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Mechanical forces exert multiple effects in cells, ranging from altered protein expression patterns to cell damage and death. Despite undisputable biological importance, little is known about structural changes in cells subjected to strain ex vivo. Here, we undertake the first transmission electron microscopy investigation combined with fluorescence imaging on pulmonary alveolar type II cells that are subjected to equibiaxial strain. When cells are investigated immediately after stretch, we demonstrate that curved cytokeratin (CK) fibers are straightened out at 10% increase in cell surface area (CSA) and that this is accompanied by a widened extracellular gap of desmosomes-the insertion points of CK fibers. Surprisingly, a CSA increase by 20% led to higher fiber curvatures of CK fibers and a concurrent return of the desmosomal gap to normal values. Since 20% CSA increase also induced a significant phosphorylation of CK8-ser431, we suggest CK phosphorylation might lower the tensile force of the transcellular CK network, which could explain the morphological observations. Stretch durations of 5 min caused membrane injury in up to 24% of the cells stretched by 30%, but the CK network remained surprisingly intact even in dead cells. We conclude that CK and desmosomes constitute a strong transcellular scaffold that survives cell death and hypothesize that phosphorylation of CK fibers is a mechano-induced adaptive mechanism to maintain epithelial overall integrity. mechanical stress; cytoskeleton; desmosome; injury

Here we report a 26- to 29-pS cation channel abundantly expressed in freshly isolated and primary cultured type II cells from rat or healthy human lungs. The channel was never spontaneously active in cell-attached patches but could be activated by cell permeabilization with [beta]-escin. Excised patch-clamp experiments revealed activation by [Ca.sup.2+] concentrations at the cytoplasmic side in the micromolar range. High concentrations of amiloride (> 10 [micro]M) at the extracellular side did not inhibit. The channel was equally permeable for K+ and Na+ but was essentially impermeable for [Cl.sup.-] , [Ca.sup.2+], and [Mg.sup.2+]. It was blocked by adenosine nucleotides (cytoplasmic side) with the following order of potency: AMP [aproximately equal to] ADP ([EC.sub.50] [less than or equal to] 10 [micro]M) > ATP [much greater than] adenosine [much greater than] cyclic AMP. The blocking effect of ATP was reproduced by its nonhydrolyzable analogs AMPPNP or ATP-[gamma]-S. GTP did not inhibit. [Cd.sup.2+] blocked the channel with an ECso [approximately or equal to] 55.5 nM. We conclude that type II cells express a [Ca.sup.2+]-dependent, nucleotide-inhibited, nonselective, and [Ca.sup.2+] impermeable cation channel (NS[C.sub.Ca/AMP]) with tonically suppressed activity. RT-PCR confirmed expression of TRPM4b, a channel with functional characteristics almost identical with NS[C.sub.Ca/AMP]. Potential physiological roles are discussed. patch clamp; calcium; adenosine 5'-monophosphate; adenosine 5'diphosphate; adenosine 5'-triphosphate; cadmium; lung; alveolus; nonselective cation channel

Using a new equibiaxial strain device, we investigated strain-induced [Ca.sup.2+] signals and their relation to lamellar body (LB) exocytosis in single rat alveolar type II (AT II) cells. The strain device allows observation of single cells while inducing strain to the entire substratum. AT II cells tolerated high strain amplitudes up to 45% increase in cell surface area ([DELTA]CSA) without release of lactate dehydrogenase or ATP. Strain exceeding a threshold of ~8% [DELTA]CSA resulted in a transient rise of the cytoplasmic [Ca.sup.2+] concentration in some cells. Higher strain levels increased the fraction of [Ca.sup.2+]-responding cells. The occurrence of strain-induced [Ca.sup.2+] signals depended on cell-cell contacts, because lone cells (i.e., cells without cell-cell contacts) did not exhibit [Ca.sup.2+] signals. Above threshold, the amplitude of the [Ca.sup.2+] signal as well as the number of stimulated LB fusions correlated well with the amplitude of strain. Furthermore, stimulated LB fusions occurred only in cells exhibiting a [Ca.sup.2+] signal; 50 [micro]M [Gd.sup.3+] in the bath affected neither [Ca.sup.2+] signals nor fusions. Intracellular [Ca.sup.2+] release was triggered at higher strain amplitudes and inhibited by thapsigargin. Removal of bath [Ca.sup.2+] completely inhibited [Ca.sup.2+] signals and fusions. We conclude that strain of AT II cells stimulates a [Ca.sup.2+] entry pathway that is highly sensitive to strain and a prerequisite for subsequent [Ca.sup.2+] release. Both mechanisms result in a graded response of fusions to strain. Our data also allow us to introduce the term 'effective strain' as the physiologically relevant portion of the strain amplitude. surfactant secretion; alveolar type II cells; mechanical strain; stretch

