Halothane Decreases Na,K-ATPase, and Na Channel Activity in Alveolar Type II Cells 

The handling procedures in this protocol conformed to the Guiding Principle in the Care and Use of Animals approved by the Council of the American Physiologic Society.

Reagents were obtained from the following sources:⁸⁶RbCl (1 - 8 mCi/mg Rb) and²²Na (0.1 mCi/mg Na) from Amersham (Amersham, United Kingdom); K²H³²PO⁴(900 - 1,000 mCi/mmol) and L-(³) H) alanine (40 - 60 mCi/mmol) from Du Pont - New England Nuclear (Boston, MA); elastase from Worthington Biochemical (Freehold, NJ); and halothane (Trofield, United Kingdom). All chemicals of the highest grade available were purchased from Sigma (St. Louis, MO). Culture media and reagents were from Gibco-BRL (Paisley, United Kingdom). Plasticware was from Costar (Cambridge, MA).

Alveolar type II cells were isolated from the lungs of adult Sprague-Dawley, specific pathogen - free rats (200 - 250 g) as previously described. [8]Pooled cells from four to six rats were prepared as follows. Rats were anesthetized with 30 mg/kg intraperitoneal sodium pentobarbital and injected intravenously with 1 U/g heparin sodium. After tracheotomies were performed, the animals were exsanguinated, and the lungs were perfused via the pulmonary artery with solution II containing (in mM) 140 NaCl, 5 KCl, 10 HEPES-Tris pH 7.5 (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffered with Tris (hydroxymethyl-aminomethane), 2 CaCl², 1.3 MgSO⁴, 2.5 sodium phosphate buffer (Na²HPO⁴and NaH²PO⁴, pH 7.5), at pH 7.4 at 22 [degree sign]C. The lungs were then removed from the thorax and the airways lavaged to total lung capacity (8 - 10 ml) five times with solution I containing (in mM) 140 NaCl, 5 KCl, 10 HEPES-Tris, 0.2 EGTA (ethylene glycol-bis-([Greek small letter beta]-aminoethyl ether), N,N'-tetra-acetic acid), 2.5 sodium phosphate buffer, 6-D-glucose, at pH 7.4 at 22 [degree sign]C to remove macrophages. The thorax and airways were then washed twice with solution II. Then the lungs were filled with 12 - 15 ml of elastase solution (4 U/ml prepared in solution II) and incubated in a water bath in air at 37 [degree sign]C for 10 min, after which additional elastase solution was instilled for another 10-min incubation. The lungs were minced in a deoxyribonuclease I solution (0.25 mg/ml), and the enzymatic reactions were stopped with 5 ml of newborn calf serum. The tissue suspension was sequentially filtered through 150- and 30-[micro sign]m nylon mesh. The filtrate was centrifuged at 130g for 8 min. The cell pellet was suspended in Dulbecco's Modified Eagle's Medium (DMEM) containing 25 mM D-glucose and plated at a density of 10⁶cells/cm²in 25-cm²plastic dishes at 37 [degree sign]C in a 5% CO²incubator for 1 h to remove macrophages by differential adherence. The unattached cells were then removed and centrifuged at 130g for 8 min. The resulting cell pellet (70% purity and 90% viability) was plated at a density of 5 x 10⁵cell/cm²in either 24- or 6-well culture dishes. Culture medium was DMEM supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 10% fetal calf serum, 50 U/ml penicillin, 50 [micro sign]g/ml streptomycin, and 10 [micro sign]g/ml gentamicin; cells were incubated in a 5% CO^2/95% air atmosphere at 37 [degree sign]C. The cell purity, assessed using fluorescence with phosphine 3R, was 90 +/- 2% after 22 h of incubation. Contaminating cells were principally macrophages.

