Voltage-gated proton channels help regulate pHi in rat alveolar epithelium

Ricardo Murphy, Vladimir V. Cherny, Deri Morgan, and Thomas E. DeCoursey

Department of Molecular Biophysics and Physiology, Rush University Medical Center, Chicago, Illinois

Submitted 9 August 2004 ; accepted in final form 25 October 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Voltage-gated proton channels are expressed highly in rat alveolar epithelial cells. Here we investigated whether these channels contribute to pH regulation. The intracellular pH (pHi) was monitored using BCECF in cultured alveolar epithelial cell monolayers and found to be 7.13 in nominally HCO3-free solutions [at external pH (pHo) 7.4]. Cells were acid-loaded by the NH4+ prepulse technique, and the recovery was observed. Under conditions designed to eliminate the contribution of other transporters that alter pH, addition of 10 µM ZnCl2, a proton channel inhibitor, slowed recovery about twofold. In addition, the pHi minimum was lower, and the time to nadir was increased. Slowing of recovery by ZnCl2 was observed at pHo 7.4 and pHo 8.0 and in normal and high-K+ Ringer solutions. The observed rate of Zn2+-sensitive pHi recovery required activation of a small fraction of the available proton conductance. We conclude that proton channels contribute to pHi recovery after an acid load in rat alveolar epithelial cells. Addition of ZnCl2 had no effect on pHi in unchallenged cells, consistent with the expectation that proton channels are not open in resting cells. After inhibition of all known pH regulators, slow pHi recovery persisted, suggesting the existence of a yet-undefined acid extrusion mechanism in these cells.

proton conductance; pH regulation; hydrogen ion; acid load; 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein; intracellular pH


ALVEOLAR TYPE II EPITHELIAL CELLS exhibit an impressive panoply of pH-regulating mechanisms, perhaps reflecting their role in extruding enormous quantities of acid in the form of CO2 during respiration as well as their exposure to an asymmetrical pH environment. Whereas the basolateral membranes face typical interstitial fluid, the apical membranes face the alveolar subphase, the fluid at the interface between air and tissues in the alveolus. This fluid is highly acidic compared with plasma or interstitial fluid, with estimates at pH 6.69 in dog lung (17), pH 6.27 in fetal lamb lung (1), and pH 6.92 in rabbit lung (31). The properties and localization of several transporters that influence pHi have been studied previously. Sodium-proton exchange, Na+/H+-antiport, is active at pHi <7.0 during recovery from an acid load (32) and appears to be localized in basolateral membranes (22, 26). Sodium-independent Cl/HCO3 exchange contributes to recovery from an alkaline load (33), and the alveolar type II epithelial isoform is restricted to the basolateral surface of alveolar epithelial monolayers (27). A Cl-independent Na+-HCO3 symporter contributes to cytosolic alkalinization and, in contrast to Na+/H+-antiport, is constitutively active at intracellular pH (pHi) >7.0 (25). The Na+-HCO3 symporter is detected in basolateral membranes (27). Evidence for a K+-H+-ATPase exists in guinea pig but not rat type II pneumocytes (24). A plasma membrane V-type H+-ATPase reportedly is active at physiological pH and may keep pHi near 7.5 (28), although Brown et al. (6) found no evidence for a V-type H+-ATPase in rat type II cells and concluded that ATP modulates Na+/H+-antiport. The possibility that Cl/OH exchange (38) might occur in rat alveolar epithelial cells was suggested by highly indirect evidence (13). Finally, voltage-gated proton channels comprise a major conductance in rat alveolar epithelial type II cells (10), are demonstrably present in the apical membranes of cultured cells (12), and might be present in all membranes. Proton channels are opened by membrane depolarization, decreased pHi, increased extracellular pH (pHo), or a combination of these factors (9, 11). Their regulation by pH and voltage ensures that these channels open only when there is an outward electrochemical gradient for protons (11). Because this gradient is normally inward (10), we predict that H+ current inhibition with Zn2+ should have no effect on pHi in unchallenged alveolar epithelial cells. In several other cells, proton channels mediate pHi recovery after an acid load. However, until now, no direct evidence for this or any other specific function for proton channels had been demonstrated in alveolar epithelial cells. Effects of Zn2+ reported here indicate that voltage-gated proton channels are closed at normal pHi but contribute to H+ extrusion after acid loading rat alveolar epithelial type II cells.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Rat alveolar epithelial cells. Type II alveolar epithelial cells were isolated from adult male Sprague-Dawley rats by enzyme digestion, lectin agglutination, and differential adherence, as described in detail elsewhere (15), with three exceptions. First, the solution used to perfuse the lungs is 40 ml of HBSS (catalog no. 14170-112; GIBCO Laboratories, Grand Island, NY). Second, we use 0.2 mg/ml elastase without trypsin to dissociate the cells. Third, the filtrate is centrifuged at 2,000 rpm (instead of 1,500 rpm). Before invasive procedures were initiated, the rats were anesthetized deeply with pentobarbital sodium. The rats were treated humanely in compliance with local law, our Institutional Animal Care and Use Committee, and with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The lungs were lavaged to remove macrophages, elastase was instilled, and then the tissue was minced and forced through fine gauze. Lectin agglutination and differential adherence further removed contaminating cell types. The preparation at first includes mainly type II alveolar epithelial cells, but after several days in culture, the properties of the cells are more like type I cells. Studies were done on monolayers of cells grown on cover glass chips for 5–25 days. Plotting the parameters defined in Fig. 2 against time in culture did not reveal any trends (data not shown).



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 2. An example of an acid-load/recovery cycle. The tissue was initially in Ringer. Addition of 30 mM NH4Cl results in a transient increase in pHi (A) associated with a rapid influx of NH3, followed by a slower fall in pHi as NH4+ enters the cells (B). On transferring the tissue to NH4Cl-free potassium Ringer (K-Ringer) solution at time (t) = 0, pHi falls rapidly (acid load) as NH3 leaves the cells (C). This is followed by a slower recovery, presumably due to proton (or proton equivalent) efflux (D). The bold curve for t > 0 is a fit of Eq. 3. For t < 0 the data were fitted with an exponential decay. This component was not used for analysis, except to establish pHi(0) (Eq. 3). pHfin, limiting value to which the pH apparently recovers as t -> {infty}; tmin, the time to reach the pH minimum at the end of phase C; pHmin, pH at that minimum; {tau}, time constant of pHi recovery.

 
Chemicals. SCH-28080 and some of the 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-acetoxymethyl ester (BCECF-AM) used here were purchased from Calbiochem (La Jolla, CA). All of the remaining chemicals, including nigericin, 5-(N,N-dimethyl)amiloride hydrochloride (DMA), bafilomycin A1, and some BCECF-AM, were obtained from Sigma Chemical (St. Louis, MO).

Measurement of pHi. Cells were loaded for 20–60 min with 10–20 µg/ml BCECF-AM, the nonfluorescent, membrane-permeant acetoxymethyl ester of BCECF dissolved in 1 ml of Ringer solution (in mM: 160 NaCl, 4.5 KCl, 2 CaCl2, 1 MgCl2, 5 HEPES; pH 7.4) or culture medium. Intracellular esterases cleave the three AM ester groups to form the charged, membrane-impermeant, pH-sensitive, fluorescent dye BCECF (30). pHi was monitored ratiometrically with a model LS50B luminescence spectrometer (Perkin-Elmer, Norwalk, CT) at excitation wavelengths of 440 and 495 nm and an emission wavelength of 525 nm. Excitation and emission slit widths were 5 and 3 nm, respectively. We took background readings before loading the dye and subtracted them from the fluorescence intensities (F{lambda}, where {lambda} is the excitation wavelength) before calculating the fluorescence ratio (R = F495/F440 measured at 525 nm). Because of a progressive detachment of cells from the cover glass (especially during solution changes), fluorescence intensities frequently declined over the course of an experiment. Data for which F440 was less than twice background were discarded.

