Department of Physiology, University of Innsbruck, A-6020 Innsbruck, Austria
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ABSTRACT |
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Pulmonary surfactant is secreted via exocytosis of lamellar bodies (LBs) by alveolar type II cells. Here we analyzed the dependence of LB exocytosis on intracellular Ca2+ concentration ([Ca2+]i). In fura 2-loaded cells, [Ca2+]i was selectively elevated by flash photolysis of a cell-permeant caged Ca2+ compound (o-nitrophenyl EGTA-AM) or by gradually enhancing cellular Ca2+ influx. Simultaneously, surfactant secretion by single cells was analyzed with the fluorescent dye FM 1-43, enabling detection of exocytotic events with a high temporal resolution (T. Haller, J. Ortmayr, F. Friedrich, H. Volkl, and P. Dietl. Proc. Natl. Acad. Sci. USA 95: 1579-1584, 1998). Exocytosis was initiated at a threshold concentration near 320 nmol/l with both instantaneous or gradual [Ca2+]i elevations. The exocytotic response to flash photolysis was highest during the first minute after the rise in [Ca2+]i and thus almost identical to purinoceptor stimulation by ATP. Correspondingly, the effects of ATP on initial secretion could be sufficiently explained by its ability to mobilize Ca2+. This was further demonstrated by the fact that exocytosis is significantly blocked by suppression of the ATP-induced Ca2+ signal below ~300 nmol/l. Our results suggest a highly Ca2+-sensitive step in LB exocytosis.
surfactant secretion; alveolar type II cells; flash photolysis; caged calcium
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INTRODUCTION |
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PULMONARY SURFACTANT, a mixture of phospholipids and lipoproteins, is secreted by alveolar type II (ATII) cells via the exocytotic release of lamellar body (LB) contents into the alveolar space. Consequently, fusion of LBs with the plasma membrane is the key step linking cellular synthesis of surfactant with its dispersion over the air-liquid interface where it lowers surface tension and increases pulmonary compliance. The regulation of secretion in ATII cells is complex, involving hormones as well as local chemical and physical factors (for reviews, see Refs. 4, 29, 32). How this multitude of different stimuli converge into a single physiological response is still incompletely understood.
Haller et al. (12) and Mair et al. (20) have recently developed a method to resolve single LB fusion events and to analyze the onset, time course, and extent of the cellular responses. This method is based on FM 1-43, a fluorescent marker of surfactant, that can be used in combination with fura 2, allowing simultaneous recordings of the intracellular Ca2+ concentration ([Ca2+]i) and exocytosis of LBs. An earlier study by Haller et al. (12) indicated that in the majority of cells stimulated with ATP plus isoproterenol, the onset of exocytosis coincides with intracellular Ca2+ release. Furthermore, Mair et al. (20) have recently shown that micromolar Ca2+ concentrations promote exocytosis when applied via the patch pipette in the whole cell patch-clamp configuration. This concurs with most investigators' opinions that elevations in Ca2+ are associated with increased secretion and support the findings of Dobbs et al. (7) and Pian et al. (26) that Ca2+ may trigger secretion, even though a decisive Ca2+ dependence of exocytosis could not be inferred from these studies. In addition, quantitative aspects, in particular, relationships between the level of [Ca2+]i and exocytosis, have not yet been investigated in these cells.
To specifically address these issues, we used three approaches to modulate [Ca2+]i and to study the early effects (within minutes after stimulation) on LB exocytosis in single cells, 1) [Ca2+]i was selectively elevated by flash photolysis of a cell-permeant caged Ca2+ compound [o-nitrophenyl EGTA-AM (NP-EGTA-AM)], 2) [Ca2+]i was gradually increased in continuously permeabilized cells, and 3) the amplitude of the ATP-induced Ca2+ signal was attenuated by the Ca2+-chelating effect of NP-EGTA. The present study is the first demonstration of simultaneous measurements of [Ca2+]i and exocytosis in single ATII cells combined with selective [Ca2+]i elevation by flash photolysis. Our findings suggest a prominent role of Ca2+ in stimulus-secretion coupling and an exocytotic mechanism with a high affinity for this messenger.
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MATERIALS AND METHODS |
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Cell preparation. ATII cells were isolated from Sprague-Dawley rats according to the procedure of Dobbs et al. (6) with the slight modifications summarized in Ref. 12. After isolation, the cells were seeded on glass coverslips at low density (40 cells/mm2), cultured in DMEM, and used 1 day thereafter.
