHi) in alveolar type II cells. Cells were exposed to halothane concentrations of 10-3 to 10-5 M for up to 120 min, and pHi was measured. Data represent the means ± SEM of three to nine experiments performed in triplicate. From 60 min exposure, all values for each concentration of halothane were significantly different from the values of the control group (dimethyl sulfoxide) and of the group exposed to halothane 10-3 M plus amiloride 1 mM (*P < 0.05).

	Discussion

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SV40-T2 cells provide an accessible source of homogeneous cells that are never contaminated with resident macrophages, in contrast to ATII cells in primary cultures, but they have the major characteristics transport properties of ATII cells in primary cultures (12). The presence of a Na⁺H⁺ antiporter has been reported in numerous cells, including ATII cells in primary cultures, but not in SV40-T2 cells (4,13). In the present study, the initial intracellular acidification (due to a reversal Na⁺H⁺ antiporter) and the Nae-dependent, amiloride-inhibited recovery of pHi demonstrate that SV40-T2 cells also express a Na⁺H⁺ antiporter. Its kinetic variables are consistent with those described in other epithelia (13).

In addition, halothane enhances the activity of the Na⁺H⁺ antiporter. This conclusion is supported by the following evidence: 1) halothane increased pHi and 2) this alkalinization was completely inhibited by amiloride 1 mM, an inhibitor of Na⁺H⁺ antiporter. At this concentration, amiloride also inhibits sodium channel activity. However, because sodium channel activity was inhibited by halothane (7), the only possible effect of amiloride 1 mM in the present study was inhibition of the Na⁺H⁺ antiporter. This effect of halothane occurred at very low concentrations (10^-5 M) and for short exposure durations (60 min), which supports the clinical relevance of our findings. It extends the evidence that halothane alters protein ion transporters, as previously described in primary ATII cells (7) and in other tissues (5,6). The lack of a clear concentration effect of halothane on the Na⁺H⁺ antiporter is consistent with previous observations on other sodium transporters, both in vivo (14) and in ATII cells in primary culture (7).

The stimulation of Na⁺H⁺ antiporter activity by halothane could be explained by an effect via membrane environment or by a direct stimulatory effect on the antiporter. Another hypothesis is that a previous decrease in the intracellular sodium concentration could have lead to activation of the Na⁺H⁺ antiporter to regulate the intracellular sodium concentration. However, this is probably not the case because halothane simultaneously decreases sodium channel and Na⁺K⁺ ATPase activities (7), which suggests that halothane-related variation in intracellular sodium concentration is weak.

In conclusion, at clinically relevant concentrations and exposure durations, halothane stimulates Na⁺H⁺ antiporter activity in ATII cells in vitro. Caution is warranted when extrapolating experimental data to a clinical setting. However, because acidification of ATII cells has been associated with alterations in the alveolar epithelial barrier (4), the effect of halothane on pHi might attenuate the potential detrimental effects of volatile anesthetics during lung injuries (2,3).

	Acknowledgments

 
This work was supported by the Société Française d’Anesthésie et Réanimation.

SV40-T2 cells were a gift from Dr. Annick Clément.

	Footnotes

 
Presented in part at the annual meetings of the American Society of Anesthesiologists, Atlanta, GA, 1995, and New Orleans, LA, 1996.

	References

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