At the end of some experiments we performed a calibration (Fig. 1A) using the nigericin technique (30, 39). Specifically, the tissue was transferred to solutions containing 80–100 mM KCl, 3–10 µM nigericin, 2 mM CaCl2, and 100 mM buffer (pH 5.5 and 6.0, MES; 6.5, bis-Tris; 7.0, N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid; 7.55, HEPES; 8.0, Tricine). Any glassware or other apparatus that came into contact with nigericin was soaked overnight in ethanol (3). Failure to observe this precaution resulted in rapid recovery from an acid load [time constant ({tau}) {approx} 4 min]. In later experiments the use of disposable cuvettes obviated the need to wash cuvettes, but tissue holders were still soaked overnight in ethanol, even when nigericin was not used (as a precaution against contamination). Calibration was not possible in all experiments because of cell detachment and the consequent fall in F{lambda}. Accordingly, a mean calibration curve was used to calculate pHi from the fluorescence-ratio data. We obtained this curve by fitting the following equation to data pooled from six experiments (Fig. 1B)

(1)



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 1. A: calibration of the absolute pH at the end of one experiment. Intracellular pH (pHi) values of 5.5–8.0 (indicated by the numbers) were obtained by incubating the tissue in "high" (80–100 mM) KCl solutions containing 10 µM nigericin, 2 mM CaCl2, and 100 mM appropriate buffers (pH 5.5 and 6.0, MES; 6.5, bis-Tris; 7.0, BES; 7.55, HEPES; 8.0, Tricine). B: a fit of Eq. 1 to data pooled from 6 experiments (represented by the different symbols) like those in A. A log transformation was applied to both sides of Eq. 1 to stabilize the residual variance while preserving the functional relationship between fluorescence ratio (R) and pH. This mean calibration curve was used to calculate pHi from R data.

 
where Rmax is the value of R as [H+] -> 0, Rmin is the value of R as [H+] -> {infty} and pKa is related to the negative log of acidic dissociation constant (pKa) of BCECF by

(2)

where Fand Fare the values of F440 as [H+] -> {infty} and [H+] -> 0, respectively. Because 440 nm is close to the isosbestic point of BCECF, the log term in Eq. 2 should be close to zero so that pKa* {approx} pKa (provided external [K+] is chosen correctly, see Refs. 4, 5). Equations 1 and 2 follow from the treatment of fluorescent calcium probes by Grynkiewicz et al. (21). The estimated parameter values were pKa* = 7.337 ± 0.071, Rmin = 1.329 ± 0.066, and Rmax = 8.83 ± 0.52.

Experimental protocol. After dye loading, cells were initially placed in 1 ml of Ringer solution and were then acid-loaded by the NH4Cl prepulse technique (35). Specifically, 250 µl of 150 mM NH4Cl solution in water was added to the 1 ml of Ringer to give a final NH4Cl concentration close to 30 mM. As shown in Fig. 2, this resulted in an abrupt rise in pHi, followed by a slower decline (phases A and B). The sharp rise in pHi is believed to reflect the rapid influx and protonation of NH3, whereas the slower decay is thought to be associated with the entry of NH4+, which then releases protons to the cytosol. When the pHi had fallen to ~7, the cells were transferred to an NH4Cl-free "recovery" solution. The ensuing efflux of NH3 then resulted in a rapid fall in pHi (acid load, phase C) followed by a slower recovery (phase D), presumably due to proton equivalent efflux. The recovery solution was either Ringer plus 100 µM DMA (an inhibitor of the Na+/H+ exchanger, Refs. 26, 28) or potassium Ringer (K-Ringer), in which NaCl is replaced by KCl (which should also prevent Na/H+ antiport). Both solutions contained 100 nM bafilomycin A1 to inhibit any H+-ATPase activity (28), 100 µM SCH-28080 to inhibit any H+/K+-ATPase activity (24, 37), and 1 mg/ml glucose. The K-Ringer also contained 1.5 µM of the K+ ionophore valinomycin as a precaution to ensure adequate charge compensation of electrogenic H+ efflux. pHo was 7.4 in Ringer and either 7.4 or 8.0 in K-Ringer; HEPES buffer was used in all solutions.

To test for Zn2+ sensitivity (an indication of the involvement of voltage-gated H+ channels) the recovery solution also contained either 10 µM ZnCl2 or 1 mM of the divalent cation chelator EGTA (plus an extra 1 mM CaCl2 to maintain normal free Ca2+). A ZnCl2 concentration of 10 µM should effectively abolish any voltage-gated H+ flux, even in cells depolarized to 0 mV (8).

During experiments, a cover glass with attached cell monolayer was held in a cover glass holder inside a spectrometer cuvette containing ~1 ml of solution, with constant stirring. We changed solutions by transferring the holder to one or two successive beakers each containing 16–18 ml of the next solution in the series and then to a cuvette containing 1 ml of that solution. When not in use, rinsing solutions were stored in an incubator at 37°C; cuvette solutions were maintained at 37°C in the spectrometer with a circulating water bath. The order of acid-load/recovery cycles with or without Zn2+ was varied between experiments (e.g., Fig. 3). In ~50% of experiments two acid-load/recovery cycles were achieved, and in ~10% three cycles were obtained. In the remaining experiments only a single acid-load/recovery cycle was possible because of cell loss.



View larger version (25K):
[in this window]
[in a new window]
 
Fig. 3. Effects of Zn2+ on pHi recovery at extracellular pH (pHo) 7.4 (A) and pHo 8.0 (B). In both we applied the NH4Cl prepulse in Ringer (R) at pHo 7.4 before transferring the tissue to K-Ringer (KR). Two successive acid-load/recovery cycles are shown, one with Zn2+, the other without. Note that the order of the treatments is reversed in B. The data were smoothed with a Fourier smoother (unsmoothed data were used for curve fitting, as in Fig. 2).

 
Data analysis. To quantify the changes in pHi following the removal of NH4Cl (i.e., on transferring the cells to the recovery solution), we fitted the data for phases C and D in Fig. 2 with the following equation by nonlinear least squares (bold curve in Fig. 2)

(3)

where, having removed the artifact associated with the solution change, we took time t = 0 as midway between the end of phase B and the start of phase C. Referring to Fig. 2, data are reported in terms of tmin (the time to reach the pH minimum at the end of phase C), pHmin (the pH at that minimum), {tau} = {tau}2 in Eq. 3 (effectively the time constant of the recovery phase, D), and pHfin (the limiting value to which the pH apparently recovers as t -> {infty}). pHfin [= pHi(0) + {Delta}pH1 + {Delta}pH2] and {tau} (= {tau}2) are obtained directly from the fits of Eq. 3. We determined tmin by setting the derivative of Eq. 3 to zero and solving for t. pHmin was then calculated by setting t = tmin in Eq. 3.

