Aims: Our overall objective is to understand the in situ cell biology of lung alveoli. Here, we will determine the role of the cytosolic free calcium concentration ([Ca2+]i) in alveolar secretion stimulated by mechanical stress and soluble stimulants.
The specific aims are to quantify for the first time, [Ca2+]i and secretion rates in type II cells of intact alveoli. Secretion will be stimulated by different amounts cf lung inflation, and different surfactant secretagogues. The new hypotheses to be tested are that (1) type II cell secretion is regulated by intercellular communication of oscillatory [Ca2+]i signals, and (2) type II cells exhibit polarity in their agonist- induced secretion responses. Procedures: (I) [Ca2+]i quantification. Fluorometric imaging of [Ca2+]i- sensitive dyes will be conducted intravitally in single alveoli of the isolated, blood-perfused rat lung, using both conventional and confocal microscopy. Amplitude and phase locking of [Ca2+]i oscillations will be analyzed in the frequency domain by fast Fourier transforms (FFT). (2) Immunoimaging. Type I and type II cells will be identified by imaging cell-specific immunofluorescence. (3) Secretion. Type II cell secretion will be determined in single cells by the loss of cell fluorescence attributable to the acidotropic dye LysoTracker Green, and by the alveolar fluorescence of the phospholipid.binding dye FM 1-43. (4) 3-D reconstruction. Dimensional changes in type I and type II cells, [Ca2+]i and secretion rates will be determined in 3-D reconstructions of alveoli inflated to different extents. Significance: This proposal addresses a new understanding of type II cell secretion. Type II cell physiology is basic to lung function, but remains inadequately understood. It is important to know the role of [Ca2+]i in type II cell secretion, because dysregulation of alveolar [Ca2+]i nay be common to many mechanisms that affect type II cell function and thereby, promote lung injury. Sustained [Ca2+]i increases, or [Ca2+]i oscillations in pathological conditions, may constitute a potent signal for gene transcription and consequently, lung remodeling. If preliminary data bear out, this research will prove for the first time that in alveolar cells, prolonged [Ca2+]i oscillations markedly extend beyond duration of stimulus. These oscillations may regulate type II cell secretion by intercellular intercommunication. The presence of polarity in type II cell responses may lead to different secretion responses depending on whether secretagogues are delivered by the blood or the airborne route. No previous understanding of such mechanisms exist in lung alveoli. These proposed studies are therefore, outstandingly novel and important.
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