Experiments were performed at 24 h of culture. At this time, cells were washed gently with DMEM to remove nonadherent cells and were incubated with 500 [micro sign]l/well DMEM in a 12-l air-tight Lwolff chamber at 37 [degree sign]C and 5% CO². The chamber atmosphere was kept continuously saturated with H²O. Culture plates were separated first into two groups according to halothane or no treatment. Halothane vapor was provided as previously described. [8]Halothane flowed through two Fluotec Mark II vaporizers (Abbott, France) placed on the entrance to the chamber, and a halogen monitor (Capnomac Datex, Helsinki, Finland) determined the concentration of the anesthetic agent exiting the chamber. The chamber was sealed when the desired concentration was obtained. After incubation, halothane concentrations in the culture medium were checked by gas-phase chromatography. [9]The effects of halothane on Na,K-ATPase activity and Na-alanine and phosphate transport were assessed after 0.5, 1.0, 2.0, and 4 h of exposure at various concentrations of halothane (1, 2, and 4%). For each concentration of halothane, a control point was obtained with cells issued from the same pool of isolated cells and incubated in the same culture conditions except exposure to halothane. Halothane time points were not matched with control, because we have previously reported that measurements of Na,K-ATPase activity and Na-alanine and phosphate transport did not change over the duration of our experiments (4 h). [10] ²²Na influx studies were performed after 45 min of exposure at a halothane concentration of 1%. Finally, the reversibility of the effects of halothane on Na transports was assessed on cells exposed for 30 min at a halothane concentration of 4% and allowed to recover after replacement of cell supernatant by fresh culture medium for 15 and 30 min.

All measurements were performed after the cells were removed from the halothane-containing chamber. To minimize evaporation of the volatile anesthetic agent, cells were immediately washed and the uptakes were started in the first minute after the end of exposure at 37 [degree sign]in a 5% CO (²) incubator without halothane.

(²²) Na Influx Studies. Influx of²²Na was assayed, as previously described, [11]after depleting the cells of Na with 45 min of incubation in a Na-free solution in the halothane-containing chamber, NaCl being substituted by N-methyl-D-glucamine. The solution was then changed to one consisting of (in mM) 14 NaCl, 35 KCl, 96 N-methyl-D-glucamine, and 1 ouabain (to block the efflux of²²Na through Na,K-ATPase), supplemented with 0.5 [micro sign]Ci/ml²²Na. Uptake was performed during 5 min in the absence or presence of 0.01 mM amiloride. Amiloride-sensitive²²Na influx was determined by the difference between the values measured in the absence or presence of amiloride.

Uptake of Phosphate and Alanine. The uptake of alanine and phosphate was performed, as previously described, [12]at 37 [degree sign]C in a buffered solution which contained (in mM) 137 NaCl, 5.4 KCl, 2.8 CaCl², 1.2 MgSO⁴, and 14 HEPES (pH 7.4). In the Na-free solution, NaCl was replaced with N-methyl-D-glucamine. After removal of culture medium, cells were washed and incubated for 10 min in the presence of K²H³²PO⁴(0.5 [micro sign]Ci/ml) or L-³H-alanine (1 [micro sign]Ci/ml), with 100 [micro sign]M of KH²PO⁴or L-alanine. All steps were performed at 37 [degree sign]C. At the end of the incubation, the uptake was stopped by washing the cells three times with 1 ml/well of ice-cold solution (in mM): 137 NaCl and 5 HEPES-Tris (pH 7.4). The Na-dependent uptake of phosphate and alanine was calculated as the difference between the uptake of phosphate or alanine in the presence and absence of Na.

(⁸⁶) Rb Influx Studies. Measurements of influx of rubidium (Rb) were performed at 37 [degree sign]C in a solution derived from Eagle's Essential Medium, which contained (in mM) 120 NaCl, 5 RbCl, 1 MgSO⁴, 0.15 Na²HPO⁴, 0.2 NaH²PO⁴, 4 NaHCO³, 1 CaCl², 5 glucose, 4 nonessential amino acid, and 20 HEPES. [13]The osmolality was adjusted by addition of mannitol to 350 mOsm/kg and adjusting the pH to 7.4. After the removal of the culture medium, cells were washed and incubated with or without ouabain (1 mM) for 5 min in the presence of⁸⁶Rb (0.5 [micro sign]Ci/ml). Uptake was stopped by removing the incubation medium and washing the cells three times with 1 ml/well of ice-cold rinsing solution containing (in mM) 140 N-methyl-D-glucamine, 1.2 MgCl², 3 BaCl², and 10 HEPES. Ouabain-sensitive influx of Rb calculated by the difference between uptake of Rb with or without ouabain was used as an indicator of Na,K-ATPase activity.

After incubations, cells were solubilized in 0.5% Triton X-100, and tracer activities were determined by liquid scintillation counting. The remainder of each sample was used for assessing the protein content per well. [14] 

Uptake of phosphate, alanine, Na, and Rb was expressed as nanomoles per milligram of protein. For evaluation of the recovery, values were expressed as a percentage of appropriate control values. Results are presented as means +/- SD of three to six independent experiments done in triplicate.