The feasibility of using the dye FM 1-43 to measure lamellar body exocytosis at high spatial and temporal resolution in alveolar type II cells was investigated. Fluorescence microscopy was combined with the whole cell patch-clamp approach to determine how the FM 1-43 fluorescence gain reflects the instance and number of fusion events or which factors affect the time course of the FM 1-43 gain. Experimental results showed that the FM 1-43 technique is highly suitable for measurement of the onset of exocytosis and amount of secretion in alveolar type II cells.

The effects of NO on L-type Ca2+ channel function were analyzed in L2 cells during perforated patch-clamp experiments and nitrate assay. Analysis of spontaneous cellular depolarizations after the administration of NO donors indicated the presence of increased levels of Sr2+-induced depolarizations. However, guanosine cyclic monophosphate (cGMP) inhibited Ca2+-dependent action potentials and spontaneous contractile activity. Furthermore, NO reduced the activity of L-type Ca2+ channels via a cGMP-dependent mechanism.

Cells of the cloned rat epithelial cell line L2 were treated with extracellular ATP to examine the mechanism by which purinoceptor stimulation by ATP increases intracellular calcium ion (Ca2+) and induces the secretion of pulmonary surfactant by alveolar epithelial cells. The results show that L2 cells express L-type Ca2+ channels, which are activated by ATP. L-type Ca2+ channels could have an important role in surfactant secretion.

The secretory pathway pulmonary surfactant from the type II pneumocyte cell to the air-liquid interface is reviewed with focus on the distinct mechanisms and regulations of lamellar body (LB) fusion and release. The situation of secreted material until it is arranged into units that finally function to reduce the surface tension in the lung is discussed.

Mucus clearance provides an essential innate defense mechanism to keep the airways and lungs free of particles and pathogens. Baseline and stimulated mucin secretion from secretory airway epithelial cells need to be tightly regulated to prevent mucus hypersecretion and mucus plugging of the airways. It is well established that extracellular ATP is a potent stimulus for regulated mucus secretion. Previous studies revealed that ATP acts via metabotropic P2Y2 purinoreceptors on goblet cells. Extracellular ATP, however, is also a potent agonist for ionotropic P2X purinoreceptors. Expression of several P2X isoforms has been reported in airways, but cell type-specific expression and the function thereof remained elusive. With this study, we now provide evidence that P2X4 is the predominant P2X isoform expressed in secretory airway epithelial cells. After IL-13 treatment of either human primary tracheal epithelial cells or mice, P2X4 expression is upregulated in vitro and in vivo under conditions of chronic inflammation, mucous metaplasia, and hyperplasia. Upregulation of P2X4 is strongest in MUC5AC-positive goblet cells. Moreover, activation of P2X4 by extracellular ATP augments intracellular Ca2+ signals and mucin secretion, whereas Ca2+ signals and mucin secretion are dampened by inhibition of P2X4 receptors. These data provide new insights into the purinergic regulation of mucin secretion and add to the emerging picture that P2X receptors modulate exocytosis of large secretory organelles and secretion of macromolecular vesicle cargo. [ABSTRACT FROM AUTHOR]

Haller, Thomas, Klaus Auktor, Manfred Frick, Norbert Mair, and Paul Dietl. Threshold calcium levels for lamellar body exocytosis in type II pneumocytes. Am. J. Physiol. 277 (Lung Cell. Mol. Physiol. 21): L893-L900, 1999.--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 [Ca.sup.2+] concentration ([[Ca.sup.2+]].sub.i]). In fura 2-loaded cells, [[Ca.sup.2+]].sub.i] was selectively elevated by flash photolysis of a cell-permeant caged [Ca.sup.2+] compound (o-nitrophenyl EGTA-AM) or by gradually enhancing cellular [Ca.sup.2+] 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 [[Ca.sup.2+]].sub.i] elevations. The exocytotic response to flash photolysis was highest during the first minute after the rise in [[Ca.sup.2+]].sub.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 [Ca.sup.2+]. This was further demonstrated by the fact that exocytosis is significantly blocked by suppression of the ATP-induced [Ca.sup.2+] signal below ~300 nmol/l. Our results suggest a highly [Ca.sup.2+]-sensitive step in LB exocytosis. surfactant secretion; alveolar type II cells; flash photolysis; caged calcium