In some cases the minimum was obscured by the solution-change artifact, and so the term in {Delta}pH1 in Eq. 3 was omitted. pHmin and tmin were then estimated as pHi and t for the first reliable data point following the solution change (i.e., after removing the artifact). Although this is somewhat arbitrary, it was done to avoid biasing the mean value of tmin toward larger values. In other cases there was insufficient curvature in phase D for a fit of the second exponential and so it was replaced with a linear term

(4)

where b is a constant. We again determined pHmin and tmin by setting the derivative of Eq. 4 to zero or from the first point in phase D if a minimum was absent (in which case the term in {Delta}pH1 was omitted); estimation of {tau} and pHfin was not possible.

From the point of view of data analysis, the ideal experiment is like those shown in Fig. 3, in which Zn2+ and control (EGTA) data are available in the same experiment. If the data from control and Zn2+-exposed cells are correlated, the use of such paired data will improve the precision of parameter-ratio estimates and hence increase the power of statistical tests. Paired data were analyzed as described in RESULTS. However, to limit the analysis to paired data would be to discard about one-third of the experiments. Hence, if paired data are not correlated it is better to use all the data (paired and unpaired) and so increase the sample size. For recovery in Ringer at a single pHo (7.4) this is easily achieved with one-way analysis of variance (ANOVA). For recovery in K-Ringer at two different pHo (7.4 and 8.0), the following model was fitted to all the data by least squares

(5a)

(5b)

(5c)

(5d)

where x represents tmin, pHmin, {tau}, or pHfin; µ is the value of x when pHo = 7.4 and [Zn2+] = 0; {alpha} is the factor by which x is changed by Zn2+ at pHo = 7.4; {beta} is the factor by which x is changed by a change in pHo (7.4 -> 8.0) when [Zn2+] = 0; and {gamma} allows for the possibility that the effect of Zn2+ is different at different pHo, and vice versa (if there is no such interaction between the effects of Zn2+ and pHo, then x = {alpha}{beta}µ when pHo = 8.0 and [Zn2+] > 0). Various reduced models were then fitted by setting {alpha}, {beta}, or {gamma} to unity. A minimum-parameter model was chosen by using F tests at the 5% significance level as described by Walpole and Myers (40). Specifically, for two models with k and k – 1 parameters (e.g., for k = 4, models with parameters µ, {alpha}, {beta}, {gamma} and µ, {alpha}, {beta}) an F value with (1, n k) degrees of freedom (where n is the number of x values) was calculated as

(6)

where RSS is the residual sum of squares. If the F value was significant the k-parameter model was retained, otherwise it was rejected in favor of the reduced model with k – 1 parameters. When nonsignificant F values were obtained with more than one reduced model, the one with the smallest RSSk – 1 was chosen for the next round of F tests (i.e., against models with k – 2 parameters).

As judged by t-tests at the 5% level and visual inspection of the data, there was no evidence that the order of the applied treatments (i.e., Zn2+ -> EGTA or EGTA -> Zn2+) affected x, and so order does not appear as a factor in Eq. 5, ad. Rather, for all types of analysis (paired, ANOVA, and Eq. 5, ad), control data obtained before and after the Zn2+ treatment were pooled, as were the Zn2+ data obtained before and after the EGTA treatments. (In this case the ANOVA is equivalent to a two-sample t-test; Ref. 40) For tmin and {tau}, a log transformation was applied to both sides of Eq. 5, ad, to stabilize the variance and reduce positive skew; this was not necessary for pHmin and pHfin (presumably because these variables are already log-transformed). Mean values are given with standard errors and, where appropriate, the number of observations in parenthesis. A significance level of 0.05 was used for all statistical tests.

Boyarsky et al. (4) concluded that in a variety of cells at around pHi 7, the high K+/nigericin calibration technique led to estimates of pHi (pHi,nig) that were 0.08–0.26 pH units above the true pHi (pHi,true). At least part of the error probably arose because [K+] in the calibration solutions was too low; this was likely true in the present study also. In a subsequent paper on smooth muscle cells, Boyarsky et al. (5) found that the error varied from ~0 to ~0.3 pH units over the pHi,nig range 6–8. Furthermore, the error increased approximately linearly with pHi,nig such that

(7)

where A and B are constants. As pointed out by Boyarsky et al. (5), such a linear relationship will mean that t-tests on pHi values are unaffected. The same can be said of F-tests; the RSS values in the numerator and denominator of Eq. 6 are simply multiplied by (1 – B)2 (40) so that the F values are unaffected. Hence, assuming any error in the present study was also linearly related to pHi,nig, the conclusions regarding pHmin and pHfin are unaltered, even though the absolute values of pHmin and pHfin may be in error. As regards tmin and {tau}, it is readily shown from Eqs. 3 and 7 that estimates of these parameters are unaffected by a linear transformation in pHi,nig.

Mathematical and statistical analyses were carried out with Mathematica v. 4.2 (Wolfram Research, Champaign, IL), the NAG Fortran Library Mark 20 (Numerical Algorithms Group, Downers Grove, IL) running under Compaq Visual Fortran v. 6.6 (Compaq Computer, Houston, TX), and with software written in True BASIC Gold Edition (True BASIC, Hartford, VT).


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Resting pHi in Ringer solution. At the beginning of each experiment the cells were placed in Ringer solution (pH 7.4) and a baseline pHi was established. The average pHi was 7.13 ± 0.17 (mean ± SD, n = 38). The average pHi measured in Ringer solution after recovery from the first acid load (pHfin) was not significantly different, 7.04 ± 0.06 (means ± SE, n = 11), indicating little drift during the 30- to 50-min experiments. After recovery in K-Ringer, average pHi was identical to that in Ringer, 7.00 ± 0.05 (means ± SE, n = 13).

To test whether proton channels help maintain the resting pHi in rat alveolar epithelial cells, we added 100 µM ZnCl2 to the Ringer solution (without EGTA) and measured pHi for 20–30 min. As illustrated in Fig. 4, there was no discernable effect. Similar results were obtained in three experiments. We routinely employ EGTA (with added CaCl2 to maintain normal free Ca2+) to eliminate polyvalent metal contaminants. Therefore, in three analogous experiments, Ringer with EGTA was used as the control solution before and after exposure to 10 µM ZnCl2 (no EGTA in the ZnCl2 solution). Again, no effect on pHi could be detected. After testing for effects of ZnCl2, we added NH4+ to verify that the cells were still viable and responsive. Evidently, proton channels are not open under resting conditions.



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 4. Lack of effect of 100 µM ZnCl2 on pHi in unchallenged alveolar epithelial cells bathed in Ringer solution. At ~24 min into the experiment, 10 µl of 10 mM ZnCl2 in water was added to the cuvette to give a final concentration of 100 µM. Later, we introduced and then removed 30 mM NH4+ (by transferring the tissue to NH4Cl-free Ringer), resulting in normal pHi changes, like those described in Fig. 2.

 
pHi recovery in K-Ringer. Most experiments were done in high-[K+] solution, K-Ringer, both to prevent Na+/H+-antiport and to depolarize the cells and thereby promote opening of voltage-gated H+ channels. Cells in normal Ringer solution are expected to maintain a more negative membrane potential, which should lower H+ channel open probability. Experiments were done at both pHo 7.4 and pHo 8.0, because high pHo promotes H+ channel opening (10). Figure 3 shows that at both pHo 7.4 (Fig. 3A) and pHo 8 (Fig. 3B), 10 µM Zn2+ slowed pHi recovery following an acid load and also appeared to delay the onset of recovery. In experiments such as these, the changes in pHi following removal of NH4+ were quantified by the parameters tmin, {tau}, pHmin, and pHfin, as illustrated in Fig. 2. Often it was possible to estimate these parameters in the presence and absence of Zn2+ within a single experiment (Fig. 3), although sometimes cell detachment meant that only a single acid-load/recovery cycle was obtained (i.e., either Zn2+ or EGTA, but not both in the same experiment).

