Exocytosis in alveolar type II cells revealed by cell capacitance and fluorescence measurements

Norbert Mair, Thomas Haller, and Paul Dietl

Department of Physiology, University of Innsbruck, A-6020 Innsbruck, Austria


    ABSTRACT
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Abstract
Introduction
Methods
Results and discussion
References

Measurement of lamellar body (LB) exocytosis at high spatial and temporal resolution was recently enabled by fluorescence of the dye FM 1-43 (FFM1-43). Here, the capabilities of this method were further examined and extended by simultaneous measurement of the cell membrane capacitance (Cm) and laser-scanning confocal microscopy. Step increases in Cm were evoked by extracellular ATP (20 µM) or an elevated pipette Ca2+ concentration (>= 3 µM). The delay between the first Cm step and the increase in FFM1-43 was <1 s, indicating ready access of FM 1-43 to exocytosed LB contents. A specific Cm of 0.88 µF/cm2 for the membrane of an exocytosed LB was calculated. Compound exocytosis was occasionally observed. Decreases in Cm, indicative of transient fusion or endocytosis, did not occur within 20 min of stimulation. Exocytosis was stimulated by 160 µM guanosine 5'-O-(3-thiotriphosphate) in the pipette, but compound exocytosis was unaffected. The comparison of methods revealed that FM 1-43 is ideally suited to measure the onset of exocytosis and amount of secretion. Patch clamp is superior in resolving fusion events with the plasma membrane.

surfactant; patch clamp; endocytosis; compound exocytosis


    INTRODUCTION
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Abstract
Introduction
Methods
Results and discussion
References

SURFACTANT IS SECRETED by type II cells via exocytosis of lamellar bodies (LBs) (see Refs. 4, 14, 19, 24 for reviews). In general, the regulation of secretion involves processes before exocytosis, such as vesicle transport, docking, or priming, but may also include events after membrane fusion, such as fusion pore expansion (18). Despite these various sites of regulation, the fusion of the vesicle membrane with the plasma membrane is a central and discrete step in the course of secretion, and its measurement by patch clamp has greatly improved our knowledge of the cellular and molecular mechanisms herein. In comparison, exocytosis of LBs is poorly understood in the type II cell, partly because measurements of cell membrane capacitance (Cm) have not yet been reported. Hence the type II cell is one of the few remaining secretory cell types where knowledge about exocytosis is essentially derived from biochemical measurements of material released into extracellular solutions. These experiments revealed that surfactant secretion is regulated by various chemical and physical factors, with extracellular ATP being one of the most potent stimulators. One way by which ATP appears to exert its effect is the release of Ca2+ from inositol 1,4,5-trisphosphate-sensitive Ca2+ stores (8). The importance of Ca2+ for surfactant secretion is supported by the findings that it is stimulated by Ca2+ ionophores (7) and that it correlates with the cytoplasmic Ca2+ concentration (17).

We have recently introduced a novel application of the fluorescent dye FM 1-43 to visualize exocytosis of single LBs and to quantify the amount of released surfactant. This method is based on the amphiphilicity of FM 1-43 and its property to emit fluorescent light in lipophilic environments but not in water (reviewed in Refs. 1, 3). Hence, in the continuous presence of FM 1-43 in the extracellular solution, LB contents become highly fluorescent as soon as FM 1-43 gets access to the lipid component of surfactant through the fusion pore. This approach to study exocytosis is quite different from conventional applications of FM 1-43 at synaptic terminals and is thus a modification of the originally described technique (2).

Despite the obvious benefits of this new method compared with conventional measurements of surfactant secretion, there is still little information about FM 1-43 with regard to permeation through fusion pores, diffusion along lipid membranes, or molecular interactions with target molecules (3). To further examine the capabilities of the FM 1-43 technique and to extend our knowledge beyond its present limits, we combined fluorescence microscopy with the whole cell patch-clamp technique. By measuring a step increase in Cm, the patch-clamp technique can be used to clearly define the time of fusion pore formation and the surface of fused vesicles. This should answer questions about how precisely the FM 1-43 fluorescence (FFM1-43) gain reflects the instance and number of fusion events or, conversely, which factors determine the time course of the FFM1-43 gain. In addition, it is yet unknown whether exocytosis of LB contents, i.e., of lipid membranes (in contrast to soluble, hydrophilic granule contents), can be measured as a Cm increase at all. Likewise, there is no information about the specific capacitance of the membrane of an exocytosed LB so far. As outlined below, the simultaneous use of these techniques in single type II cells yielded further information about compound exocytosis (i.e., exocytosis by vesicle-vesicle fusion), endocytosis, and transient fusion. In summary, an integrative view of fusion events and membrane dynamics in response to physiological stimuli is presented.