Optical setup and ultraviolet flash. Excitation light was generated by a polychromatic illumination system (TILL Photonics) as previously described (21). Excitation bands centered at 340 or 380 (for fura 2) and 475 nm (for FM 1-43), each of 10 ms duration, were consecutively applied after dark intervals of 1 s (sample interval of 1 Hz). Excitation light was directed through a quartz glass fiber, attenuated by a gray filter to 10% transmission, and fed laterally into a two-port condenser unit (TILL Photonics) attached to the epifluorescence port of an inverted microscope (Axiovert 35, Zeiss). The source of the ultraviolet (UV)-flash light was a pulsed xenon arc lamp (pulse length 0.5 ms, wavelength ~320-390 nm) coupled by a quartz-light guide into the rear port of the same condenser unit. Both beams were centered at a sapphire window by which they were made parallel to each other and to the epifluorescence path of the microscope. Alignment of the sapphire allowed transmission of 92% of the UV flash and reflection of 8% of the excitation light intensity into an achromatic UV condenser. Incident light was further deflected by a 500-nm dichroic mirror into a Zeiss Fluar ×40 1.3-numerical aperture oil objective. With this optical configuration, the discharged flash energy of 80 J yields ~2 × 1023 photons/m2 in the specimen plane. The intensity of the excitation light, however, was below the level at which Ca2+ release due to photolysis of NP-EGTA could be observed. The emitted fluorescence was directed through a 520-nm long-pass filter and a Viewfinder (TILL Photonics) to a photomultiplier tube (Hamamatsu). The Viewfinder contained adjustable field stops by which the collected fluorescence could be restricted to the area of one single cell. Correspondingly, excitation and flash light were aligned by separate field stops to the same area in the specimen plane (~20 µm). Cellular fluorescence was simultaneously recorded with a charge-coupled device video camera (Variocam, PCO Computer Optics). Control of excitation light and data acquisition were performed with the fura extension of the Pulse software (Heka). Data were analyzed with DatGraf (Cyclobios) and SigmaPlot (Jandel Scientific). Data are reported as arithmetic means ± SE.
Fluorescence measurements. Cells were preincubated at 37°C in DMEM with fura 2-AM (1.2 µmol/l, 15 min) and NP-EGTA-AM (1-10 µmol/l, 5-30 min; both from Molecular Probes). Coverslips with stained cells were mounted into a perfusion chamber placed on the stage of the microscope and rinsed at 25°C with bath solution A (in mmol/l: 140 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 5 glucose, and 10 HEPES, pH 7.4). Exocytosis was recorded in the continuous presence of 0.5 µmol/l of FM 1-43 (Molecular Probes) in the bath (12). Although interference between the FM 1-43 fluorescence (FFM1-43) and fura 2 fluorescence (Ffura2) intensity ratios is generally small, the fura 2 signal was corrected off-line for the progressive increase in FFM1-43 (see [Ca2+]i calculation).
As an alternative approach to flash photolysis, we aimed at gradually raising [Ca2+]i from resting levels up to micromolar concentrations. We found that this is most effectively achieved by exposing ATII cells to a linear perfusion gradient ranging from 100% of control conditions (solution A; see above) to 100% of a "maximum Ca2+" solution B (65-125 mmol/l of NaCl, 5-25 mmol/l of CaCl2, and 10-50 nmol/l of ionomycin; other constituents were the same as in solution A). FM 1-43 was permanently present at 0.5 µmol/l in solutions A and B. Both solutions were mixed by means of a self-made gradient pump and supplied to the cells. Gradient perfusion typically lasted 10 min.
Dose-response relationships between ATP and LB exocytosis were determined with a video-imaging system as previously described (12). In each experiment, the mean cellular FFM1-43 of up to 50 cells was measured before and 10 min after stimulation with ATP and corrected for the respective baseline value.
[Ca2+]i
calculation.
[Ca2+]i
was calculated as previously described (11, 13). At 380-nm excitation,
FM 1-43 contributes, to a small extent, to the fura 2 emission, which
leads to a slight underestimation of the actual
[Ca2+]i.
To minimize this effect, FM 1-43 was applied at the lowest possible
concentration but was still sufficient for detection (0.5 µmol/l).
Furthermore, before background subtraction and ratio (R) calculation
(340-to-380 nm), the Ffura2 at 380 nm was corrected by subtracting
(FFM1-43 × 0.0127). Because
we found that overestimation of
Ffura2 at 380 nm is proportional
to FFM1-43, we corrected the former by subtraction of this empirically determined value. A dissociation constant
(Kd) of 224 nmol/l was used for the fura 2-Ca2+ complex (11).