Mean values of tmin, {tau}, pHmin, and pHfin before, during, and after the Zn2+ treatment are summarized in Fig. 5. (The "Before" and "After" solutions contained 1 mM EGTA, and so Zn2+ should have been essentially absent.) In considering these mean values, we find the overall impression to be that Zn2+ slows recovery (i.e., increases tmin and {tau}; Fig. 5, A and B) but does not reduce the final level of recovery (pHfin, Fig. 5D). Also the acid load itself is more extreme (i.e., pHmin is lower; Fig. 5C) in the presence of Zn2+. These effects of Zn2+ were reversible (compare Fig. 3, A and B, as well as the Before and After mean values in Fig. 5). To test the statistical significance of these apparent effects, we subjected the data to the model-selection procedure described in the MATERIALS AND METHODS (Eq. 5, ad). This analysis, which is summarized in Table 1, essentially confirms one's subjective impression of the data. Thus Zn2+ produced an approximately twofold increase in tmin and {tau} at both pHo (Fig. 5, A and B). Although the {tau} data at pHo 8.0 in Fig. 5B are fitted almost as well by a model in which there is no effect of Zn2+ (the effect at pHo 7.4 is significant), this was evidently due to variability in the control {tau} values, as can be seen in Fig. 6. If we select those experiments (e.g., Fig. 3) in which pH recovery time constants both in the presence and absence of Zn2+ ({tau}Zn and {tau}EGTA, respectively) were obtained in the same experiment, then a clear effect of Zn2+ at pHo 8.0 emerges. For these paired data, {tau}Zn and {tau}EGTA were significantly correlated (Fig. 6), which means that the errors on estimates of {tau}Zn/{tau}EGTA can be reduced relative to those obtained from unpaired data. Estimates of {tau}Zn/{tau}EGTA obtained from paired data were significantly greater than unity at both pHo 7.4 (2.34 ± 0.36, n = 4) and pHo 8.0 (2.52 ± 0.40, n = 5).



View larger version (45K):
[in this window]
[in a new window]
 
Fig. 5. Mean values of tmin, {tau}, pHmin, and pHfin for pHi recovery in K-Ringer in the presence and absence of Zn2+ (see Fig. 2 for the meaning of these parameters). Statistical analyses (Table 1 and Fig. 6) showed that Zn2+ significantly increased tmin and {tau} (A and B) and significantly reduced pHmin (C). Zn2+ had no significant effect on pHfin (D). Zn2+ solutions contained 10 µM ZnCl2, whereas Zn2+-free solutions ("Before" and "After") contained 1 mM EGTA. The numbers above each bar show the number of observations; error bars are standard errors; *significant differences at a given pHo in the presence and absence of Zn2+.

 

View this table:
[in this window]
[in a new window]
 
Table 1. Results of the statistical analysis to determine the effects of Zn2+ and pHo on tmin, {tau}, pHmin and pHfin for tissue in K-Ringer

 


View larger version (10K):
[in this window]
[in a new window]
 
Fig. 6. The pHi recovery time constants in the presence and absence of Zn2+ ({tau}Zn and {tau}EGTA, respectively) were significantly correlated (P < 0.001). The correlation is still significant if the extreme point (*) is omitted (P < 0.02). Each point represents a different experiment. The mean ratios {tau}Zn/{tau}EGTA were 2.34 ± 0.36 and 2.52 ± 0.40 for pHo 7.4 and 8.0, respectively (both significantly greater then unity). Pooling the data for both pHo gave a value 2.44 ± 0.26.

 
The statistical analysis also confirmed that pHmin is reduced in the presence of Zn2+, at least at pHo 7.4, and there was a clear effect of pHo, such that pHmin was higher at pHo 8.0 than at pHo 7.4 (Fig. 5C). There was no statistically significant effect of Zn2+ on pHfin, i.e., pHi recovered to about the same level in the presence or absence of Zn2+ (albeit over a longer time course in the presence of Zn2+). As one might expect, pHfin was significantly higher at pHo 8.0 than at pHo 7.4 (Fig. 5D).

The outcome of statistical analysis of the effects of Zn2+ and pHo on acid loading and pHi recovery is illustrated by the graphs in Fig. 7, A and B, which shows plots of pHi vs. t predicted by the best-fit models in Table 1 (see Fig. 7 legend for details). The curves in Fig. 7A illustrate that Zn2+ caused a similar relative slowing of recovery (increase in {tau}) at both pHo 7.4 and pHo 8.0 but had no significant effect on the final level of pH recovery (pHfin). In Fig. 7B the early portions of the curves in Fig. 7A are shown on an expanded time scale. The increased acid load (lower pHmin) and delayed recovery (larger tmin) in the presence of Zn2+ are evident.



View larger version (22K):
[in this window]
[in a new window]
 
Fig. 7. Idealized pHi recovery time courses reconstructed statistically from all of the experimental data. A and B: plots of pHi vs. t predicted by the best-fit models in Table 1. To produce these plots, we generated predicted mean values of tmin, {tau}, pHmin, and pHfin by substituting the parameter values from Table 1 into Eq. 5, a–d. Using these values, expressions for tmin, pHmin, and pHfin derived from Eq. 3 were then solved simultaneously for {tau}1, {Delta}pH1, and {Delta}pH2, and {tau}2 was set equal to {tau}. Finally, pH(0) was set to its mean value, and the curves in A and B were generated with Eq. 3. The curves in A illustrate how Zn2+ caused a similar relative slowing of recovery (increase in {tau}) in K-Ringer at both pHo (7.4 and 8.0) but had no significant effect on the final level of pH recovery (pHfin). In B the early portions of the curves in A are shown on an expanded time scale. The increased acid load (lower pHmin) and delayed recovery (larger tmin) in the presence of Zn2+ are evident. C and D: similar plots for recovery in Ringer (pHo 7.4); for these plots pH(0) was set to its mean value and the values of tmin, {tau}, pHmin, and pHfin were taken from Fig. 10 ("Before" and "After" data were pooled).