    METHODS
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Abstract
Introduction
Methods
Results and discussion
References

Cell preparation. Alveolar type II cells were isolated from anesthetized (thiopental sodium) male Sprague-Dawley rats (approx 200 g) according to the procedure by Dobbs et al. (6) as previously outlined (11). In this study, type II cells grown on untreated glass coverslips at low density (40 cells/mm2) were used for the experiment 1 day after isolation from the lungs.

Measurement of FFM1-43. The details were published recently (11). In short, coverslips with the cells were mounted in a perfusion chamber placed on the stage of an inverted microscope equipped for epifluorescence and photometry (10). The cells were rinsed at 25°C with a bath solution (in mM: 140 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 5 glucose, and 10 HEPES, pH 7.4). Exocytosed surfactant was stained in the continuous presence of 1-4 µM FM 1-43 (Molecular Probes) in the nonperfused bath. The number of exocytosed LBs before and after stimulation of the cells was counted from the images taken on-line with a charge-coupled device camera during each patch-clamp experiment, and quantitative analysis of released material was made throughout the experiment by continuously measuring the emitted FFM1-43 with a photomultiplier tube. Excitation light of 475-nm wavelength, directed through a 520-nm dichroic mirror, was applied for 30 ms, followed by 0.45 s of dark, resulting in an illumination rate of approx 2.2 Hz. During illumination, emitted light from a single cell under study was sampled at a rate of 1 kHz and averaged.

Laser-scanning confocal microscopy (LSM) images were acquired with a Zeiss LSM 510 fitted with a Plan-Apochromat ×63/1.4 numerical aperture oil objective. Excitation light (argon-ion laser) was 488 nm, and the emitted light passed through a 585-nm long-pass filter (for FFM1-43) and a 505- to 530-nm band-pass filter [for LysoTracker Green DND-26 (LTG) fluorescence; see Ref. 11]. LTG was used to visualize the LBs before membrane fusion.

Measurement of Cm. Cm measurements were made with an EPC-9 patch-clamp setup (22) with the "sine + dc" method originally described by Lindau and Neher (12). In short, patch pipettes (between 3- and 5-MOmega tip resistance) were made from borosilicate glass and filled with the following control pipette solution (in mM): 135 potassium gluconate, 10 NaCl, 1 MgCl2, 0.1 EGTA, and 10 HEPES, pH 7.3 (with KOH). The "elevated-Ca2+" pipette solutions omitted EGTA and contained either no additional Ca2+, resulting in ~3 µM free Ca2+, or 500 µM Ca2+ with the addition of Ca2+. Because Haller et al. (9) found that surfactant secretion is completely elicited at submicromolar Ca2+ concentrations and both pipette Ca2+ concentrations were equally potent to initiate exocytosis, these data were pooled. When indicated, 160 µM 5'-O-(3-thiotriphosphate) (GTPgamma S) was added to the elevated-Ca2+ (3 µM) pipette solution.

A holding potential of -60 mV was superimposed by a 1.01-kHz sine wave, with a peak-to-peak amplitude of 20 mV, and the cell membrane conductance (Gm), Cm, and series resistance (<12 MOmega ) were calculated by the Pulse + PulseFit version 8.11 lock-in-amplifier software (HEKA). Cm and Gm measurements over 100 ms were averaged, sampled at a final rate of approx 2.2 Hz, and stored on the hard disk of a personal computer (Pentium). The value of a Cm step, indicative of LB exocytosis, is occasionally expressed as (Cm step)3/2. This transformation was performed when the LB volume rather than the LB surface was of interest. Because Cm is a parameter of surface, the surface-to-volume transformation of a sphere yields <FR><NU>3</NU><DE>2</DE></FR> as the exponent. Gm was measured to assess ion channel activity throughout the exocytotic process because there is no information about the electrical behavior of type II cells during exocytosis. Data are reported as means ± SE.


