Departments of Medicine and Physiology and Cellular Biophysics, College of Physicians and Surgeons; and St. Luke's-Roosevelt Hospital Center, Columbia University, New York, New York 10019
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ABSTRACT |
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To determine whether lung capillary pressure regulates surfactant secretion, we viewed alveoli of the constantly inflated, isolated blood-perfused rat lung by fluorescence microscopy. By alveolar micropuncture we infused fura 2 and lamellar body (LB)-localizing dyes for fluorescence detection of, respectively, the alveolar cytosolic Ca2+ concentration ([Ca2+]i) and type II cell exocytosis. Increasing left atrial pressure (Pla) from 5 to 10 cmH2O increased septal capillary diameter by 26% and induced marked alveolar [Ca2+]i oscillations that abated on relief of pressure elevation. The rate of loss of LB fluorescence that reflects the LB exocytosis rate increased fourfold after the pressure elevation and continued at the same rate even after pressure and [Ca2+]i oscillations had returned to baseline. In alveoli pretreated with either 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, the intracellular Ca2+ chelator, or heptanol, the gap junctional blocker, the pressure-induced exocytosis was completely inhibited. We conclude that capillary pressure and surfactant secretion are mechanically coupled. The secretion initiates in a Ca2+-dependent manner but is sustained by Ca2+-independent mechanisms.
surfactant; intracellular calcium; intercellular communication; gap junction; pulmonary hypertension; mechanical stretch; LysoTracker; heptanol; BAPTA-AM
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INTRODUCTION |
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LUNG SURFACTANT, a constitutive feature of the alveolar air-liquid interface, is secreted by alveolar type II cells and is critical for maintenance of physiological lung compliance (4). This physiological role is best illustrated by the facts that decreased surfactant in premature neonates precipitates neonatal respiratory distress syndrome and that surfactant replacement alleviates the condition (2, 6). Surfactant secretion is induced by lung hyperinflation (3, 10, 15) and stretch of type II cells (18). Among cellular mechanisms implicated are increases in the cytosolic calcium concentration ([Ca2+]i) of type II cells (5, 11, 18) that couple the mechanical stimulus to the secretory response. A previous report from our laboratory indicated that hyperinflation-induced secretion results from the communication of [Ca2+]i signals from type I to type II cells (1). Although microvascular pressure (Pmv) elevation increases [Ca2+]i in endothelial cells of lung capillaries (14), alveolar responses are undefined.
We addressed these issues in our in situ imaging model of the isolated blood-perfused rat lung that allows real-time quantification of type II cell exocytosis in the intact alveolus (1). This quantification is achieved by imaging the fluorescence of the acidophilic LysoTracker dyes that localize to lamellar bodies (LB) of type II cells (1, 8). LysoTracker-loaded type II cells appear as brightly fluorescent spots. Loss of fluorescence reflects exocytosis of LB, hence surfactant secretion. An advantage of this approach is that both alveolar pressure and Pmv may be set to target levels, and exocytosis can be followed in real time. We report experiments in which we raised Pmv and determined time-dependent responses of [Ca2+]i and exocytosis in single type II cells of intact alveoli.
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METHODS |
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Fluorescence probes and agents. We used the following: 1) the fluorophores fura 2-AM, LysoTracker green (LTG), LysoTracker red (LTR), and fluo 4 (Molecular Probes) in a solution of 2% dextran-HEPES containing 1% fetal bovine serum; 2) 3% reconstituted bovine surfactant (Survanta, Ross) in Ringer lactate; 3) Ca2+-free buffer containing (in mM) 150 NaCl, 5 KCl, 10 glucose, and 20 HEPES (pH 7.4); and 4) the agents 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM (Calbiochem, San Diego, CA) and heptanol (Sigma).
Lung microscopy. We used the isolated blood-perfused rat lung prepared as previously described (1, 17). Briefly, lungs excised from anesthetized male adult Sprague-Dawley rats (470-700 g) were pump perfused with 14 ml/min of autologous rat blood at 37°C. At baseline, pulmonary artery left atrial (Pla) and alveolar pressures were held constant at 12.5, 5, and 5 cmH2O, respectively. Lungs were positioned on a vibration-free table, and saline at 37°C was infused on the surface to prevent drying. We viewed the diaphragmatic lung surface through a fluorescence microscope (AX70, Olympus) or a confocal (LSM 5 Pascal, Zeiss) microscope and recorded and analyzed images by means of our imaging system, which we have previously described in detail (1, 19).