Values obtained in cells exposed to halothane were compared with those obtained in control cells for each experiment by analysis of variance followed by Fisher's correction for multiple comparisons. A two-way analysis of variance was used to assess the effects of halothane and Ca-free culture conditions or 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA) addition.

Na,K-ATPase activity was evaluated by the ouabain-sensitive component of the influx of⁸⁶Rb (OsRb), calculated as the difference between influx of Rb in the presence and absence of ouabain, a specific inhibitor of the Na,K-ATPase. Exposure to halothane reduced the influx of OsRb in ATII cells (Figure 1), whereas the ouabain-insensitive part of the influx of ouabain, which represents 50% of the total influx, was unchanged. The maximal decrease of OsRb occurred at 30 min of exposure, did not change for longer exposure times, and was of similar magnitude for concentrations of 1, 2, and 4%, representing 50, 42, and 56% of the control values, respectively.

The Na channel activity was evaluated by the amiloride-sensitive component of the influx of²²Na, calculated as the difference between uptake of Na in the presence of ouabain and ouabain plus amiloride. Exposure of the cells to 1% halothane for 45 min induced a 46% decrease in amiloride-sensitive influx of²²Na (Figure 2). Exposure to halothane had no effect on the amiloride-independent component of the uptake, which represented 55% of the total influx.

Uptake of phosphate and alanine was not modified by halothane, regardless of the concentration or the duration of exposure (Figure 3).

When ATII cells were allowed to recover from exposure to halothane,²²Na amiloride-sensitive and OsRb influx returned to control values within 30 min (Figure 4).

In attempt to evaluate whether halothane-induced inhibition of Na,K-ATPase activity could be attributed to an increase in intracellular Ca, two experiments were performed. First, control cells were incubated with a Ca ionophore ionomycin (10^-6M). Ionomycin induced a 40% decrease in Na,K-ATPase activity and a 60% decrease in amiloride-sensitive influx of²²Na, but it had no effect on Na-dependent uptake of phosphate and alanine (data not shown). These results suggested that an increase in concentration of intracellular Ca could inhibit Na,K-ATPase and Na channel activities. Second, we tested whether the decrease in Na,K-ATPase activity produced by exposure to halothane was dependent on the presence of external Ca or on the release on intracellular Ca. As shown in Table 1, the removal of the extracellular Ca or chelation of intracellular Ca by BAPTA increased influx of OsRb in control cells by 74% and 82%, respectively, but did not prevent the halothane-induced decrease in Na,K-ATPase activity.

This study demonstrates that exposure of alveolar epithelial type II cells to halothane inhibits Na ion vectorial transport in vitro. The Na,K-ATPase and Na channels are the major targets of the halothane effect, and their activities were impaired for short-duration exposures at low concentrations of halothane.

Na,K-ATPase and Na channel are the two major proteins involved in Na transport in alveolar epithelial cells. In our study, halothane induced a decrease in Na,K-ATPase activity and amiloride-sensitive influx of Na. The finding that the decrease in amiloride-sensitive influx was related to a decrease in Na channel activity rather than to Na-H⁺inhibition is supported by a recent preliminary report, which indicates that halothane initiated a significant intracellular alkalinization in an alveolar rat cell line. [15]Because halothane induced a concomitant decrease in influx of OsRb and amiloride-sensitive Na, it is not possible to determine whether the inhibition of Na,K-ATPase is the cause or the consequence of the reduced rate of Na entry through Na channels. [16]In the current study, several lines of evidence indicate that halothane-induced Na,K-ATPase and Na channel activity impairment was not the consequence of irreversible and nonspecific damage:(1) Release of lactate dehydrogenase (LDH), reflecting cell viability, did not increase in cells exposed to halothane [8];(2) other pathways of Na transport, Na-phosphate and Na-alanine cotransporters, were not affected by halothane; and (3) the inhibitory effect of halothane on Na transport was rapidly and completely reversible after cessation of delivery of anesthesia washout. The observation that, in ATII cells, halothane induced a decrease in Na,K-ATPase differs from the results obtained in other cell preparations, in which pharmacologically relevant concentrations of volatile anesthetic agents have little or no effect on Na,K-ATPase activity. [17–19]In human erythrocytes, Halsey et al. have shown that the Na-K pump was not modified by clinical concentrations of volatile anesthetic agents. [17]In accordance with this latter study, a recent study performed on cerebral synaptic plasma membranes indicated that halothane exposure did not modify Na,K-ATPase activity. [18]Fomitcheva and Kosk-Kosicka, however, compared the effects of halothane on various ATPase activities in synaptosomal or erythrocyte membranes and found that a minimum alveolar concentration of 3 was required to half-maximally inhibit the Na,K-ATPase activity, but a minimum alveolar concentration of only 0.75 - 1.15 was necessary for the same inhibition of calcium-transporting ATPase. [19]In this study, Na,K-ATPase activity was strongly inhibited by halothane at a minimum alveolar concentration 1. These data may represent evidence of functional and pharmacologic differences between Na pumps among different tissues.

