1Division of Pulmonary and Critical Care Medicine, Department of Medicine, Medical College of Wisconsin, Milwaukee 53226; 2Zablocki Veterans Affairs Medical Center, Milwaukee 53295; and 3Department of Biomedical Engineering, Marquette University, Milwaukee, Wisconsin 53233
Submitted 18 November 2002 ; accepted in final form 31 March 2003
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ABSTRACT |
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air space acidification; intracellular pH; ammonium; bicarbonate; acetate
Joseph et al. (9) used a
fluorescent pH sensitive dye,
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), to
conduct studies of intracellular pH on alveolar epithelial cell monolayers.
These monolayers were bathed with a nonbicarbonate solution buffered with 6 mM
HEPES. Replacement of apical fluid with acidic (pH 6.4) or basic (pH 8.0)
solutions had little effect on intracellular pH. In contrast, changes in
basolateral fluid pH caused rapid responses in intracellular pH. Intracellular
alkalinization was blocked 80% by dimethyl amiloride, an inhibitor of the
Na+/H+ exchanger. No measurements were provided for the
movement of specific buffers such as HCO3- across the
apical membranes, leaving the possibility that alteration of air space pH
could result in rapid changes in cellular pH in vivo. Furthermore, movement of
HEPES buffer across basolateral membranes was not ruled out.
We have investigated whether the polarity in permeability of the apical cells to the movement of acid-base equivalents observed in vitro can be demonstrated in intact lungs. The pH of the air space and cellular compartments of the lung were monitored with a surface fluorescence approach modified from that introduced by Carter et al. (4). These studies support the conclusions of Joseph et al. (9), by showing that the apical membranes of the pulmonary epithelium are less permeable to ionized buffers than the basolateral surfaces. The apical barrier presumably permits maintenance of relatively acid fluid in the air space compartment and may also serve to protect the underlying pulmonary tissues from transient acid challenges.
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METHODS |
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The protocol was approved by the Animal Studies Subcommittee (Institutional Animal Care and Use Committee) of the Zablocki Veterans Affairs Medical Center.
Fluid-filled lungs (air space pH). Sprague-Dawley rats (n
= 26, 390 ± 71 g body wt) were anesthetized with an intraperitoneal
injection of 0.7 ml of a 50 mg/ml pentobarbital sodium solution. The chest was
opened, and catheters were placed in the trachea, pulmonary artery, and left
atrium. Blood was flushed from the vasculature at 37°C with 20 ml of
perfusion fluid (see below), and the air spaces were filled with 5 ml of
perfusate containing 0.2 mg/ml FITC-dextran (mol wt 2 x 106)
or 0.1 µg/ml BCECF. The lungs were then perfused at 10 ml/min for 1 h.
A trifurcated fiber-optic bundle (Oriel) was placed against the surface of the
lungs (Fig. 1). The surface of
the lungs was alternately illuminated at 2-s intervals with monochromatic
excitation beams at wavelengths of 490 and 420 nm, and the emission from the
surface of the lungs was monitored at 515 nm. When BCECF was used, the lungs
were illuminated alternately at 490 and 440 nm, and emission was monitored at
515 nm. The change in excitation wavelength was accomplished by alternately
opening the shutters of two monochromatic light sources at these wavelengths
at 2-s intervals. The fluorescent signals were monitored with a
photomultiplier (Hamamatsu), the output of which was amplified, filtered, and
recorded on a spreadsheet in a computer. No change in the drift of the
fluorescence was observed when the light source was kept off during most of
the experiment, indicating that exposure to light did not affect air space pH
measurements.
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Air-filled lungs (intracellular pH). In these experiments, the lungs were inflated with air at a constant pressure of 5 cmH2O, and the lungs were perfused for 10 min with a solution of 2 µg/ml BCECF-AM. Perfusate containing 20 mM NH4Cl was infused for 2 min as described above.