 
Maximum H+ fluxes in K-Ringer. The curves in Fig. 7A allow us to calculate a quantity qJH (Fig. 8) that is proportional to the Zn2+-sensitive H+ flux (JH), which we attribute to H+ transport through voltage-gated proton channels (q is the apical surface area to volume ratio of the monolayer {approx}1/monolayer thickness). qJH was calculated as

(8)



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 8. The quantity qJH was calculated from the curves in Fig. 7, A and C, according to Eq. 8. JH is the Zn2+-sensitive transapical membrane H+ efflux (considered negative), and q is the apical surface area to volume ratio ({approx}1/monolayer thickness). Calculations were restricted to times t > 3{tau}1 to ensure that dpHi/dt reflects mainly the proton efflux. KR, solid curves; R, dashed curve.

 
where (dpHi/dt)Zn and (dpHi/dt)EGTA are the rates of change of pHi in the presence and absence of Zn2+ respectively, and Bi is the (pHi-dependent) intracellular buffer capacity. In this formulation, proton efflux is a negative quantity. To employ Eq. 8, (dpHi/dt)Zn and (dpHi/dt)EGTA were determined at the same pHi, and calculations were restricted to times t > 3{tau}1 (see Eq. 3) so as to be clear of the pHi minimum (so that changes in pHi reflect JH rather than NH3 and NH4+ fluxes). Values of Bi were taken from Fig. 1 in Lubman and Crandall (26). Although these values pertain to 22°C rather than 37°C, we are really only seeking an order of magnitude calculation here. In Fig. 8 qJH is plotted against pHi (solid curves). It can be seen that at both pHo, the maximum qJH approaches 2 mM/min. If one assumes a monolayer thickness of 1–5 µm (i.e., q = 0.2–1/µm), this implies a maximum equivalent proton-current density across the apical membrane of 3–16 fA/µm2. This calculation assumes that 1) the JH observed in the presence of Zn2+ persists unaltered and simply adds to JH when Zn2+ is removed and 2) the rate of production of H+ by metabolism is the same in the presence and absence of Zn2+.

Boyarsky et al. (4, 5) concluded that the high K+/nigericin calibration technique leads to overestimates of pHi. As discussed in the MATERIALS AND METHODS, this error does not affect the conclusions regarding tmin, {tau}, pHmin, and pHfin but would affect the estimates of qJH. From Boyarsky et al. (5) the true value of qJH is given by

(9)

where qJH,nig is the value of qJH calculated under the assumption that the high-K+/nigericin calibration technique is unbiased, and pHerror is the amount by which pHi is overestimated. Assuming pHerror = 0.0–0.3 (5), the calculated maximum current density should be increased by a factor of 1–2. So instead of 3–16 fA/µm2 as calculated above, a reasonable range might be 3–30 fA/µm2. Assuming a membrane capacitance of 1 µF/cm2, we can translate this to a normalized H+ current (IH) = 0.3–3 pA/pF. As a frame of reference, IH is typically 10–20 pA/pF during large depolarizations in rat alveolar epithelial cells over a wide pH range (9). The IH required to produce the pHi recovery in this study thus requires activating only a small fraction of the maximum available H+ conductance.

SCH-28080, bafilomycin A1, Zn2+, and 4,4'-dibenzamidostilbene-2,2'-disulfonic acid did not prevent pHi recovery in K-Ringer. As a precaution, we routinely included 100 µM SCH-28080 in the K-Ringer recovery solutions to inhibit any H+/K+-ATPase activity (37). In a subset of experiments this inhibitor was omitted. No effect of SCH-28080 was detected (Table 2), suggesting an absence of H+/K+-ATPase activity in rat alveolar epithelial cells. This corroborates the results of Kemp et al. (24), who found evidence for an H+/K+-ATPase in type II pneumocytes of guinea pig but not those of rat. The K-Ringer also contained 100 nM bafilomycin A1, a specific inhibitor of V-type H+-ATPases. Despite the presence of 100 nM bafilomycin A1, 10 µM ZnCl2, and no external Na+ (hence no Na+/H+ exchange), pHi recovery still occurred following an acid load. As pointed out by Richard D. Vaughan-Jones (personal communication), the presumed membrane depolarization in K-Ringer would lead to an accumulation of intracellular Cl, which might then result in pHi recovery via Cl/OH exchange. To test whether this mechanism might contribute under the present conditions, we conducted a separate set of experiments with the anion-transporter inhibitor 4,4'-dibenzamidostilbene-2,2'-disulfonic acid (DBDS, Ref. 38). Adding 0.2 mM DBDS to the recovery solution containing all other inhibitors (K-Ringer containing 100 nM bafilomycin A1, 100 µM SCH-28080, 10 µM ZnCl2, and 1.5 µM valinomycin) had no significant effect on pHi recovery (Table 3). Given the variability of the data, we cannot exclude a small effect, but it is clear that cells still recovered from an acid load in the presence of DBDS. Hence the residual pHi recovery is apparently not primarily associated with a DBDS-sensitive anion transporter.


View this table:
[in this window]
[in a new window]
 
Table 2. H+/K+-ATPase inhibitor SCH-28080 had no detectable effect on {tau} or pHfin in the absence of Zn2+, or on pHmin

 

View this table:
[in this window]
[in a new window]
 
Table 3. Anion-transport inhibitor DBDS had no detectable effect on pHi recovery following an NH4Cl prepulse

 
pHi recovery in Ringer with normal [K+]o. To test the possibility that the residual (Zn2+-insensitive) pHi recovery was associated with elevated external K+ (e.g., a K+/H+ exchanger; Ref. 2), we conducted experiments in Ringer (pHo 7.4) using 100 µM DMA to block the Na+/H+ exchanger (26, 28). The Ringer solution also contained 100 nM bafilomycin A1 and 100 µM SCH-28080 (but no valinomycin). It was also of interest to examine the effects of 10 µM ZnCl2 in this more physiological medium. If the resting membrane potential were more negative in Ringer, this would be expected to decrease Zn2+-sensitive JH via voltage-gated H+ channels (9). As usual, Zn2+-free Ringer contained 1 mM EGTA plus an additional 1 mM CaCl2. Fig. 9 shows example pHi records with ZnCl2 applied before (Fig. 9A) or after (Fig. 9B) the EGTA control. Surprisingly, 10 µM Zn2+ slowed recovery in Ringer solution. As in K-Ringer, recovery still occurred in Ringer despite addition of the entire gamut of inhibitors.



View larger version (24K):
[in this window]
[in a new window]
 
Fig. 9. Two experiments (A and B) in which the tissue was bathed in Ringer (pHo 7.4) containing 0.1 mM 5-(N,N-dimethyl)amiloride (DMA), 0.1 µM bafilomycin A1, and 0.1 mM SCH-28080. In both cases, 2 successive acid-load/recovery cycles were achieved, one with Zn2+, the other without. The order of the treatments is reversed in B. The data were smoothed with a Fourier smoother (unsmoothed data were used for curve fitting, as in Fig. 2).

 
Figure 10 summarizes the mean values of tmin, {tau}, pHmin, and pHfin for experiments in Ringer solution. As with the K-Ringer data, these mean values were used to generate the pH(t) plots in Fig. 7, C and D, and the qJH plot (dashed curve) in Fig. 8 (see legends to Figs. 7 and 8 for details). The overall impression is that recovery in Ringer is generally similar to recovery in K-Ringer at pHo 7.4, both in kinetics and effects of Zn2+ (compare Figs. 5 and 10; Fig. 7, A, B and C, D, and the pHo 7.4 curves in Fig. 8). One difference was that Zn2+ had no detectable effect on pHmin in Ringer. As with K-Ringer, there was no effect of Zn2+ on pHfin (Fig. 10D). One-way ANOVA, as well as analysis of paired data, shows that Zn2+ significantly increased both tmin (Fig. 10A) and {tau} (Fig. 10B), although the slowing of {tau} was less profound than in K-Ringer. In summary, the effects of Zn2+ were less pronounced in normal-Na+ Ringer solution, but both the Zn2+-sensitive and Zn2+-insensitive components of pHi recovery following an acid load still occurred.



