MICROSCOPIC IMAGING OF ALVEOLAR FUNCTION

St. Luke'S-Roosevelt Inst For Hlth Scis, New York NY

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Abstract

DESCRIPTION (provided by applicant): Aims: Our overall objective is to understand the role of the cytosolic Ca2+ in the regulation of type 2 cell exocytosis of lamellar bodies (LB) in the intact alveolus. Here, we will determine the role of cell-cell communication and mitochondria in the regulation of type 2 cell Ca2+. The specific aims are to quantify for the first time, regulation of type 2 cell exocytosis in relation to Ca2+ increase in the type 1 cell (Specific Aim 1), paracrine signaling by juxta-alveolar capillaries (Specific Aim 2) and mitochondrial reactive oxygen species (ROS) production (Specific Aim 3). The new hypotheses to be tested are that type 1 cell communicates signals to the type 2 cell and that Ca2+ increase in the type 2 cell induces LB exocytosis by inducing mitochondrial ROS production. Procedures: (1) Ca2+ quantification. Fluorometric imaging of Ca2+-sensitive dyes will be conducted intravitally in single alveoli of the isolated, blood-perfused rat lung, using both conventional and confocal microscopy using methods that have been developed in the current cycle. (2) Immunoimaging. Type 1 and type 2 cells will be identified by imaging cell-specific immunofluorescence. (3) LB exocytosis. Type 2 cell exocytosis will be determined in single cells by the loss of cell fluorescence of to the acidotropic dye LysoTracker Green and by the alveolar fluorescence of the phospholipid-binding dye FM 1-43. (4) Ca2+ increase. In targeted type 1 cells physiological Ca2+ increases will be induced by (i) photo-excited intracellular uncaging (PEIU) in situ and (ii) by infusing Ca2+ agonists. Responses in Ca2+ and exocytosis rates will be determined with respect to duration and extent of cell conduction of signals in the presence of appropriate inhibitors. In one set of experiments we will test hypotheses in a mouse lacking components of NADPH-oxidase to determine the role of non-mitochondrial ROS production. Significance: This proposal addresses a new understanding of type 2 cell exocytosis of LB. This exocytosis, which leads to secretion of surfactant and surfactant proteins critical to lung function, remains inadequately understood. It is important to know the role of Ca2+ in type 2 cell exocytosis, because dysregulation of alveolar Ca2+ may be common to many mechanisms that affect type 2 cell function and thereby, promote lung injury. Sustained Ca2+ increases 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 Ca2+ increases in type 2 cells are generated by signaling from type 1 cells, endothelial cells and mitochondria. No previous understanding of these mechanisms exists. These proposed studies are therefore, outstandingly novel and important.