    RESULTS AND DISCUSSION
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Abstract
Introduction
Methods
Results and discussion
References

Changes in Cm and Gm in response to physiological stimuli. Unstimulated type II cells (i.e., cells "dialyzed" with a pipette solution containing 100 µM EGTA in the absence of Ca2+) exhibited a whole cell capacitance of 6.57 ± 0.22 pF (n = 71), which remained stable for the period observed (several minutes). This value corresponds to a spherical cell diameter of 14 µm (using the generally assumed specific cell capacitance of 1 µF/cm2). When ATP (20 µM) was added to the bath solution, Cm started to increase in steps after delays of various lengths (between 18 and 192 s; n = 6 cells). A similar response (Fig. 1) was observed with elevated pipette Ca2+ concentrations in the absence of the agonist (between 14 and 227 s; n = 22 cells). This corresponds well with the effect of Ca2+ ionophores on surfactant secretion (7, 17) and the response time previously assessed with the FM 1-43 technique (11). Consecutive Cm steps followed, with greatly varying numbers and declining frequency for several minutes, an example of which is shown in Fig. 1. Because ATP-induced LB exocytosis persists for >30 min (11), it was, for technical reasons (loss of gigaseal, change in series resistance due to clogging of the pipette tip, diffuse FM 1-43 staining due to increased cell permeability), usually not possible to pursue the entire secretory response.


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Fig. 1.   Original tracings of time course of cell membrane capacitance (Cm) and conductance (Gm) made under whole cell patch-clamp conditions with an elevated-Ca2+ pipette solution. Time 0 indicates rupture of cell membrane.

As shown in Fig. 1, stepwise Cm increases were not accompanied by changes in Gm. This indicates that insertion of active ion channels from the limiting LB membrane into the plasma membrane during LB exocytosis was, if present, below the detection limits of the whole cell patch-clamp technique. Hence insertion of Cl- channels in response to intracellular Ca2+ release, as recently suggested in an alveolar cell line (L2) stimulated with endothelin-1, does not appear to play a significant role in native type II cells (13). So far, the role of ion channels and membrane potential on LB exocytosis is still entirely unknown.

Relationship between Cm and FFM1-43. Haller et al. (11) previously showed that FFM1-43 correlates well with the number of exocytosed LBs. In other words, FFM1-43 is a good parameter for the amount of secreted LB material. This is based on two unique properties of surfactant: 1) it is brightly stained by FM 1-43 and 2) it remains in an aggregated and closely cell-attached state in aqueous solutions, resulting in a small loss of fluorescence. As outlined in the introduction, the access of FM 1-43 to LB material through the fusion pore is the underlying principle for the estimation of the instance of exocytosis. Hence the time course of FFM1-43 could well be limited by diffusion of the dye, which may be dissected into three steps: 1) diffusion from the plasma membrane along the limiting LB membrane after fusion, 2) diffusion through the fusion pore, and 3) diffusion within the lamellar layers of surfactant. These theoretical considerations raise the question of how precisely the time course of FFM1-43 reflects the time course of fusion pore formation or, more specifically, 1) what is the delay between fusion pore formation (measured as the Cm step) and the onset of the FFM1-43 increase and 2) is the time course of FFM1-43 related to the volume of an exocytosed LB as would be expected for a diffusional process?

The relationship between the onset of the FFM1-43 increase and the first Cm step in an individual cell is exemplified in Fig. 2A. In all experiments, this first Cm step was strictly coupled with the onset of the FFM1-43 change, its delay being <1 s. Due to the relatively low sampling rate of approx 2.2 Hz (for both recordings), which was due to limited hard- and software capabilities of our system, an exact value could not be determined. But given the extremely slow time constant (tau ) of the entire exocytotic response (tau  = 14 min after stimulation with ATP) (11), this short delay does not lead to a significant underestimate of the secretory time course. Hence these observations confirm earlier speculations that extracellular FM 1-43 has very fast access to surfactant once the fusion pore has opened (11) and prove that the FM 1-43 technique is ideally suited to measure the exocytotic onset. Less reproducible than the onset of the FFM1-43 increase, however, is its tau . This may be due to different LB sizes imposing different diffusion spaces for FM 1-43 to incorporate into the entire LB content. According to this assumption, tau  should be related to the LB volume, which can be expressed as (Cm step)3/2, the surface-to-volume transformation of the surface parameter Cm. Consistently, there is a clear correlation between (Cm step)3/2 and tau  (Fig. 2B). These data support the above hypothesis that the time course of FFM1-43 is mainly determined by dye diffusion.