All other calibration factors [background fluorescence (Fb), minimum ratio
(Rmin), maximum ratio
(Rmax), and proportionality coefficients for fluorescence of free to Ca2+-bound dye
(Sf2/Sb2)]
were determined at the end of each individual experiment as previously
described (13). Where calibration was not feasible or indefinite, the
calibration values from other experiments on the same day were pooled
and the resulting average values were applied. Determination of
Rmin is particularly difficult in
ATII cells because even in the absence of
Ca2+ and the presence of high
concentrations of both EGTA (5 mmol/l) and ionomycin (20 µmol/l) in
the bath, Ca2+ is only slowly
extruded from the cells. The decline in the 340- to 380-nm ratio,
however, could be fitted by a single exponential in most cases, and
Rmin was determined from
extrapolation of the fitted ratio calculation. In experiments applying
flash photolysis, the fura 2 calibration factors had to be corrected
for the flash artifacts. In a separate set of experiments, these
flash-induced changes in calibration factors were analyzed in detail
and were as follows (expressed as mean percent change from the preflash values; n = 15 measurements):
Fb at 340 nm, 3.6 ± 1.1%; Fb at 380 nm,
2.1 ± 1.4%; Rmin,
3.0 ± 1.0%; Rmax,
5.2 ± 1.3%; and Sf2/Sb2,
4.4 ± 1.1%. The flash-induced change in
Kd was estimated with the following protocol: fura 2- and
Ca2+-preloaded cells (10 µmol/l
of ionomycin plus 5 mmol/l of
Ca2+) were exposed to a solution
with a measured Ca2+ activity of
447 nmol/l (in mmol/l: 7 NaCl, 130 KCl, 1 MgCl2, 7.906 CaCl2, 10 HEPES, 10 EGTA, 2 × 10
5 ionomycin, and
0.01 nigericin, pH 7.25). As a result,
[Ca2+]i
slowly decreased within 30-60 min. When stable
[Ca2+]i
readings in the range of the expected
Kd (224 nmol/l)
were reached, the cells were exposed to the UV flash, and the change in
the fura 2 ratio [R to postflash R
(Rpost)] was analyzed.
With the assumption of identical
[Ca2+]i
values before and after flash and with the respective pre- and
postflash calibration factors, the postflash
Kd-to-Kd
ratio can be calculated according to Grynkiewicz et al. (11) as
{1/(postflash Sf2/postflash
Sb2) × [(postflash Rmax
Rpost)/(Rpost
postflash Rmin)]}/{1/(Sf2/Sb2) × [(Rmax
R)/(R
Rmin)]}. In
our experiments, the postflash
Kd increased by
10.6 ± 2.1% of the preflash value (n = 15 measurements). Based on the
reported Kd of
224 nmol/l, this yields 248 nmol/l, which was subsequently used for
calculating postflash
[Ca2+]i.
The significance of applying corrected calibration values for
[Ca2+]i
is shown in Fig. 1.
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RESULTS |
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Stimulation of LB exocytosis by photolysis of caged Ca2+. To selectively and rapidly increase [Ca2+]i in intact ATII cells, we used the photoactivable (caged) Ca2+ compound NP-EGTA. Photolytic cleavage of the molecule (uncaging) is accomplished by a short UV illumination, resulting in an immediate and uniform rise in [Ca2+]i (Fig. 1). Cells were loaded with the cell-permeant NP-EGTA-AM. Within the cell, the ester groups are removed by esterases and free cytosolic Ca2+ is bound by complexation. On flash, Ca2+ is rapidly released from NP-EGTA, leading to [Ca2+]i spikes of up to 5 µmol/l. After the peak (which was, in fact, a short plateau in most cases), [Ca2+]i declined, presumably due to sequestration into intracellular organelles and concomitant Ca2+ extrusion into the extracellular space (Fig. 1).
The flash-induced [Ca2+]i transients were sufficient to stimulate a pronounced exocytotic activity, which was continuously recorded as FFM1-43, starting to increase, with a delay of several seconds after the flash (Fig. 1). In a previous report, Haller et al. (12) have shown that the increase in FFM1-43 correlates well with the number of exocytosed LBs and hence with the amount of secreted material. In cells not loaded with NP-EGTA, an increase in FFM1-43 could not be observed after the UV flash, excluding the possibility that UV illumination by itself stimulates secretion (data not shown). These observations emphasize the role of Ca2+ as a potent effector of secretion, in particular because it acts even after short periods of Ca2+ elevation.Defining the
Ca2+ threshold
for exocytosis.