Perfusion Solutions
The lungs were perfused with solutions containing the following solutes: 30 g/dl dextran (mol wt 7 x 104), 1 g/l glucose, 118 mM NaCl, 4.7 mM KCl, 1.1 mM KH2PO4, 2.5 mM CaCl2, and 1.15 mM MgSO4. In all but one group of experiments, 5 mM HEPES (pKa = 7.55) and 5 mM MES (pKa = 6.15) were included in the solution. This mixture of buffers provided quite linear buffering capacity over the range of pH values that were observed in the air spaces of these experiments (Fig. 2A). The amount of buffer was intentionally increased in one group of experiments to 25 mM HEPES. As indicated in Fig. 2, this solution was better buffered than the HEPES-MES solution. Although the buffer curve was more curvilinear, it was fairly linear between pH 7.4 and 7.0, values encountered in these experiments. The pH of the perfusion solutions was adjusted to 7.4 with small volumes of 1 N NaOH.
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Calculation of Alveolar pH
When fluorescein-dextran (pKa = 6.4) or BCECF
(pKa = 6.98) is excited at 490 nm, the emission at 515 nm
decreases as pH decreases. In contrast, emission from FITC-dextran is
insensitive to pH when it is excited at 420 nm, and emission of BCECF is
insensitive to pH when it is excited at 440 nm. As indicated in Figs.
3,
4,
5, the excitation signal at 490
nm exceeded that at 420 nm, making it possible to distinguish the emission
signals at these two wavelengths. The lung was illuminated alternately at 490
and 420 nm for 2-s intervals, and the recorded fluorescent signals were
interpolated to provide values every 2 s. The voltage of the signal at 490 nm
was then divided by that at 420 nm at each point along the curve to yield the
fluorescence ratio (R). R fell as pH decreased. R0 at the beginning
of each experiment was assumed to be representative of pH 7.4, because the pH
of the FITC-dextran solution that was originally instilled into the lungs was
7.4. Similarly, the fluorescence ratio at the end of the experiment
(Rend) was assumed to be representative of the pH that was measured
in the air space fluid recovered from the trachea at the end of the
experiment. A linear relation was assumed between fluorescence and air space
pH between the beginning and end of the studies, and the values of air space
pH were calculated from the ratios on the basis of the linear equation
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Selection of the maximum pH changes after each of the infusions of weak acids and bases, hypotonic solutions, and FITC-dextran was facilitated by using the PeakFit program (Jandel).
Detection of Directional Changes in Intracellular pH
BCECF-AM is the esterified, nonfluorescent form of BCECF. BCECF-AM is lipophilic and readily crosses cell membranes. Once inside the cells, it is rapidly deesterified to BCECF, which is fluorescent and lipophobic. It remains trapped within the cells, where it can be used to monitor changes in intracellular pH. Standardization of intracellular pH with this dye in isolated cell preparations is conventionally accomplished by treating the cells with nigericin and 100150 mM K+, which equalizes intracellular and extracellular pH. It was not possible to perfuse lungs with these solutions without disrupting the pulmonary vasculature. Although absolute values of intracellular pH could not be calculated, directional changes in pH after injections of NH4Cl could be determined by following changes in fluorescence of lungs that had been loaded with BCECF-AM. Characteristically, NH4Cl challenges result in an initial alkalinization for the cell, which is followed by acidification. Studies of the ability of the cells to extrude excess acid are based on the rate at which pH is restored to normal. For this purpose, the fluorescence of the lungs was plotted on a logarithmic ordinate against the time elapsed, and the slope of the regression of the return of the signal to baseline levels was calculated.
Chemical Analysis
Na+, K+, and Cl- concentrations were measured with ion-specific electrodes. Glucose concentrations were determined by the glucose oxidase Trinder method (catalog no. 315, Sigma, St. Louis, MO), and lactic acid was determined with lactic dehydrogenase (catalog no. 726-UV/826-UV, Sigma). These determinations were made in air space and perfusate samples at the beginning and end of the studies.