View larger version (37K):
[in this window]
[in a new window]
 
Fig. 10. Mean values of tmin, {tau}, pHmin, and pHfin in the presence and absence of Zn2+ for tissue bathed in Ringer plus 0.1 mM DMA, 0.1 µM bafilomycin A1, and 0.1 mM SCH-28080. One-way ANOVA showed that Zn2+ significantly increased tmin and {tau} (* in A and B) but had no significant effect on pHmin and pHfin (C and D). However, large standard errors on at least 2 of the pHmin estimates may have precluded detection of a Zn2+ effect (C). Zn2+ solutions contained 10 µM ZnCl2 while Zn2+-free solutions (Before and After) contained 1 mM EGTA. The numbers above each bar show the number of observations; error bars are standard errors.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
pHi in unchallenged alveolar epithelial cells. The average baseline pHi was 7.13, within the lower range of values in previous studies of cultured rat alveolar type II epithelial cells in nominally HCO3-free solutions, 7.17–7.36 (6, 19, 36); a higher value of 7.50 has also been reported (25, 28).

Given the strong regulation of their gating by pH, one would predict that proton channels would not be open under resting conditions. Voltage-gated proton channels open only positive to the Nernst potential for H+ (EH) (9, 11), with the result that they always extrude acid. Because EH is normally positive to the resting membrane potential in alveolar epithelial cells (10), proton channels would be expected to be closed in an unchallenged cell. The data confirm this expectation. Addition of 10–100 µM ZnCl2 had no effect on the baseline pHi. Although a variety of alternative explanations could be suggested, the simplest interpretation is that proton channels are not open under resting conditions.

Zn2+-sensitive pHi recovery: voltage-gated proton channels. Several types of evidence support the conclusion that voltage-gated proton channels contribute to pHi recovery after acid loading of rat alveolar epithelial cells. In Ringer and K-Ringer, respectively, pHi recovery was slowed on average 1.50-fold and 2.44-fold by the addition of 10 µM ZnCl2 (Figs. 5B, 6, and 10B). Although Zn2+ has effects on many proteins, including many ion channels, it is a potent inhibitor of voltage-gated proton channels. At pH ≥7, 10 µM Zn2+ severely inhibits proton currents (8) but has relatively little effect on several other ion channels (11), including voltage-gated K+ channels in type II cells (V. V. Cherny and T. E. DeCoursey, unpublished) and cGMP-activated cation channels in type II cells (23). We thus attribute the slowing of pHi recovery by ZnCl2 to inhibition of proton channels. These effects of Zn2+ were observed under conditions designed to prevent the operation of other pH regulating transporters. Two more subtle effects of Zn2+ also suggest the involvement of the proton conductance. The time to reach the pHi nadir (tmin) increased (Figs. 5A and 10A), and, at least in K-Ringer, pHmin was lower in the presence of Zn2+ (Fig. 5C). Both of these effects indicate that the proton conductance was activated rapidly, before tmin was reached. The effects of Zn2+ were reversible; t-tests on parameter estimates obtained before and after treatment with Zn2+ were never significant. Finally, the H+ efflux was substantially larger at a given pHi when pHo was 8.0 than 7.4 (Fig. 8), which is consistent with the well-established pHo dependence of proton channels (9, 11). A qualitatively similar effect of pHo would also be expected for any transporter that is driven by the pH gradient. In summary, the data strongly support the interpretation that the Zn2+-inhibitable component of pHi recovery is mediated by voltage-gated proton channels.

Zn2+ had no significant effect on pHfin (Figs. 5D and 10D). That is, although Zn2+ slowed recovery, it did not alter the final level of recovery. This is not surprising given the effects of transmembrane pH gradients on H+ channel gating and the lack of effect of Zn2+ on unchallenged cells (Fig. 4). H+ channels open only at membrane potentials positive to EH, and the threshold voltage (Vthreshold) for opening is described by the following empirical relation (9)

(10)

where {Delta}pH = pHo – pHi. For the K-Ringer experiments in the absence of Zn2+, pHfin was 7.040 ± 0.040 (n = 21) and 7.456 ± 0.051 (n = 14) at pHo 7.4 and 8.0, respectively. So on setting pHi = pHfin, Eq. 10 gives Vthreshold values of 5.6 mV and –1.8 mV at pHo 7.4 and 8.0, respectively. Thus at pHfin and if it is assumed the cells were depolarized to near zero, any contribution of voltage-gated proton channels to H+ efflux will be small, even in the absence of Zn2+. In other words, as H+ efflux increases pHi, the resulting dissipation of the pH gradient shuts off the proton conductance. Accordingly, pHfin will be determined mainly by any Zn2+-insensitive H+ transport and the rate of production of H+ by metabolism. For the same reasons, proton channels do not contribute to pHi in resting, unchallenged cells (Fig. 4); Zn2+ did not change baseline pHi in cells in Ringer solution. Proton channels are expected to come into play when cells are challenged by periods of intense metabolic activity, membrane depolarization, acid loading, or other stressful conditions. Thus we cannot blithely extend the conclusion regarding proton channel activity to the in vivo situation, because the environment of cultured alveolar epithelial cells in these experiments differs radically from that in vivo, in which there is continuous CO2 flux as well as a large pH gradient across the epithelium.

The Zn2+-sensitive component of pHi recovery (Fig. 8) represents proton currents of ~0.3–3 pA/pF or less. Is this flux consistent with known properties and magnitude of the voltage-gated proton conductance? From whole cell patch-clamp studies, the expected normalized IH is given to a first approximation by

(11)

where V is the membrane potential, R is the universal gas constant, T is the absolute temperature, F is Faraday’s constant, a {approx} 40 mV, b {approx} –40 mV/pH, the slope factor Vs {approx} 10 mV, and the normalized maximum H+ conductance (GH,max) is ~100 pS/pF at 20°C (9). GH,max at 37°C is at least 3.5 times larger (14). Assuming {Delta}pH {approx} 1 and V = 0 in K-Ringer, Eq. 11 predicts a proton current, IH {approx} RTGH,max/F {approx} 10 pA/pF, under "typical" conditions at the peak of the experimental acid load. The Zn2+-sensitive IH calculated above from the observed rate of pHi change (Fig. 8) is smaller than this value. However, it is necessary to consider the dynamic nature of events during the acid load and recovery processes. Before the acid load, the K+ conductance may successfully clamp the membrane potential near 0 mV. However, when pHi drops to pHmin, EH will shift to –53 mV (for pHo 7.4) or –77 mV (for pHo 8.0), and proton channels will open. In a nonvoltage-clamped cell in vivo, any IH will tend to drive the membrane potential toward EH, and thus the proton current is in a sense self-limiting. If no other electrogenic processes intervene, the resting potential during recovery will fall between the Nernst potentials for K+ and H+. Because these two conductances are of similar magnitude (10), they will compete for the privilege of controlling the resting potential. As pHi recovers, {Delta}pH will dissipate, decreasing the open probability of H+ channels and removing the driving force for H+ current.