The amplitude of flash-induced
[Ca2+]i
transients depends primarily on the uncaging efficiency of the actinic
light and the amount of Ca2+ bound
to the intracellular chelator. The latter can be modulated by varying
the extracellular concentration of NP-EGTA-AM. Using 1-10
µmol/l, we were able to generate
[Ca2+]i
peaks from several nanomoles per liter up to a few micromoles per
liter. As evident from Fig.
2A,
most cells responded when [Ca2+]i
peaked above ~300 nmol/l. A considerable portion of the cells could
not be activated at all. Responding cells could be clearly discriminated from such nonresponders by observation of FM 1-43-stained spots after termination of the measurements. Although we presently have
no explanation for these nonresponders, which are always present in our
primary culture of ATII cells irrespective of the method of stimulation
(12), we can rule out that they are simply dead cells: they are
identical to responding cells with respect to cell morphology, calcium
homeostasis, plasma membrane integrity (Haller, unpublished
observations), and acidification of LBs (12).
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[Ca2+]i
threshold and agonist-induced exocytosis.
ATP is a potent stimulus in ATII cells, acting via cAMP formation and
P2 receptor-dependent
phosphoinositide hydrolysis, with subsequent
Ca2+ mobilization (8, 27, 28). The
importance of the transient rise in
[Ca2+]i
on ATP-induced surfactant secretion, however, is still a matter of
debate. Based on the above findings, we speculated that the effects of
ATP on early exocytosis might be sufficiently interpreted by its
Ca2+-mobilizing action. We tested
this hypothesis by modulating ATP-induced [Ca2+]i
rises in NP-EGTA-loaded cells at "low" (nominally
Ca2+-free) or "high" (1 mmol/l) extracellular Ca2+. By
acting as a high-affinity Ca2+
buffer, cytosolic NP-EGTA binds
Ca2+ and diminishes the
ATP-induced
[Ca2+]i
increase. In the low-Ca2+ bath,
the intracellular concentration of NP-EGTA was sufficient to completely
block the ATP-induced
[Ca2+]i
signal (Fig.
5B),
whereas ATP at 1 mmol/l of Ca2+
exceeded the buffer capacity of this chelator, resulting in a delayed
and small
[Ca2+]i
increase (Fig. 5A). Although these
[Ca2+]i
peaks were much smaller than ATP-evoked
Ca2+ signals without NP-EGTA
(usually >500 nmol/l), they were close to the threshold concentration
in every single case and were sufficient to stimulate exocytosis.
Subsequent photolysis of NP-EGTA increased [Ca2+]i
severalfold but had no additional stimulatory effect on exocytosis. In
the nominally Ca2+-free solution
(Fig. 5B), ATP failed to create a
detectable increase in
[Ca2+]i,
and exocytosis was significantly, although not completely, inhibited.
The remaining exocytotic activity could be a result of constitutive
exocytosis, which always contributes ~10% to the total observed
exocytosis on stimulation (12). The subsequent Ca2+ release by flash photolysis
of NP-EGTA significantly enhanced or initiated exocytosis in all
responding cells under study. The results from all three protocols
(flash photolysis, gradient perfusion, and ATP stimulation) strongly
support the above hypothesis and suggest that in ATII cells the
Ca2+ threshold is the major
determinant for early exocytotic activity, with other factors playing a
negligible role during that phase.
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DISCUSSION |
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Evidence for the involvement of Ca2+ in LB exocytosis. Ca2+ has long been recognized as a modulator of surfactant secretion. This notion emerged from observations that various kinds of stimulation (e.g., ATP) increased [Ca2+]i and phospholipid secretion similarly (27, 33) and that some of them require a certain basal [Ca2+]i (26, 30). Furthermore, mechanical stretch promotes secretion by an apparently Ca2+-dependent process (33). The interpretation of these studies with regard to the role of Ca2+, however, is limited because the use of agonists does not allow an accurate dissection of the relative contribution of branching signaling pathways, and physical treatments such as stretch also modulate Ca2+ by processes not clearly defined. So far, the most compelling support for [Ca2+]i in surfactant secretion comes from measurement with Ca2+ ionophores (7, 26, 30). In addition, Mair et al. (20) have recently shown that micromolar [Ca2+]i values promote exocytosis when applied via the patch pipette in the whole cell patch-clamp configuration.
