[Ca²⁺]_i oscillations regulate type II cell exocytosis in the pulmonary alveolus

Abstract

Pulmonary surfactant, a critical determinant of alveolar stability, is secreted by alveolar type II cells by exocytosis of lamellar bodies (LBs). To determine exocytosis mechanisms in situ, we imaged single alveolar cells from the isolated blood-perfused rat lung. We quantified cytosolic Ca(2+) concentration ([Ca(2+)](i)) by the fura 2 method and LB exocytosis as the loss of cell fluorescence of LysoTracker Green. We identified alveolar cell type by immunofluorescence in situ. A 15-s lung expansion induced synchronous [Ca(2+)](i) oscillations in all alveolar cells and LB exocytosis in type II cells. The exocytosis rate correlated with the frequency of [Ca(2+)](i) oscillations. Fluorescence of the lipidophilic dye FM1-43 indicated multiple exocytosis sites per cell. Intracellular Ca(2+) chelation and gap junctional inhibition each blocked [Ca(2+)](i) oscillations and exocytosis in type II cells. We demonstrated the feasibility of real-time quantifications in alveolar cells in situ. We conclude that in lung expansion, type II cell exocytosis is modulated by the frequency of intercellularly communicated [Ca(2+)](i) oscillations that are likely to be initiated in type I cells. Thus during lung inflation, type I cells may act as alveolar mechanotransducers that regulate type II cell secretion.

Purinergic P2X7 receptor regulates lung surfactant secretion in a paracrine manner

“…It has been suggested that AEC I contribute to the regulation of surfactant secretion in AEC II (Ashino et al, 2000;Isakson et al, 2003;Patel et al, 2005). However, the factors in AEC I that mediate AEC I and AEC II communication are unknown.…”

“…In situ fluorescence imaging has shown that interalveolar Ca 2+ conductance initiates in AEC I and stimulates extrusion of lamellar bodies from AEC II (Ichimura et al, 2006). Moreover, Ca 2+ oscillations from AEC I communicate with AEC II during lung inflation, suggesting a role for AEC I in surfactant secretion (Ashino et al, 2000). However, the mediators for AEC I and AEC II communication are unclear.…”

The use of alveolar epithelial type I cell-selective markers to investigate lung injury and repair

“…The gas exchange barrier is composed of ATI and endothelial cells joined by fused basement membranes. ATI cells are important in the regulation of alveolar fluid balance and surfactant secretion by ATII cells in response to stretch [7][8][9][10]. ATII cells cover the remaining 2-5% of the lung9s surface area.…”

Connexins as therapeutic targets in lung disease

“…Along with the work in [29][30][31][32], this manuscript highlights the role of Cx43 in mediating calcium waves. The paper shows that Cx43-made gap junctions contribute to endothelial cell expression of adhesion molecules for leukocytes.…”

“…It is important to note that transmission of calcium waves from type I to type II cells can also occur via paracrine stimulation of purinergic receptors through ATP release [30,31]. These calcium signals may also help to adjust surfactant production to stimuli such as changes in pulmonary blood pressure [32,33]. Furthermore, GJIC also enables calcium wave propagation from one alveolus to the next [29], as demonstrated by in situ fluorescence microscopy analysis of the intact lung.…”