Submitted on December 28, 2007
Accepted on August 3, 2008
Long term exposure to LPS enhances the rate of stimulated exocytosis and surfactant secretion in alveolar type II cells and upregulates P2Y2 receptor expression
Ignacio Garcia-Verdugo1, Andrea Ravasio2, Elvira Garcia de Paco1, Monique Synguelakis1, Nina Ivanova3, Jean Kanellopoulos1, and Thomas Haller2*
1 IBBMC, University of Paris-Sud, France
2 Physiology and Medical Physics, Innsbruck Medical University, Innsbruck, Austria
3 General Physiology, University of Ulm, Germany
* To whom correspondence should be addressed. E-mail: Thomas.Haller{at}i-med.ac.at.
Bacterial lipopolysaccharide (LPS1) is a potent pro-inflammatory molecule. In the lungs, LPS induces alterations in surfactant pool sizes and phospholipid (PL) contents, though direct actions of LPS on the alveolar type II cells (AT II) are not yet clear. For this reason, we studied short- and long-term effects of LPS on basal and agonist stimulated secretory responses of rat AT II by using Ca2+ microfluorimetry, a microtiter-plate based exocytosis assay, by quantitating PL and [3H]-labeled choline released into cell supernatants, and by using quantitative PCR and Western blot analysis. Long-term, but not short-term exposures to LPS lead to prolonged ATP-induced Ca2+ signals and an increased rate in vesicle fusions with an augmented release of surfactant PL. Most notably, the stimulatory effect of LPS was ATP-dependent and may be mediated by the upregulation of the purinergic receptor subtype P2Y2. Western blot analysis confirmed higher levels of P2Y2, and suramin, a P2Y-receptor antagonist, was more effective in LPS treated cells. From these observations, we conclude that LPS, probably via TLR-4, induces a time-dependent increase in P2Y2 receptors which, by yet unknown mechanisms, leads to prolonged agonist-induced Ca2+ responses that trigger a higher activity in vesicle fusion and secretion. We further conclude that chronic exposure to endotoxin sensitizes AT II to increase the extracellular surfactant pool which aids in the pulmonary host defence mechanisms.
