Threshold calcium levels for lamellar body exocytosis in type II pneumocytes

Thomas Haller, Klaus Auktor, Manfred Frick, Norbert Mair, and Paul Dietl

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Stimulation of exocytosis by photolysis of caged Ca2+. Ultraviolet (UV) flash (arrow)-induced transient rise in intracellular Ca2+ concentration ([Ca2+]i) and simultaneous recording of FM 1-43 fluorescence intensity (FFM1-43) in a single type II cell are shown. During flash, connection to photomultiplier tube was switched off (dotted line). Importance of correcting fura 2 calibration factors for flash artifacts is shown by [Ca2+]i calculated without such corrections at 3 conditions (open circle ) during measurement. arb, Arbitrary. Left inset: enlargement of [Ca2+]i peak showing single data points sampled at 1 Hz. Right inset: background-corrected fura 2 ratio image (340-to-380 nm) of a single cell acquired 850 ms after flash, demonstrating homogeneous Ca2+ elevations throughout cell.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 2.   Determination of [Ca2+]i threshold for lamellar body (LB) exocytosis. A: peak values of UV flash-induced [Ca2+]i transients vs. FFM1-43 increase (Delta FFM1-43) determined before and 10 min after UV flash in single cells as in Fig. 1 (n = 9 preparations). Responding cells () were discriminated from nonresponding cells (open circle ) by observation of FM 1-43-stained spots after termination of measurements (10 min). Inset: peak values of UV flash-induced [Ca2+]i (binning interval = exponent 0.2) from responding cells in A vs. delay between UV flash and onset of Delta FFM1-43. * Delay is significantly higher at high compared with low [Ca2+]i, P < 0.05. B: peak values of UV flash-induced [Ca2+]i transients (data in A) were binned as in A, inset, and plotted against percentage of responders. Data were fitted according to y = a - b/(1 + cx)(1/d), where a, b, c, and d are constants. Threshold [Ca2+]i was defined at 90% of extrapolated asymptote (arrow), yielding 320 nmol/l.

The frequency histogram in Fig. 2B comprises the data from Fig. 2A. The peak [Ca2+]i values from both responding and nonresponding cells were binned in logarithmic intervals and plotted against the percentage of responders within the respective [Ca2+]i interval. The data in Fig. 2A are consistent with the idea that initiation of exocytosis is dependent on whether a certain threshold of [Ca2+]i is reached or not. Therefore, the binned data were fitted by a hyperbolic function. Due to the varying degree of nonresponders, 100% is never reached, and threshold [Ca2+]i was defined on reaching 90% of the asymptote (Fig. 2B, arrow) rather than a lower value. This type of analysis yielded a [Ca2+]i threshold of 320 nmol/l.

Gradient perfusion. Flash photolysis is an adequate technique to study Ca2+-dependent processes but has two major drawbacks: 1) because the photomultiplier had to be shut off during flash illumination (<1 s; Fig. 1, dotted line), the actual [Ca2+]i peak is difficult to determine due to the following decay in [Ca2+]i, and the calculated threshold could possibly be lower than the actual one; and 2) UV exposure alters the biophysical properties of fura 2. Although [Ca2+]i calculations were thoroughly corrected for the flash-induced artifacts (see MATERIALS AND METHODS and Fig. 1), inaccuracies cannot be entirely excluded. We therefore analyzed the [Ca2+]i threshold with a second independent method. A uniform and gradual increase in [Ca2+]i above threshold should initiate exocytosis and should allow it to relate its onset with the simultaneously measured [Ca2+]i in individual cells. By gradient perfusion, increasing amounts of ionomycin (0-20 nmol/l) were mixed with the perfusate to slowly permeabilize the cells. This had the major advantage that [Ca2+]i transients, which would occur in response to a single, fast application of this Ca2+ ionophore due to Ca2+ release from intracellular stores, were not observed (see Fig. 3A). In addition to ionomycin, it was necessary to concomitantly raise extracellular Ca2+ from 2 to 20 mmol/l to enhance cellular Ca2+ influx. This kind of perfusion, as shown by a representative single cell measurement in Fig. 3A, induced a slow and monotonic increase in [Ca2+]i by which secretion was initiated. As indicated in Fig. 3A, [Ca2+]i at the exocytotic onset (FFM1-43 increase above baseline; *) was noted, and the frequency of these values derived from single-cell experiments is shown in Fig. 3B. The histogram reveals that these values are distributed within Ca2+ concentrations of 0.2-1 µmol/l, exhibiting a maximum near 460 nmol/l.