The mechanisms by which halothane alters Na transport protein remain unknown. Inhibition of the Na pump and Na channels may result from indirect modifications of intracellular mediators, such as ATP, [19,20]or cytosolic free calcium, [21,22]or from direct qualitative or quantitative alterations of the proteins.

Halothane was reported previously to reduce ATP content in ATII cells. [8]Therefore, the possibility that depletion of intracellular ATP in halothane-exposed cells might be responsible for the inhibition of Na,K-ATPase activity was examined. Several studies have reported that Na,K-ATPase is directly dependent on cellular concentrations of ATP. [20,21]When ATII cells were exposed to a halothane concentration of 4% for 1 h, ATP was depleted by only 10%. [8]In a previous study, we reported that depletion of ATP in these cells to a greater degree, achieved by metabolic inhibitors, did not alter influx of OsRb. [13]Taken together, these results suggest that depletion of ATP did not play a major role in halothane-induced inhibition of Na,K-ATPase activity.

Volatile anesthetic agents increase cytoplasmic Ca in several cells and tissues, including hepatocytes, mouse brain synaptosomes, peripheral blood mononuclear cells, and L6 muscle cells. [24]In most studies, this effect is attributed to the release of Ca from intracellular stores or an increase in Ca entry. [24]The intracellular concentration of free Ca ions is one of the most important regulatory systems in the cell. Our results suggest that, in ATII cells, Na,K-ATPase activity is modulated by intracellular concentrations of Ca, inasmuch as ionomycin, a Ca ionophore, reduced influx of OsRb, whereas the absence of extracellular Ca increased influx of OsRb. Halothane-induced inhibition of Na,K-ATPase activity was not prevented, however, by removal of external Ca or by chelating internal Ca by BAPTA. These results suggest that the reduction of Na,K-ATPase activity is not likely related to an increase in concentration of intracellular Ca. Finally, it can not be excluded that halothane induced changes of other intracellular mediators, which could in turn inhibit transport indirectly.

The decrease in the activity of Na,K-ATPase and Na channel may also result from a direct action of halothane on Na transport proteins by binding to the channel or the pump protein itself or by modifying the lipid environment of the protein. [25,26]Volatile anesthetic agents could induce modification in membrane fluidity, an effect that might affect the activity of numerous membrane proteins, among which are transport systems. A certain degree of fluidity has been shown to be essential for Na,K-ATPase activity. In ATII cells and other epithelial cells, Na-phosphate contransporter was reported to be very sensitive to the modification of the lipid environment. Because Na-phosphate transport was not affected by halothane, in this study, the decrease in Na,K-ATPase and Na channel activity was probably attributable to changes in the protein itself.

This study demonstrates that halothane, at clinically relevant concentrations and for short-exposure duration, impairs Na transport in ATII cells, in vitro, by decreasing Na,K-ATPase and Na channel activity. Na coupled transport is unchanged regardless of the concentration of halothane or the duration of exposure. These cotransporters are a common route of amino acids and phosphate entry into the cells but only account for 13% of total Na influx into the cells in freshly prepared ATII cells in suspension. [27]Although the direct measurement of circuit short current was not done, the decrease in Na,K-ATPase activity along with the decrease in Na channel activity strongly suggests that halothane reduces the transepithelial Na flux. In alveolar epithelium, transepithelial Na transport plays a major role in the regulation of the alveolar fluid balance. Alveolar edema is obviously associated with a serious impairment of gas exchange, because it increases distance for gas diffusion and severely impairs the ventilation-perfusion relationship. Therefore, inhibition of Na transport by halothane in alveolar epithelium might be of clinical relevance, particularly in patients with pulmonary edema. A recent in vivo study in rats confirmed that halothane causes a reversible decrease in net alveolar epithelial Na and fluid transport. [28]Further studies are needed to investigate the effects of anesthetic agents on alveolar fluid clearance on in vivo models of pulmonary edema.