Alveolar methods. We followed our previously described methods for alveolar microinfusion (1). Briefly, we micropunctured single alveoli with micropipettes (5-µm tip diameter), then gently injected different solutions to fill 7-10 adjacent alveoli. Only nonmicropunctured alveoli were selected for image analysis. The procedures were: 1) Alveolar [Ca2+]i quantification. We loaded alveoli by microinfusions of fura 2-AM (10 µM, 25 min) under dark room conditions. Fluorophore-loaded alveoli were washed with Ringer solution containing Survanta and then excited alternatingly at 340 and 380 nm every 10 s. [Ca2+]i was determined based on computer-generated 340/380 ratios with fura-Ca2+ Kd of 224 nmol/l and appropriate calibration parameters (19). 2) Type II cell exocytosis. To quantify LB exocytosis in alveoli, we gave 15-min microinfusions of the LB localizing fluorophores LTG or LTR (50 nM) and imaged alveoli at excitations of 488 and 545 nm, respectively. 3) Infusions of inhibitors. We infused BAPTA-AM (40 µM), which chelates the cytoplasmic Ca2+, and heptanol, which blocks gap junctional communication. Each inhibitor was infused for 15 min. BAPTA-AM was infused in Ca2+-free HEPES, and heptanol in unmodified HEPES.
Data are means ± SE; n = number of lungs. Significance was accepted at P < 0.05. ![]() |
RESULTS |
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Alveolar fluorescence.
As viewed by confocal microscopy, vascular diameters varied in the
range 5-7 µm in septal capillaries and 10-20 µm in
venular capillaries forming the first postseptal generation of vessels (Fig. 1). A Pla increase from 5 to 10 cmH2O increased the diameters of septal capillaries and
venules by 26 ± 8 and 8 ± 3%, respectively (for each:
n = 6, P < 0.05), the increase being
greater for the septal capillaries (P < 0.05).
Concomitantly, alveolar diameter decreased by 6 ± 2%
(n = 15, P < 0.05). These findings
indicated that the pressure increase distorted the alveolar wall.
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Alveolar [Ca2+]i.
The [Ca2+]i oscillations were immediately
induced and dominated by waves of amplitude >40 nM (Fig.
3). The [Ca2+]i
level that formed the nadir of the waves increased after a delay of
3-5 min, reaching levels that were 44 ± 9 nM higher than baseline in the final minute of the 10-min pressure elevation period
(n = 6, P < 0.05) (Fig. 3).
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Imaging alveolar type II cells.
Previously, we used conventional fluorescence microscopy to image
LTG-loaded type II cells in alveoli (1). Here we applied in situ confocal microscopy that provided clearer images of LB in
single type II cells (Fig. 4). Under
baseline conditions, optical sectioning at high magnification revealed
LB distributed in a necklace-like orientation that circumscribed a
central nonfluorescent zone that may have contained the nucleus and
other perinuclear structures (Fig. 4A). Onset of LB
exocytosis after pressure elevation was evident as a highly polarized
loss of fluorescence (Fig. 4, B and C),
consistent with the expected apical location of the secretion.
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DISCUSSION |
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We show here for the first time that increase of Pla induces increased LB exocytosis in alveolar type II cells, indicating that septal hemodynamics determine surfactant secretion. We determined the secretion in terms of the exocytosis of LTG from single cells that we previously confirmed to be alveolar type II cells by immunofluorescence colocalization methods (1). LTG-loaded cells seen at high magnification by in situ confocal microscopy revealed LB distributed in a circular orientation (Fig. 2). These first real-time images of LB distribution in an intact alveolus further confirmed the strong localization of LTG to type II cells. Consequently, type II cells stood out as the most highly fluorescent cells in an LTG-loaded alveolus and could be identified easily. Secretion was evident in the loss of fluorescence from these cells and also by the disappearance of lamellar bodies in cells seen at high magnification (Fig. 2).
We determined alveolar [Ca2+]i by the fura 2 method as before (1), using alveolar micropuncture to load the dye in alveolar cells. We have shown previously (14) that fura 2 loading of alveoli by the micropuncture approach does not cause cross leakage of dye into endothelial cells of capillaries adjacent to the experimental alveolus. This was evident in that, in fura 2-loaded alveoli, fluorescence increased when a calcium agonist was given directly in the alveolar lumen, but not when the agonist was injected into the capillary (13). Furthermore, as we point out in RESULTS, in the present experiments a steady nonoscillating fluorescence at baseline signified that the fluorescence was of alveolar, not capillary, origin.
