Exocytosis in alveolar type II cells

The main purpose of respiration is the supply of oxygen and the elimination of carbon dioxide. To achieve this, proper ventilation of the terminal structures of the airways (alveoli) and proper diffusion of gas between alveoli and perfused blood are required. Surfactant, a phospholipid-rich secretory product of alveolar type II (AT II) cells, plays a fundamental role for these processes: By lowering the surface tension of the air-liquid-interface, it prevents alveolar collapse and fluid accumulation (lung edema). Failure or dysfunction of surfactant results in respiratory insufficiency and death. Surfactant secretion is stimulated by various hormones as well as physicochemical factors, and inhibited by at least one of its own secretory products, surfactant protein A. Surfactant is stored in secretory organelles of AT II cells, termed lamellar bodies (LB). It is secreted by fusion of LB with the plasma membrane (exocytosis). Exocytosis of LB is a highly regulated event and thus a key step for secretion. It is enhanced by Ca2+, cAMP and protein kinase C, but the precise mode of action of these second messengers is yet unclear. Likewise, mechanisms and molecular controls of the exocytotic process are largely unknown. This lack of information is mainly due to methodological difficulties resolving surfactant release at the level of single cells. Based on the surfactant-staining properties of the fluorescent dye FM 1-43, we have recently developed a method to observe real time exocytosis in single AT II cells (Haller et al., Proc. Natl. Acad. Sci. (USA), in press). This method also enables us to define the moment and cellular location of single exocytotic events as well as to quantify the amount of material released. This project aims at a better understanding of the cellular and molecular mechanisms and controls of LB exocytosis. By simultaneous use of FM 1-43 and fura-2 fluorescence in combination with the patch clamp technique, stimulus-secretion-coupling will be studied in single living AT II cells. The roles of Ca2+. other second messengers and surfactant protein A during LB exocytosis will be explored. Steps of LB processing and functional pools of LB should be revealed. In addition, Ca2+ stores and Ca2+ entry pathways will be investigated. We believe that this project is an important basis for future therapeutical approaches to enhance surfactant secretion.

In this project, cellular mechanisms and physical forces involved in the release of surface active agents (surfactant) from cells of the pulmonary alveolus were disclosed. It was shown that this process consists of distinct phases, whereof the opening of "fusion pores" is the limiting step for the release of surfactant. Fusion pores are mechanical barriers for the exit of surfactant from the cell into the alveolus. Exocytosis is a fundamental biological process, in which substances stored in intracellular vesicles are released from the cell by fusion of these vesicles with the plasma membrane. In the lung, exocytosis is essential for the release of surfactant from alveolar type II cells into the lumen of the alveolus. Vesicles that store surfactant are called lamellar bodies (LBs). Released surfactant is absorbed at the air-liquid-interface, thereby reducing the amount of force required for inspiration. Surfactant deficiency, as for example the case in some premature neonates, leads to respiratory distress and death. It was the aim of this project to elucidate, by use of innovative cellphysiological techniques, the processes involved in surfactant release by single cells and their regulation. Experiments were performed in cells of the rat lung grown in primary culture. For the measurement of exocytosis, a new fluorescence technique, which had been developed in the lab of the project leader in a previous project, was used. This innovative technique is based on the diffusion principle of a special dye (FM1-43) through the fusion pore. The fusion pore is a structure evolving immediately upon vesicle fusion with the plasma membrane and representing the open access to the vesicle interior from outside. In this project, a mathematical diffusion model for FM1-43 was established, by which the diameter of the fusion pore can be calculated from continuous measurements of the FM1-43 fluorescence intensity. By means of this method we could show for the first time that fusion pores are structures which open slowly and discontinuously and which are regulated by the intracellular messenger Ca2+. An elevation of the intracellular Ca2+ concentration, which takes place in response to various stimuli, opens the fusion pores, resulting in an accelerated surfactant release. The general importance of these findings in the field of cell biology is demonstrated by the fact that the results were published in the Journal of Cell Biology, the most renowned international journal of cell biology. Further key results of this project were the elucidation of terminal steps leading to the fusion of LBs with the plasma membrane. The analysis of multiple, in part simultaneously measured parameters in response to various types of cell stimulation lead to the establishment of a "minimal fusion model", in which the terminal signaling cascade can be explained by a simple common pathway for all types of stimulation. These results were published in various high-ranking journals and reviewed in additional articles (such as in the News in Physiological Sciences). Methodological development in the area of nanotechnology ("single cell stretcher") lead to an Austrian patent and a German petty patent (Gebrauchsmuster). The results of this project also resulted in various prices and decorations, such as the Novartis price to Dr. Dietl and the Aventis price to Dr. Haller.