Statistical Analysis
The significance of differences between mean values was calculated with a one-way analysis of variance, and Tukey's test was used to compare individual mean values.
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RESULTS |
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Changes in baseline fluorescence, pH, and solute concentrations of the air space compartment. There was initially a tendency for baseline fluorescence to increase when the lungs were excited at 490 nm, followed by a decline in baseline values (Figs. 3, 4, 5). Baseline fluorescence at 420 nm consistently increased in the FITC-dextran but not the BCECF experiments. Calculated, ratiometric values of pH invariably fell. Direct measurements of the pH of the air space fluid indicated that air space pH fell from 7.40 to 6.61 ± 0.03 (mean ± SE) over the course of the 1 h when the buffer in the solutions was kept low (5 mM HEPES-5 mM MES). This is equivalent to an increase of 2.02 ± 0.07 µeq of H+ in each milliliter of the air space solution. Significantly more H+ was transported in the high-buffer (25 mM HEPES) experiments (P < 0.05). The pH of the air spaces fell from 7.40 to 7.13 ± 0.025 in the high-buffer studies; this is equivalent to an addition of 3.32 ± 0.29 µeq of H+ into each milliliter of the air space fluid. Acidification of the air spaces was not due to accumulation of lactic acid, which remained <1 mM in all but one of these experiments.
K+ concentrations in the air spaces exceeded those in the perfusate by 41 ± 10% at the end of 1 h (P < 0.05). In contrast, no differences were observed in airway and perfusate concentrations of Na+ or Cl-. Glucose concentrations of the air space solution fell to 60 ± 2% of those in the perfusate (P < 0.05).
Response of air space fluorescence and pH to transient infusions of buffers. After a baseline infusion of unlabeled perfusate, the lungs were perfused for 2 min with a total of 20 ml of each solution (Table 1, Figs. 3, 4, 5, 6). Infusions of perfusate containing FITC-dextran increased the emission signal when the lungs were illuminated at 490 and 420 nm. However, the signal at 490 nm decreased when the pH of the injection solutions was decreased. Injections of NH4Cl increased the lung fluorescence at 490 nm but had no effect at 420 or 440 nm, and an increase in air space pH was calculated. This response was greater when the perfusate contained lower concentrations of buffer (5 mM HEPES-5 mM MES, rather than 25 mM HEPES), despite the fact that more NH4Cl was injected in the latter experiments (Fig. 5). Injections of NaHCO3 decreased the fluorescence at 490 nm but had no effect at 420 nm and decreased the calculated air space pH. This effect was less (P < 0.05) when the injection pH was increased from 7.4 to 8.0. Acetazolamide, an inhibitor of carbonic anhydrase, had no effect on air space fluorescence or pH. The effects of sodium acetate injections were similar to those of sodium bicarbonate: fluorescence at 490 nm decreased, but there was no effect at 420 nm, and the calculated air space pH decreased. In all the experiments with NH4Cl, NaHCO3, and sodium acetate, the change in pH was "monotonic," in other words, after the injection fluid had been washed from the vasculature, pH returned to normal without an overshoot in the opposite direction. Furthermore, the effects of these weak acids and bases was less pronounced when the air space fluid contained more buffer (25 mM HEPES, rather than 5 mM HEPES-5 mM MES; cf. Fig. 6; P < 0.05). Injections of acidified perfusate (pH 6.0) had little effect on air space pH. Hypotonic injections decreased the fluorescence of the air space fluid at 490 and 420 nm but had no effect on the air space pH.
Similar observations were made when the extracellular pH indicator BCECF (1 µg/ml), rather than FITC-dextran, was instilled into the air spaces. Infusions of NH4Cl were followed by monophasic increases in the fluorescence at 490 nm, and infusions of NaHCO3 resulted in monophasic decreases in fluorescence (Figs. 4 and 5).