Mechanisms. Initiation of secretion was through Ca2+-dependent mechanisms as indicated by the concomitant induction of [Ca2+]i oscillations in alveolar cells and the inhibition of secretion by BAPTA-AM, which chelates cytosolic Ca2+. Although we are not certain of the precise mechanisms by which an increase of septal capillary pressure mechanically distorts alveolar cells, the possibilities are as follows. First, increased tensile stress on the capillary wall decreases the interstitial pressure (7) and could thereby induce distension stress on adjacent alveolar cells. Second, hydrostatically increased capillary filtration could cause alveolar liquid entry, leading to inhibition of surfactant function (12) and alveolar collapse. Third, pressure-induced increase of endothelial [Ca2+]i in lung capillaries (14) could induce endothelial secretion of soluble factors that could initiate surfactant secretion by a paracrine effect. Fourth, septal congestion could cause compressive stress on alveolar cells. Some support for the fourth possibility is indicated in that confocal images revealed not alveolar collapse, but a small decrease of alveolar diameter that would be consistent with alveolar compression due to vascular congestion. The pressure-induced increase of capillary diameter was by 8% in venular capillaries, which is consistent with our previous data (16), but by 26% in septal capillaries. This difference is consistent with the higher compliance of septal capillaries and supports the view that capillary dilatation compressed alveolar cells.
We expected that capillary congestion would directly compress the type II cell and initiate secretion by inducing distortional effects similar to those of cell indentation (11). Furthermore, localized compression of type II cells should be independent of communicated signals from other cells; hence inhibition of cell communication should not block exocytosis stimulated by localized compression. To test this possibility we infused heptanol, the gap junctional blocker, which in our previous study blocked type II cell exocytosis by blocking the communication of [Ca2+]i signals from adjacent type I cells (1). We also showed previously that although heptanol blocks intercellular communication of [Ca2+]i signals (1, 19), it does not block exocytosis in type II cells stimulated directly (1). However, in the present experiments, heptanol completely inhibited the Pla-induced exocytosis. This unexpected result indicated that the secretion response was evoked not by local stimulation of type II cells, but by signals communicated from an adjacent cell that was most likely a type I cell. These findings indicate that type I, but not type II, cells were targeted for vascular compression. The reasons underlying this selectivity remain unclear. However, other considerations may apply, such as the possibility that the inhibitory effect of heptanol on cell communication in capillaries (19) blocked the endothelial release of a diffusible factor that stimulated type II cell exocytosis. This and other possibilities require further investigation.Role of [Ca2+]i. Our findings also provide novel insights into the role of [Ca2+]i in surfactant secretion. This role has been considered from the standpoint of the second messenger effect of [Ca2+]i (reviewed in Ref. 1). Both in mechanically and in secretagogue-stimulated type II cells, [Ca2+]i increases associate with secretion (5, 8, 9, 18). However, in our previous study, although [Ca2+]i oscillations accompanied the induction of type II cell exocytosis by a 15-s hyperinflation, the responses to the Ca2+ chelator BAPTA-AM differed, depending on whether the chelator was given pre- or posthyperinflation (1). Given before hyperinflation, BAPTA-AM completely inhibited the secretory response. Given posthyperinflation, namely after initiation of secretion, the chelator did not modify the exocytosis rate. We interpreted these results to mean that a [Ca2+]i increase was required for initiation but not maintenance of the secretion rate.
We propose a similar interpretation for our present findings based on the following considerations. If the exocytosis were [Ca2+]i dependent throughout, in the postchallenge period in which Pla and [Ca2+]i responses returned to baseline, the exocytosis rate should have decreased. However, no such decrease occurred, and the postchallenge exocytosis rate remained identical to that during pressure elevation. This result indicates that Ca2+-independent processes maintained the secretion. We interpret these results, together with the exocytosis inhibitory effect of BAPTA-AM, to mean that, as in the case of hyperinflation (1), the [Ca2+]i increase that occurred immediately after capillary distension was the initiating stimulus for secretion. However, unidentified [Ca2+]i-independent mechanisms sustained the secretion subsequently. In conclusion, we show here that even a modest increase of lung capillary pressure induces surfactant exocytosis. We believe that this is the first evidence that the vascular system is coupled to alveolar surfactant secretion through capillary pressure. A better understanding of the mechanisms underlying this coupling and of the physiological implications of the coupling requires further investigation. ![]() |
ACKNOWLEDGEMENTS |
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This research was supported by National Heart, Lung, and Blood Institute Grants HL-64896 and HL-57556 to J. Bhattacharya and HL-10142 to P. M. Wang.
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. Bhattacharya, St. Luke's-Roosevelt Hospital Center, 1000 10th Ave, New York, NY 10019 (E-mail: jb39{at}columbia.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published January 4, 2002;10.1152/ajplung.00303.2001
Received 3 August 2001; accepted in final form 14 December 2001.
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