The effects of 10 µM Zn2+ strongly implicate voltage-gated proton channels in pHi recovery from an acid load in rat type II alveolar epithelial cells in K-Ringer. Surprisingly, Zn2+ inhibited pHi recovery in Ringer solution, although to a lesser extent than it did in K-Ringer. One would expect the cells to be depolarized in K-Ringer, but hyperpolarized in Ringer. Because hyperpolarization decreases the open probability of voltage-gated H+ channels, one would expect a smaller IH in Ringer than in K-Ringer. Two early estimates of the resting membrane potential of rat and rabbit alveolar type II epithelial cells are –27 mV (7) and –63 mV (18), respectively, based on K+ or Rb+ distribution. However, the assumption that the membrane potential is equivalent to the K+ gradient neglects the fact that the voltage-gated K+ channels identified in rat alveolar epithelial type II cells are predominantly Kv1.3 (20), which open only with depolarization above roughly –40 mV (15, 34). If Kv1.3 channels set the resting membrane potential, then it is likely to be in the vicinity of –30 to –40 mV. Then, if one assumes {Delta}pH {approx} 1, Eq. 11 gives IH = 0.04–0.15 pA/pF, much less than calculated above for V = 0. Yet Fig. 8 indicates that the Zn2+-sensitive JH (qJH ~ IH) was similar in Ringer and K-Ringer at pHo 7.4. These calculations are based on whole cell patch-clamp studies; conceivably the gating of H+ channels in intact cells might be different. This raises the intriguing possibility that voltage-gated proton channels may be active in these cells under a wider range of conditions than previously supposed. A more mundane explanation is that the membrane potential may have become depolarized during the acid-loading procedure in Ringer solution. Because decreasing pHi inhibits many ion channels including K+ channels (16, 29), some depolarization in response to decreased pHi would not be surprising. Finally, part of the explanation must be the hyperpolarization produced by proton current at low pHi, as discussed above. These questions should be addressed in future studies using membrane potential-sensitive probes and kinetic modeling.

Zn2+-insensitive pHi recovery. To isolate the contribution of proton channels to pHi regulation in alveolar epithelial cells, we created conditions to eliminate other transporters that might influence pH, especially those that might contribute to cytoplasmic alkalinization. The absence of Na+ in K-Ringer prevents Na+/H+-antiport, at least in its normal mode of operation. Bafilomycin A1 was used to inhibit the plasma membrane H+-ATPase (28). We included SCH-28080 (37) to inhibit any possible H+-K+-ATPase activity, although this drug had no detectable effect under our conditions. This observation is consistent with the report that H+-K+-ATPase activity can be detected in guinea pig, but not rat, alveolar epithelium (24). We used nominally HCO3- and CO2-free conditions to avoid HCO3 transport. The Cl/HCO3 exchanger (33) and Cl/OH exchange (38) both normally acidify the cytoplasm and thus would not normally contribute to produce recovery from an acid load. However, if intracellular Cl were elevated, for example due to depolarization of the membrane potential by the high [K+] in K-Ringer, then reverse operation could conceivably produce alkalinization using environmental HCO3 or OH as a substrate. To circumvent this possibility, we also added DBDS, which inhibits anion exchangers in general and Cl/OH exchange specifically (38). Finally, to minimize any K+/H+ exchange (2), we monitored pHi recovery in Ringer plus DMA (to inhibit Na+/H+ antiport). Yet, even when all known transporters were inhibited or prevented from working, pHi recovery was not prevented. Recovery was slow, with an average time constant of ~20 min in K-Ringer, but complete recovery still occurred.

The mechanism for this residual slow alkalinization is not known. Incomplete inhibition of channel-mediated H+ efflux by Zn2+ is unlikely. Zn2+ inhibits H+ currents by shifting Vthreshold to more positive voltages. The shift of Vthreshold by 10 µM Zn2+ would be 59 mV at pHo 7.4 and 66 mV at pH 8.0 (8). Given the values of pHmin (Figs. 5C and 10C), {Delta}pH = 1.0 and 1.3 at pHo 7.4 and 8.0, respectively. From Eq. 10, the corresponding values of Vthreshold are –20 and –32 mV. Hence Vthreshold would be shifted by 10 µM Zn2+ to +39 mV at pHo 7.4 and +34 mV at pH 8.0. Assuming that the high [K+]o clamped the membrane potential close to 0 mV, Vthreshold for activating the proton conductance would be well positive to the membrane potential, and hence little IH should occur. Significant H+ permeation through the lipid bilayer is also exceedingly unlikely given the small, pH-independent leakage current measured in these cells (13). Because Cl was present at ~160 mM in both Ringer and K-Ringer, a recovery mechanism involving Cl cannot be ruled out, although any such mechanism was apparently insensitive to DBDS.

Conclusions. Voltage-gated proton channels in rat alveolar epithelial cells contribute to pHi recovery after an acid load in normal and high-[K+]o solutions. Slow recovery still occurred after all known transporters were inhibited, suggesting the existence of a yet-unidentified acid extrusion mechanism. Whether this mysterious transporter is the same as that deduced by Joseph et al. (22) is an open question. Zn2+ does not change resting pHi, indicating that proton channels are closed under resting conditions, presumably because the membrane potential is negative to EH. The classical property of proton channels opening only with an outward electrochemical gradient for protons ensures that there is no proton influx under normal conditions; the fundamental problem of pH regulation is acid extrusion.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported in part by Heart, Lung, and Blood Institute Grants HL-52671 and HL-61437 (to T. E. DeCoursey).


    ACKNOWLEDGMENTS
 
The authors thank Tatiana Iastrebova for excellent technical assistance.


    FOOTNOTES
 

Address for reprint requests and other correspondence: T. E. DeCoursey, Dept. of Molecular Biophysics and Physiology, Rush Univ. Medical Center, 1750 W. Harrison, Chicago, IL 60612 (E-mail: tdecours{at}rush.edu)