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Fig. 2.   Relationship between Cm and fluorescence of the dye FM 1-43 (FFM1-43). A: original tracings of Cm and FFM1-43 [expressed in arbitrary (arb) units] during 1st exocytosis in a cell stimulated by an elevated-Ca2+ pipette solution. FFM1-43 trace (bullet ) is superimposed by an exponential fit (solid line) according to y(t) = A + Bexp(-t/tau ), where A and B are constants, t is time, and tau  is time constant. B: correlation between (Cm step)3/2, a parameter of lamellar body (LB) volume calculated from the measured LB-surface parameter Cm and respective tau  values of FFM1-43 signals as determined in A. Measurements were made in 14 cells, and 1st fusion events were evaluated. Correlation coefficient = 0.95.

The relatively smooth FFM1-43 increase after fusion pore formation compared with the concrete change in Cm makes the former an unreliable parameter to assess fusion events that succeed the very first one, particularly when the interval between successive fusions is small. An example thereof is shown in Fig. 3A. The reason for the precise determination of the first fusion but the inability to make out successive events is evident: whereas the first FFM1-43 gain adds to a very low and steady preexisting FFM1-43, successive gains add to a high and drifting FFM1-43. Nevertheless, the total amount of secreted material [expressed as the sum of (Cm step)3/2] correlates with the total increase in FFM1-43 (Fig. 3B), confirming earlier conclusions that the latter indicates the number of exocytosed LBs (11).


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Fig. 3.   Cumulative Cm and FFM1-43 signals. A: original tracings of Cm and FFM1-43 during several fusion events in a cell stimulated with 5'-O-(3-thiotriphosphate) (GTPgamma S). B: correlation between a parameter of total secreted volume {sum of (Cm step)3/2 [<LIM><OP>∑</OP></LIM>(Cm step)3/2]; compare with Fig. 2B} and cumulative FFM1-43 increase (Delta FFM1-43) in individual cells (n = 26) as determined in A at various times after initiation of whole cell configuration (at end of each patch-clamp experiment). Correlation coefficient = 0.88.

We would like to emphasize here that this limitation (i.e., the inability to resolve the times of subsequent LB fusions after the first one) only exists when FFM1-43 is measured over the area of an entire cell. It can easily be overcome, for example, by using a two-dimensional imaging system, LSM, or other methods that allow a spatial definition of distinct areas of interest (described in detail in Ref. 11).

Effect of GTPgamma S. As shown in Fig. 4A, GTPgamma S increased the cumulative Cm increase as measured 2 min after the onset of exocytosis (as noted in Changes in Cm and Gm in response to physiological stimuli, it was not possible for technical reasons to track the full exocytotic response with the patch-clamp technique). In eosinophils, GTPgamma S at a high concentration stimulates granule-granule fusion, resulting in compound exocytosis of large degranulation sacs (20). In the patch-clamp experiment, this is seen as large Cm steps. We therefore examined the effect of 160 µM GTPgamma S in the pipette on the distribution of unitary Cm-step amplitudes (Fig. 4B). Evidently, GTPgamma S did not significantly affect this distribution, indicating that it did not induce granule-granule fusion. Hence GTPgamma S appeared to stimulate exocytosis in type II cells, which has also been described in other cell types (5, 16), without affecting intracellular LB fusion. This suggests that the effect of GTPgamma S on intracellular granule fusion is specific to some cell types but does not represent a feature common to all secretory cells.


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Fig. 4.   Effects of GTPgamma S. A: <LIM><OP>∑</OP></LIM>(Cm step)3/2 (cumulative exocytosis) measured for 2 min after 1st fusion event in cells treated with elevated Ca2+ in absence (-) and presence (+) of GTPgamma S. n, No. of cells. B: Cm-step size distribution of cells stimulated with elevated Ca2+ in absence and presence of 160 µM GTPgamma S. Overlapping histograms suggest that GTPgamma S had no effect on intracellular LB fusion.

