Ca²⁺ entry is essential for cell strain-induced lamellar body fusion in isolated rat type II pneumocytes

Abstract

Using a new equibiaxial strain device, we investigated strain-induced Ca2+ 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 approximately 8% Delta CSA resulted in a transient rise of the cytoplasmic Ca2+ concentration in some cells. Higher strain levels increased the fraction of Ca2+-responding cells. The occurrence of strain-induced Ca2+ signals depended on cell-cell contacts, because lone cells (i.e., cells without cell-cell contacts) did not exhibit Ca2+ signals. Above threshold, the amplitude of the Ca2+ 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 Ca2+ signal; 50 microM Gd3+ in the bath affected neither Ca2+ signals nor fusions. Intracellular Ca2+ release was triggered at higher strain amplitudes and inhibited by thapsigargin. Removal of bath Ca2+ completely inhibited Ca2+ signals and fusions. We conclude that strain of AT II cells stimulates a Ca2+ entry pathway that is highly sensitive to strain and a prerequisite for subsequent Ca2+ 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.

Mechanical strain of alveolar type II cells in culture: changes in the transcellular cytokeratin network and adaptations

“…The characteristic surfactant containing lamellar bodies (some indicated with arrows) and other organelles were clearly visible under both conditions. As shown in earlier studies, mechanical stretch stimulates LB fusion with the plasma membrane (12,41), and also we found fusion events in the transmission electron microscopy (TEM) only in stretched cells (see vertical section in Fig. 2, C and D).…”

“…The range of the stretch with a maximum of 30% CSA increase was based on the similar increases in vivo of the basal cell membrane at 100% total lung capacity (2,36) and on the range used in other studies (12,28,35). However, ATII cells may experience considerably less deformation than alveolar type I cells in vivo (29).…”

Interfacial stress affects rat alveolar type II cell signaling and gene expression

“…On the other hand, mechanical stress can also have an immediate effect, depending on the cell type and the nature of the forces that are acting on them. In AT II cells, for example, cell stretch is a potent stimulus for surfactant secretion and proceeds via Ca 2ϩ -dependent pathways (23,73). In most studies, the intracellular Ca 2ϩ elevation is mediated by a transmembrane Ca 2ϩ influx, which, in turn, activates a secondary Ca 2ϩ -induced intracellular Ca 2ϩ release mechanism.…”

“…On the other hand, their higher tolerance could be the result of a constitutive release of surfactant. Removal of bath Ca 2ϩ , known to completely inhibit agonist and stretch-induced exocytosis in AT II cells (23,24), indeed rendered their susceptibility comparable to that of the fibroblasts, whereas direct addition of purified surfactant to 3T3 cells clearly prolonged the time to cytotoxic damage. A further evidence for the role of Ca 2ϩ -induced surfactant secretion was observed by preincubation of AT II cells with gadolinium.…”

“…In particular, tissue stretch (ϭ tensile strain) has already been shown to be an important determinant of surfactant secretion in alveolar type II (AT II) cells (2,15,36,47,71,73). Studies on single cells revealed that stretch induces an elevation of the intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) (23,73), perhaps via mechanosensitive ion channels like the wellcharacterized transient receptor potential (TRP) vanilloids (reviewed in Refs. 16,77).…”