Methods to modulate [Ca2+]i. The present study is a further step forward in the analysis of the physiological role of Ca2+ in LB exocytosis with experimental protocols that modulate [Ca2+]i in a most selective way in single cells. This was achieved by flash photolysis of caged Ca2+, which has been used for studies of stimulus-secretion coupling in a variety of other secretory cells (14, 15, 31). With this technique, activation of Ca2+-independent signaling pathways are bypassed. Furthermore, it can be assumed that Ca2+ is uniformly elevated within the cytosol, excluding the possibility of spatially restricted Ca2+ gradients (31). This is a crucial point because agonist stimulation could lead to high local [Ca2+]i in the vicinity of LBs despite a moderate or even low overall [Ca2+]i signal in the cytosol, which would lead to a severe underestimation of the actual Ca2+ requirement for exocytosis. The same problem may also arise during pulsed addition of ionophores and in stretch-induced Ca2+ mobilizations, creating high local Ca2+ gradients beneath the plasma membrane or close to intracellular compartments. The issue of localized versus global Ca2+ effects in secretion and the problem to relate exocytosis with [Ca2+]i in a quantitative manner are discussed elsewhere in great detail (23, 24). The experiments with gradient perfusions were similarly designed to yield a uniform but slow increase in [Ca2+]i.
[Ca2+]i threshold for LB exocytosis. Both methods effectively triggered LB exocytosis irrespective of whether [Ca2+]i was elevated instantaneously (UV flash) or slowly (gradient experiments). This is in contrast to some nonexcitable cells (e.g., mast cells) where Ca2+ was suggested merely as a cofactor of exocytosis, probably not sufficient to trigger this process by itself (1, 9, 22). Because secretion in our cells was stimulated by photolytic Ca2+ increase, which is a transient event, we conclude that a sustained [Ca2+]i is not a prerequisite for stimulation. Furthermore, both methods of calculating [Ca2+]i thresholds revealed a consistently and unexpectedly low value. This finding is corroborated, although indirectly, by attenuating the rise in [Ca2+]i below that value during cellular stimulation with ATP, which blocked exocytosis (Fig. 5B). From all these observations, strong evidence emerges that in ATII cells the Ca2+ threshold is a major determinant for the early exocytotic activity during the first minutes of stimulation.
To some extent, however, the importance of Ca2+ in agonist-induced exocytosis is controversially discussed. Chander et al. (5) suggested that the major part of ATP-induced surfactant secretion is mediated via activation of protein kinase C (PKC). In line with their conclusion, Rice et al. (27) doubted that Ca2+ mobilization is a necessary step for ATP-induced surfactant secretion. Instead, a prominent role for PKC has been proposed. Their conclusion is primarily based on measurements with bis-(2-amino-5-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester-loaded cells where ATP-induced elevations in [Ca2+]i were inhibited 90% by this EGTA analog, whereas surfactant secretion was not affected. This result is in accordance with the present data, demonstrating that secretion is inhibited when the ATP-induced [Ca2+]i increase is reduced below ~300 nmol/l, whereas partial inhibition has no effect. Because Rice et al. measured a basal [Ca2+]i of ~250-300 nmol/l, even small elevations above this level would exceed the threshold for secretion in their experiments. Studies on surfactant secretion are generally done by directly measuring phosphatidylcholine release from whole cell populations into the cell supernatant (29). The observation time in these studies is usually longer (up to hours) than in the present one, whereas Ca2+ mobilization is a short event, occurring immediately after the addition of the agonist (within seconds). ATP is well known to exert a complex cellular reaction, including increasing [Ca2+]i, inositol 1,4,5-trisphosphate, cAMP, and diacylglycerol and activating PKC (5, 10). Therefore, it is conceivable that persistent ATP-induced surfactant secretion does not primarily depend on a sustained high [Ca2+]i but on the action of alternative signaling cascades. Because Rice et al. (27) measured the amount of phosphatidylcholine after 3 h in the continued presence of ATP, it is likely that the late effects of ATP, mediated by PKC or other factors, significantly contribute to late exocytosis, thereby compensating for a suppressed early exocytotic activity in the presence of the chelator. It is important to emphasize that the relative contributions of Ca2+ and other intracellular messengers could differ considerably during the early and late stages of secretion, an issue that has not yet been adequately addressed. By dissecting early from late responses, we found that the ATP-evoked pattern of exocytotic onsets matches perfectly that of the UV-flash experiments during early stimulation. However, whereas Ca2+-induced exocytosis levels off with a fast decay, ATP exhibited some prolonged effects (Fig. 4). In general, exocytotic delays can be explained by generating additional cytosolic messengers, by removing cytosolic barrier proteins, or by conveying vesicles from the depth of the cell to the plasma membrane (1). A broad distribution of exocytotic delays most likely reflects LBs at different stages during vesicle processing. Because this was the case for both kinds of stimulation (Fig. 4), we suggest that a "pool" of ready releasable vesicles either does not exist in ATII cells or comprises a few LBs only. This is also supported by the significant cell-to-cell variability of exocytotic responses to [Ca2+]i levels above threshold (Fig. 2A), a feature also shared by some other secretory cell types (14). As noted above, the most plausible explanation is the presence of a varying number of fusion-competent vesicles in individual cells.Comparative aspects of
Ca2+ in
stimulus-secretion coupling.