Air-Filled Lungs: Intracellular pH
The intracellular compartments of the isolated lungs were labeled by perfusion with BCECF-AM. Injections of 20 mM NH4Cl for 2 min yielded a "biphasic" response in fluorescence (at 490 nm excitation): fluorescence increased above baseline levels and then decreased below baseline levels after the NH4Cl had been flushed from the lungs (see Fig. 8). No change in fluorescence was observed when the lungs were illuminated at 440 nm, the pH-insensitive wavelength for this indicator. The return of fluorescence to baseline levels after injections of NH4Cl was slowed in each of five studies (by >90% in 4 of 5 experiments, P < 0.05) after the lungs were perfused for 5 min with perfusate containing the Na+-H+ inhibitor dimethyl amiloride at 0.1 mM (Fig. 7).
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DISCUSSION |
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The initial pH of the air spaces fell progressively in these experiments. Acidification of the baseline pH of the air space fluid was initially rapid but subsequently slowed (Figs. 3, 4, 5). This slowing could be due to back-diffusion of acid out of the air spaces. Backdiffusion could also be responsible for the observation that the net movement of H+ into the air spaces was greater when the air spaces were more effectively buffered with 25 mM HEPES than when less buffer was used (5 mM HEPES-5 mM MES). Decreases in air space pH could not be attributed to accumulation of lactic acid in the preparation. There was an increase in K+ of the air space fluid that was consistent with previous observations (1, 2). Secretion of K+ and H+ into the air spaces could play a role in the reabsorption of Na+ from the air spaces (11). Glucose concentrations in the air space fell significantly relative to those in the plasma. This could represent local consumption or transport out of the air spaces (3). Consistent increases in fluorescence of FITC-dextran at 420 nm presumably reflect fluid reabsorption from the air spaces. These increases were not reproducible in the studies with BCECF, which is a smaller molecule that may leak out of the air spaces during the course of the experiments.
Monophasic Response of Air Space pH to Infusions of Acids and Bases (Fluid-Filled Lungs)
Exchange of weak acids and bases across the barriers separating the perfusate and the air spaces was virtually confined to the nonionized forms of the acid-base pairs. For example, alkalinization of the air spaces with infusions of NH4Cl at pH 7.4 was presumably due to the selective diffusion of NH3, rather than NH4+, into the air spaces. The initial alkalinization with NH4Cl has also been reported in individual cells loaded with pH-sensitive dyes (13), but there is an acidic overshoot after the exposure to NH4Cl ends. This was not observed in air space pH, which returned to normal without an acidic overshoot after the NH4Cl injection ended. In this respect, the pH response of the air spaces appears to differ from that of the cellular pH. As indicated in Fig. 7, intracellular pH (as judged from fluorescence after cellular loading with BCECF-AM) initially increased after the injections of NH4Cl but then fell below baseline levels. This overshoot has been attributed to some movement of NH4+ into the cells when they are exposed to NH4Cl (Fig. 8). After the NH4Cl fluid is washed out of the vasculature, NH4+ remaining in the cells dissociates into H+ and NH3. NH3 diffuses out of the cell, leaving excess H+. Excess H+ within the cells are then extruded by transport from the cells. The rate at which intracellular pH returned to normal was significantly slowed when the lungs were perfused with 0.1 mM amiloride, an inhibitor of Na+/H+ exchange. This observation is in accord with the observations of Joseph et al. (9).
Failure to observe an overshoot when the pH-sensitive indicators were
placed in the air spaces suggests that the membranes separating the
vasculature and air spaces are more impermeable to NH4+
than the basolateral surfaces. Because the pKa of
NH4+ is 9.23, the amount of NH4+
in the perfusaste exceeds that of NH3 by a factor of 67 at pH 7.4.
It can therefore be concluded that the barrier is 67 times less permeable
to NH4+ than to NH3.