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Fig. 3.   A: stimulation of exocytosis by a gradual rise in [Ca2+]i in a single cell. Cellular Ca2+ influx was slowly enhanced by cell perfusion with a continuous rise in ionomycin (0-20 nmol/l) and Ca2+ (2-20 mmol/l; gradient was started at time 0; FM 1-43 was continuously present at 0.5 µmol/l; see MATERIALS AND METHODS). [Ca2+]i (*) at initiation of exocytosis (right dotted line) was noted for histogram in B. [Ca2+]i threshold values were determined 30 s before onset of Delta FFM1-43 (left dotted line and arrow), corresponding to peak of delay histogram in Fig. 4. B: distribution of [Ca2+]i values determined in A. Data were binned in intervals of 50 nmol/l [Ca2+]i and plotted against no. of observations (n = 10 preparations) C: data corrected for a constant exocytotic delay of 30 s in A, arrow. Both histograms were fitted by a 6th-order polynomial. Frequency peak in C yielded a [Ca2+]i threshold of 330 nmol/l.

This mean [Ca2+]i required to initiate exocytosis is apparently higher than the threshold concentration determined by the flash experiments but could be explained by a delay between the stimulus and exocytosis after stimulation (note that by gradient perfusion, [Ca2+]i at the time of membrane fusion but not at the Ca2+ threshold is determined; compare with delay after UV flash in Fig. 1). For this reason, we sought to determine the Ca2+ threshold in the gradient perfusion experiments by correcting for this delay, using a constant time factor as depicted in Fig. 3A. To determine this factor, the exocytotic response-time distribution had to be assessed. This was done by UV flash, yielding defined time points of stimulation. The analysis revealed an exocytotic onset that is delayed for up to 1 min in most cells, with a few cells responding even later (Fig. 4; note also that the early response to UV flash is almost identical to that obtained by stimulation with 10 µmol/l of ATP). The peak value (30 s) of the frequency distribution in Fig. 4 (equivalent to the most frequently observed delay) was then used to individually correct all single-cell measurements for this constant factor. The frequency maximum of these delay-corrected values (330 nmol/l; Fig. 3C) matches well with that of the Ca2+ threshold obtained from flash photolysis experiments (320 nmol/l).


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Fig. 4.   Comparison of delay histograms in response to UV flash and ATP (10 µmol/l). Delay denotes time between first detectable onset of [Ca2+]i increase (measured by photometry) and onset of exocytosis (determined by observation with a charge-coupled device video camera). Cells were binned in intervals of 20 s and are expressed as percentage of total measurements (n = 36 for UV flash and 70 for ATP). Inset: dose-response curve for ATP (n = 15 measurements, with each n representing mean of up to 50 cells from 11 preparations). Data were fitted by a logistic dose-response function.

[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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Fig. 5.   Effects of ATP on [Ca2+]i and exocytosis (FFM1-43) in single alveolar type II cells in presence of cytosolic Ca2+ chelator o-nitrophenyl EGTA (NP-EGTA). Cells were preincubated for 15 min at 37°C with 2 µmol/l of fura 2-AM and 5 µmol/l of NP-EGTA-AM. Before measurement, cells were exposed for 5 min to bath solution A (see MATERIALS AND METHODS) containing 0.5 µmol/l of FM 1-43, 10 mmol/l of NP-EGTA-AM, and no added CaCl2. Flash photolysis caused a bleaching artifact of FFM1-43 (down deflection). A: 10 µmol/l of ATP and 1 mmol/l of external Ca2+ (+Ca2+) were added (straight arrow). By this treatment, [Ca2+]i increased to levels (411 ± 63 nmol/l; note that by averaging individual measurements, resulting [Ca2+]i peak is lower than individual values) still sufficient to stimulate exocytosis. Subsequent photolysis with NP-EGTA (flash arrow) increased [Ca2+]i severalfold but had no additional effect on exocytosis. B: in absence of external Ca2+ (-Ca2+), stimulation by 10 µmol/l of ATP did not lead to a detectable [Ca2+]i increase, and exocytosis was negligible. However, flash induced Ca2+ release beyond threshold-initiated exocytosis. max, Maximum. Before individual FFM1-43 tracings were averaged, data were normalized between 0 (before addition of ATP) and 100% (maximum increase). Tracings are arithmetic means (thick lines) ± SE (thin lines); n = 8 measurements from 4 preparations. Nonresponding cells were omitted. During breaks, photomultiplier was switched off.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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.


    ACKNOWLEDGEMENTS

We thank G. Siber, I. Öttl, and H. Heitzenberger for skillful technical assistance and Dr. B. Flucher for critical comments.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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