Ca2+ acts as a second
messenger-coupling stimulus to secretion in a variety of cells (15, 25,
31). In ATII cells, LB exocytosis is triggered by a mechanism with a
high affinity for Ca2+. Compared
with synaptic vesicle fusion, where
Ca2+ has to reach levels of
hundreds of micromoles per liter, LB exocytosis is effectively
initiated near 320 nmol/l. In fact, this threshold is among the lowest
known for mammalian cells, comparable with human neutrophils or
pancreatic -cells (3, 19). The differences in
Ca2+ requirements to initiate
exocytosis in various cell types could imply different secretory
mechanisms or different regulatory controls and seem to be inversely
related to vesicle size and the time course of secretion (2, 25). LBs
have diameters in the micrometer range (20), and the exocytotic
activity of ATII cells is among the slowest known, corresponding to the
above hypothesis (1, 12). From a physiological point of view, the time
needed to release the secretory products corresponds to different
cellular functions. In ATII cells, the time-limiting step is probably
not exocytosis itself but the subsequent slow release of the
hydrophobic surfactant material and its transformation into molecular
forms that are able to adsorb to the air-liquid interface in the
alveoli. The high Ca2+ sensitivity
of LB exocytosis could reflect the physiological function of ATII cells
to release surfactant on a variety of different signals. In particular,
this sensitivity toward Ca2+ could
be explained by its role in responding to alveolar distension with
effective secretion. As shown by Wirtz and Dobbs (33), a
single stretch of ATII cells causes a transient increase in [Ca2+]i
followed by a sustained stimulation of surfactant secretion. Both
Ca2+ mobilization and exocytosis
exhibited a dose dependence to the magnitude of the stretch, whereas
cAMP was not enhanced, supporting the concept of a sensitive,
Ca2+-mediated mechanism. Because
Lee et al. (18) found gap junction complexes between type I and type II
cells and because intercellular propagation of
Ca2+ waves with declining
amplitude are described for monolayers comprising both cell types (17),
the high Ca2+ sensitivity could be
an effective mechanism to sense alveolar distension remote of the
stimulus itself. Proteins that have been suggested to mediate secretion
in ATII cells in a Ca2+-dependent
fashion are annexin II and annexin VII (29). A likely candidate for a
high-affinity Ca2+ sensor is also
calmodulin, which has been reported to be reversibly associated with
LBs (16). Nevertheless, the Ca2+
sensor(s) in ATII cells, which participates in the regulation of the
final steps of LB exocytosis, and the role of soluble
N-ethylmaleimide-sensitive factor
attachment protein receptor proteins in this highly
Ca2+-sensitive process remain to
be determined.
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ACKNOWLEDGEMENTS |
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We thank G. Siber, I. Öttl, and H. Heitzenberger for skillful technical assistance and Dr. B. Flucher for critical comments.
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FOOTNOTES |
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This work was supported by the Fonds zur Förderung der Wissenschaftlichen Forschung Grants P-11533 and P-10963 (both to P. Dietl) and Österreichische Nationalbank Grant 7413 (to T. Haller).
Preliminary results were presented in abstract form (J. Gen. Physiol. 112: 43a, 1998 and FASEB J. 12: A177, 1998).
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 and other correspondence: T. Haller, Dept. of Physiology, Univ. of Innsbruck, Fritz-Pregl-Str. 3, A-6020 Innsbruck, Austria (E-mail: thomas.haller{at}uibk.ac.at).
Received 21 December 1998; accepted in final form 13 July 1999.
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