Single or compound exocytosis in the absence of GTPgamma S? As mentioned previously (11), FM 1-43-stained spots frequently appear as clusters, apparently by simultaneous fusion of several LBs with the plasma membrane. This raises the question of whether these clusters are the result of fusion of individual LBs beneath predestined areas of the plasma membrane ("pits"; 21) or of compound exocytosis, i.e., fusion of only one LB with the plasma membrane, which is, in turn, coupled to other LBs by intracellular LB-LB fusion. Because FM 1-43-stained spots and Cm can be measured in the same cell, it is easy to determine whether a unitary Cm step is coupled to the appearance of one or more than one exocytosed LB. The latter is in strong support of compound exocytosis because the likelihood that several LBs independently fuse with the plasma membrane at the very same time and in close proximity is extremely low. In the majority of experiments, a single Cm step was accompanied by the appearance of one single fluorescent spot. Only occasionally, two or more spots were seen in response to one large Cm step. Figure 5A illustrates an example where three subsequent Cm steps were accompanied by the appearance of seven FM 1-43-stained spots. This strong evidence for compound exocytosis in type II cells is supported by observations with LSM (Fig. 5B): two confocal images show a type II cell (through the central portion of the cell) where intracellular (preexocytotic) LBs are stained with LTG (green) as previously described (11). Images were taken ~10 min after stimulation with ATP, as reflected by the presence of FM 1-43-stained surfactant material (red). The transition of two preexocytotic LBs (Fig. 5B, left) to postexocytotic LBs (Fig. 5B, right, arrow) within 2 min is shown. A major argument in favor of compound exocytosis is that these postexocytotic LBs are located close to each other, one being deeply inside the cell where contact to the plasma membrane is hardly conceivable. Much more likely, these LBs were prefused, and only the upper LB underwent fusion with the plasma membrane (another example of clustered disappearance of LTG-stained LBs can be viewed as a time-lapse video animation in our homepage at URL http://138.232.233.31/respiratory-cellphysiology.htm).


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Fig. 5.   Compound exocytosis. A: original tracing of Cm in a cell stimulated with GTPgamma S. Three large Cm steps are seen, indicating 3 fusion events with plasma membrane. Inset: drawing (by hand) of same cell viewed by a charge-coupled device camera at end of depicted Cm trace. Seven exocytosed LBs are clearly discernible, although there was no exocytosis before beginning of experiment. B: 2 laser-scanning confocal microscopy images of a single cell during stimulation with ATP (given ~10 min before images were taken). Transition of 2 preexocytotic LBs (green; stained with LysoTracker Green DND-26; Ref. 11) to postexocytotic LBs (red; stained with FM 1-43; arrow) is shown. Right image was corrected for fluorescence intensity loss due to photobleaching. Hue shifts (red to yellow) resulted from incomplete separation of emitted light by the 2 channels. Diffuse red staining might result from out-of-focus surfactant on cell surface. Image size, 25 × 25 µm. Simultaneous exocytosis of LB clusters can also be viewed as a time-lapse video animation in our homepage at URL http://138.232.233. 31/respiratory-cellphysiology.htm.

Taken together, there is strong evidence for an occasional occurrence of compound exocytosis in type II cells, although definite proof will require resolution of the plasma membrane, including the fusion pore itself.

What is the specific capacitance of the membrane of an exocytosed LB? Because surfactant is not readily released from exocytosed LBs and thus represents a lipid environment along its limiting membrane, the specific capacitance of this membrane could differ greatly from that of a lipid bilayer like the plasma membrane. For its assessment, LB surfaces were calculated from measured LB diameters obtained from Normarski differential interference contrast images of unstimulated cells. The distribution of these vesicular areas is shown in Fig. 6B. This distribution matched well with the Cm distribution (Fig. 6A), indicating that there was no preference for fusion with regard to LB size. The specific capacitance of the membrane of an exocytosed LB was calculated by dividing the peak amplitude of the fitted Cm-step distribution (75 fF; data from all experimental protocols were pooled) by the peak of the fitted LB-surface distribution (8.47 µm2). The calculated value of 0.88 µF/cm2 is close to the generally assumed specific Cm of 1 µF/cm2. This would suggest that the lipid content of LBs does not affect determination of the area of its limiting membrane; in other words, surfactant-filled granules behave electrically like granules filled with hydrophilic contents. Solsona et al. (23), however, recently argued that in mast cells the Cm may, in fact, be as low as 0.5 µF/cm2.