Infusions of 10 mM sodium acetate (pKa = 5.23) and 25
mM NaHCO3 (pKa = 6.1) acidified the air space
solution, and no alkaline overshoot was observed with either of these
solutions. This again suggests that the nonionized forms of the acid-base
pairs diffuse through the membranes much more rapidly than the ionized forms.
In the case of acetate, the membranes were 148 times more permeable to
acetic acid than to acetate. Acidification of the air spaces with
NaHCO3 was greater at pH 7.4 than 8.0. This presumably reflects the
fact that the concentration of CO2 is 1.2 mM at pH 7.4 and only
0.31 mM at pH 8.0. The observation that, even at pH 8.0, only acidification is
observed indicates that the air space-perfusate barrier must be
79 times
more permeable to CO2 than to HCO3-.
Differences between permeability of the barrier to ionized and neutral forms
of these weak acids are probably much greater than the minimum values
calculated from the fluorescent data. In studies using
14CO2 and H14CO3- with
more extreme changes in pH, it was possible to show that the barrier is
600 times more permeable to CO2 than to
HCO3-
(7). Acetazolamide failed to
cause a significant decrease in acidification of the air spaces. This suggests
that the delivery of CO2 in the perfusate is sufficiently rapid
that CO2 production from HCO3- does not
significantly limit CO2 movement into the air spaces.
DeCoursey (5) recently suggested that movement of CO2 from the blood to the air spaces is facilitated by the secretion of acid across alveolar epithelial cells through H+ channels. This hypothesis assumes that the diffusion of HCO3- into the epithelial lining fluid is rapid compared with that of CO2. The present study and an earlier study with H14CO3- suggest that the transport of HCO3- across the apical membranes of the lung is considerably slower than that of CO2 (6). Any facilitation of CO2 transport by H+ secretion would presumably be modest.
An infusion of only 2 mM NH4Cl resulted in a pH increase of 0.142 unit, whereas 10 mM sodium acetate resulted in a pH decrease of 0.089 unit. In other words, for equal concentrations of NH4Cl and sodium acetate, changes in pH are 8 times greater with the NH4Cl infusions than with sodium acetate. This suggests that the membranes separating the vasculature from the air spaces are more permeable to NH3 than to acetic acid.
Our observations concerning air space pH differed in some respects from those reported by Carter et al. (4) in the original work describing the surface fluorescence in one mouse. They observed a rapid decrease in air space fluorescence after 5-min infusions of perfusate at pH 5.6. Infusion of bicarbonate caused an initial acidification that was followed by alkalinization. Neither of these phenomena were observed in the present study. It is possible that the more prolonged infusion of a more acidic solution resulted in leakage of ions across the epithelium in this species. However, it is difficult to interpret the data, because the lung was illuminated at a single wavelength, and alterations in pH cannot be directly calculated.
Intracellular pH is commonly measured by fluorescent microscopy in individual cells with a variety of indicators. Alternatively, it can be measured in suspensions of cells using conventional fluorometry (13). As indicated in this study, it is also possible to measure changes in compartmental pH (air space and cellular) with these indicators by using an appropriate optical bundle placed on the surface of an organ. This approach permits verification of measurements made in cellular monolayers with observations made in intact organs. The studies of Joseph et al. (9) in monolayers and these studies in intact lungs show that the apical membranes of the lungs are more impermeable to ionized buffers than the basolateral membranes. This may maintain the normally acidic environment in the air spaces and insulate the lung parenchyma from abrupt changes in pH induced by aspiration of small amounts of acid from the lungs or inflammatory cells in the air spaces. These studies are also in accord with earlier studies of the extravascular pH of the lung, which indicated that tissue pH is more closely linked to vascular than to air space pH (6). This barrier presumably fails when larger amounts of acid are aspirated. The present study and the study of Joseph et al. (9) also suggest that the Na+/H+ exchanger mediates extrusion of excess acid from basolateral surfaces of the lung cells.
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ACKNOWLEDGMENTS |
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DISCLOSURES
This work was supported by National Institutes of Health Grants HL-60057 and DC-03191.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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