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.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Adamson TM, Boyd RDH, Platt HS, and Strang LB. Composition of alveolar liquid in the foetal lamb. J Physiol 204: 159–168, 1969.[ISI][Medline]
  2. Attmane-Elakeb A, Boulanger H, Vernimmen C, and Bichara M. Apical location and inhibition by arginine vasopressin of K+/H+ antiport of the medullary thick ascending limb of rat kidney. J Biol Chem 272: 25668–25677, 1997.[Abstract/Free Full Text]
  3. Bevensee MO, Bashi E, and Boron WF. Effect of trace levels of nigericin on intracellular pH and acid-base transport in rat renal mesangial cells. J Membr Biol 169: 131–139, 1999.[CrossRef][ISI][Medline]
  4. Boyarsky G, Hanssen C, and Clyne LA. Inadequacy of high K+/nigericin for calibrating BCECF. I. Estimating steady-state intracellular pH. Am J Physiol Cell Physiol 271: C1131–C1145, 1996.[Abstract/Free Full Text]
  5. Boyarsky G, Hanssen C, and Clyne LA. Inadequacy of high K+/nigericin for calibrating BCECF. II. Intracellular pH dependence of the correction. Am J Physiol Cell Physiol 271: C1146–C1156, 1996.[Abstract/Free Full Text]
  6. Brown SES, Heming TA, Benedict CR, and Bidani A. ATP-sensitive Na+-H+ antiport in type II alveolar epithelial cells. Am J Physiol Cell Physiol 261: C954–C963, 1991.[Abstract/Free Full Text]
  7. Castranova V, Jones GS, and Miles PR. Transmembrane potential of isolated rat alveolar type II cells. J Appl Physiol 54: 1511–1517, 1983.[Abstract/Free Full Text]
  8. Cherny VV and DeCoursey TE. pH-dependent inhibition of voltage-gated H+ currents in rat alveolar epithelial cells by Zn2+ and other divalent cations. J Gen Physiol 114: 819–838, 1999.[Abstract/Free Full Text]
  9. Cherny VV, Markin VS, and DeCoursey TE. The voltage-activated hydrogen ion conductance in rat alveolar epithelial cells is determined by the pH gradient. J Gen Physiol 105: 861–896, 1995.[Abstract]
  10. DeCoursey TE. Hydrogen ion currents in rat alveolar epithelial cells. Biophys J 60: 1243–1253, 1991.[Abstract]
  11. DeCoursey TE. Voltage-gated proton channels and other proton transfer pathways. Physiol Rev 83: 475–579, 2003.[Abstract/Free Full Text]
  12. DeCoursey TE and Cherny VV. Voltage-activated proton currents in membrane patches of rat alveolar epithelial cells. J Physiol 489: 299–307, 1995.[Abstract]
  13. DeCoursey TE and Cherny VV. Deuterium isotope effects on permeation and gating of proton channels in rat alveolar epithelium. J Gen Physiol 109: 415–434, 1997.[Abstract/Free Full Text]
  14. DeCoursey TE and Cherny VV. Temperature dependence of voltage-gated H+ currents in human neutrophils, rat alveolar epithelial cells, and mammalian phagocytes. J Gen Physiol 112: 503–522, 1998.[Abstract/Free Full Text]
  15. DeCoursey TE, Jacobs ER, and Silver MR. Potassium currents in rat type II alveolar epithelial cells. J Physiol 395: 487–505, 1988.[Abstract]
  16. Deitmer JW and Rose CR. pH regulation and proton signalling by glial cells. Prog Neurobiol 48: 73–103, 1996.[CrossRef][ISI][Medline]
  17. Effros RM and Chinard FP. The in vivo pH of the extravascular space of the lung. J Clin Invest 48: 1983–1996, 1969.[ISI][Medline]
  18. Gallo RL, Finkelstein JN, and Notter RH. Characterization of the plasma and mitochondrial membrane potentials of alveolar type II epithelial cells by the use of ionic probes. Biochim Biophys Acta 771: 217–227, 1984.[ISI][Medline]
  19. Gerboth GD, Effros RM, Roman RJ, and Jacobs ER. pH-induced calcium transients in type II alveolar epithelial cells. Am J Physiol Lung Cell Mol Physiol 264: L448–L457, 1993.[Abstract/Free Full Text]
  20. Grunnet M, Rasmussen HB, Hay-Schmidt A, and Klaerke DA. The voltage-gated potassium channel subunit, Kv1.3, is expressed in epithelia. Biochim Biophys Acta 1616: 85–94, 2003.[ISI][Medline]
  21. Grynkiewicz G, Poenie M, and Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260: 3440–3450, 1985.[Abstract]
  22. Joseph D, Tirmizi O, Zhang XL, Crandall ED, and Lubman RL. Polarity of alveolar epithelial cell acid-base permeability. Am J Physiol Lung Cell Mol Physiol 282: L675–L683, 2002.[Abstract/Free Full Text]
  23. Kemp PJ, Kim KJ, Borok Z, and Crandall ED. Re-evaluating the Na+ conductance of adult rat alveolar type II pneumocytes: evidence for the involvement of cGMP-activated cation channels. J Physiol 536: 693–701, 2001.[Abstract/Free Full Text]
  24. Kemp PJ, Roberts GC, and Boyd CA. Identification and properties of pathways for K+ transport in guinea-pig and rat alveolar epithelial type II cells. J Physiol 476: 79–88, 1994.[Abstract]
  25. Lubman RL and Crandall ED. Na+-HCO3 symport modulates intracellular pH in alveolar epithelial cells. Am J Physiol Lung Cell Mol Physiol 260: L555–L561, 1991.[Abstract/Free Full Text]
  26. Lubman RL and Crandall ED. Polarized distribution of Na+-H+ antiport activity in rat alveolar epithelial cells. Am J Physiol Lung Cell Mol Physiol 266: L138–L147, 1994.[Abstract/Free Full Text]
  27. Lubman RL, Danto SI, Chao DC, Fricks CE, and Crandall ED. Cl-HCO3 exchanger isoform AE2 is restricted to the basolateral surface of alveolar epithelial cell monolayers. Am J Respir Cell Mol Biol 12: 211–219, 1995.[Abstract]
  28. Lubman RL, Danto SI, and Crandall ED. Evidence for active H+ secretion by rat alveolar epithelial cells. Am J Physiol Lung Cell Mol Physiol 257: L438–L445, 1989.[Abstract/Free Full Text]
  29. Moody W. Effects of intracellular H+ on the electrical properties of excitable cells. Annu Rev Neurosci 7: 257–278, 1984.[CrossRef][ISI][Medline]
  30. Negulescu PA and Machen TE. Intracellular ion activities and membrane transport in parietal cells measured with fluorescent probes. Methods Enzymol 192: 38–81, 1990.[Medline]
  31. Nielson DW, Goerke J, and Clements JA. Alveolar subphase pH in the lungs of anesthetized rabbits. Proc Natl Acad Sci USA 78: 7119–7123, 1981.[Abstract]
  32. Nord EP, Brown SES, and Crandall ED. Characterization of Na+-H+ antiport in type II alveolar epithelial cells. Am J Physiol Cell Physiol 252: C490–C498, 1987.[Abstract/Free Full Text]
  33. Nord EP, Brown SES, and Crandall ED. Cl/HCO3 exchange modulates intracellular pH in rat type II alveolar epithelial cells. J Biol Chem 263: 5599–5606, 1988.[Abstract/Free Full Text]
  34. Peers C, Kemp PJ, Boyd CAR, and Nye PCG. Whole-cell K+ currents in type II pneumocytes freshly isolated from rat lung: pharmacological evidence for two subpopulations of cells. Biochim Biophys Acta 1052: 113–118, 1990.[CrossRef][ISI][Medline]
  35. Roos A and Boron WF. Intracellular pH. Physiol Rev 61: 296–434, 1981.[Free Full Text]
  36. Sano K, Cott GR, Voelker DR, and Mason RJ. The Na+/H+ antiporter in rat alveolar type II cells and its role in stimulated surfactant secretion. Biochim Biophys Acta 939: 449–458, 1988.[ISI][Medline]
  37. Scott CK and Sundell E. Inhibition of H+K+ATPase by SCH 28080 and SCH 32651. Eur J Pharmacol 112: 268–270, 1985.[CrossRef][ISI][Medline]
  38. Sun B, Leem CH, and Vaughan-Jones RD. Novel chloride-dependent acid loader in the guinea-pig ventricular myocyte: part of a dual acid-loading mechanism. J Physiol 15: 65–82, 1996.
  39. Thomas JA, Buchsaum RN, Zimniak A, and Racker E. Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry 18: 2210–2218, 1979.[ISI][Medline]
  40. Walpole RE and Myers RH. Probability and Statistics for Engineers and Scientists. New York: Macmillan, 1978, p. 580.




This Article
Abstract
Full Text (PDF)
All Versions of this Article:
288/2/L398    most recent
00299.2004v1
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Murphy, R.
Articles by DeCoursey, T. E.
Articles citing this Article
PubMed
PubMed Citation
Articles by Murphy, R.
Articles by DeCoursey, T. E.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2005 by the American Physiological Society.