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Fig. 6.   A: Cm-step size histogram. Data from all experimental protocols were pooled. Forty-three cells were studied: 6 cells were stimulated with ATP, 22 with elevated pipette Ca2+, and 15 with GTPgamma S. B: LB surface histogram. LB surfaces were calculated from measured LB diameters obtained from Normarski differential interference contrast images of unstimulated cells. Vesicle diameters were measured with Adobe Photoshop 4.0 based on a calibrated pixel-to-micrometer relationship and binned at 0.22-µm intervals. Varying bin width results from conversion of diameter into surface area. Dotted line, vesicle diameters below this value could not be accurately determined and data were omitted. Specific capacitance of membrane of an exocytosed LB was calculated from these data (see text).

Exocytosis without endocytosis? As shown in Fig. 3A, type II cells may increase their Cm values and thus their cell surface by up to 50% during degranulation. Notably, this is almost entirely a result of exocytotic Cm steps because compensation of this surface gain by subsequent endocytosis, which should be visible as a gradual downward drift of the Cm "plateau" after a Cm-step increase, was not observed. Naturally, the whole cell patch-clamp configuration represents a pseudophysiological condition, and results should be interpreted with caution, particularly because we did not add ATP to our patch-clamp solution (we omitted ATP because leakage of ATP out of the patch pipette might be sufficient to stimulate type II cells before the beginning of the experiment, resulting in poorly defined experimental conditions). Nevertheless, endocytosis was never observed, even during early exocytosis when the cellular ATP content was certainly still high. Hence we favor the interpretation that type II cells do not show compensatory endocytosis strictly coupled to LB fusion with the plasma membrane but instead increase their cell surface. This idea is in agreement with observations made with LSM (Fig. 5B), which revealed that exocytosed LB contents remain located inside the cell, resembling invaginations of the cell membrane but leaving the cell volume unchanged. Hence there is no apparent need to retrieve cell surface for the sake of cell volume regulation.

Transient or permanent fusion? In neuroendocrine cells, the fusion pore may flicker between the closed and open states unless it fully expands (permanent fusion) or closes again (transient fusion). Because granule contents may be released during transient fusion, this phenomenon is of potential physiological importance (reviewed in detail in Ref. 15). By analogy, it is also conceivable that LBs release only part of their contents and then are retrieved back into the cell interior. Transient exocytosis should result in a downward Cm step. Although we cannot exclude fusion pore flickering during the first milliseconds (due to our low sampling rate), downward Cm steps were never observed once FFM1-43 started to increase. Thus, even if very early transient fusion exists, it is certainly not important for partial surfactant release.

In summary, both the FM1-43 and patch-clamp techniques yield highly consistant and nonconflicting results. The patch-clamp technique, although technically more demanding, has its major advantage in accurately defining the number and instance of fusion events over a defined period of time. Due to the slow rate of exocytosis in type II cells, however, this technique will hardly ever become a routine method for studies on surfactant secretion. The FM1-43 method offers several important advantages compared with the patch-clamp technique. It is noninvasive and easy to perform, may be used over a long time, and is almost equally potent to define the time of exocytosis. Moreover, the amount of released material can be quantified and postexocytotic events can be studied.


    ACKNOWLEDGEMENTS

We thank H. Niederstätter and Prof. B. Pelster for use of the laser-scanning confocal microscope and G. Buemberger for reading the manuscript. The skillful technical assistance of I. Öttl, G. Siber, and H. Heitzenberger is gratefully acknowledged.


    FOOTNOTES

This work was supported by Fonds zur Förderung der Wissenschaftlichen Forschung Grants P11533-MED and P12974-MED.

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. §1734 solely to indicate this fact.

Address for reprint requests: P. Dietl, Dept. of Physiology, Univ. of Innsbruck, Fritz-Pregl-Str. 3, A-6020 Innsbruck, Austria.

Received 10 August 1998; accepted in final form 26 October 1998.


    REFERENCES
Top
Abstract
Introduction
Methods
Results and discussion
References

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Am J Physiol Lung Cell Mol Physiol 276(2):L376-L382
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