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
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ABSTRACT |
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 |
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 |
Cell preparation. Alveolar type II
cells were isolated from anesthetized (thiopental sodium) male
Sprague-Dawley rats (
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
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-M
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) (GTP
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 M
) 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
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
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 |
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.
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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
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 (
) of the entire exocytotic response (
= 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
. 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,
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
(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 ( ) is
superimposed by an exponential fit (solid line) according to
y(t) = A + Bexp( t/ ),
where A and
B are constants,
t is time, and 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
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.
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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)
(GTP S). B: correlation between a
parameter of total secreted volume {sum of
(Cm
step)3/2
[ (Cm
step)3/2]; compare
with Fig. 2B} and cumulative
FFM1-43 increase
( 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.
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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 GTP
S. As shown in Fig.
4A,
GTP
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, GTP
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 GTP
S in the pipette on
the distribution of unitary
Cm-step
amplitudes (Fig. 4B). Evidently, GTP
S did not significantly affect this distribution, indicating that
it did not induce granule-granule fusion. Hence GTP
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 GTP
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 GTP S. A:
(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 GTP S.
n, No. of cells.
B:
Cm-step size
distribution of cells stimulated with elevated
Ca2+ in absence and
presence of 160 µM GTP S. Overlapping histograms suggest
that GTP S had no effect on intracellular LB fusion.
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Single or compound exocytosis in the absence of
GTP
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 GTP 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.
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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 GTP 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).
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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.
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