Long-term exposure to LPS enhances the rate of stimulated exocytosis and surfactant secretion in alveolar type II cells and upregulates P2Y2 receptor expression.
Bacterial LPS is a potent proinflammatory molecule. In the lungs, LPS induces alterations in surfactant pool sizes and phospholipid (PL) contents, although 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 Ca(2+) microfluorimetry, a microtiter plate-based exocytosis assay, by quantitating PL and (3)H-labeled choline released into cell supernatants and by using quantitative PCR and Western blot analysis. Long term, but not short term, exposures to LPS led to prolonged ATP-induced Ca(2+) 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 P2Y(2). Western blot analysis confirmed higher levels of P2Y(2), and suramin, a P2Y receptor antagonist, was more effective in LPS-treated cells. From these observations, we conclude that LPS, probably via Toll-like receptor-4, induces a time-dependent increase in P2Y(2) receptors, which, by yet unknown mechanisms, leads to prolonged agonist-induced Ca(2+) 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 defense mechanisms.
Garcia-Verdugo
Ignacio
I
Department of Physiology and Medical Physics, Innsbruck Medical University, Innsbruck, Austria.
Ravasio
Andrea
A
de Paco
Elvira Garcia
EG
Synguelakis
Monique
M
Ivanova
Nina
N
Kanellopoulos
Jean
J
Haller
Thomas
T
D000199:Actins / Q000235:genetics; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D017931:DNA Primers; D005089:Exocytosis / Q000187:drug effects*; D005786:Gene Expression Regulation / Q000187:drug effects; D007700:Kinetics; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008297:Male; D051379:Mice; D016133:Polymerase Chain Reaction; D011650:Pulmonary Alveoli / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D011663:Pulmonary Surfactants / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D018048:Receptors, Purinergic P2 / Q000235:genetics*; D058473:Receptors, Purinergic P2Y2
D000199:Actins; D017931:DNA Primers; D008070:Lipopolysaccharides; C546386:P2ry2 protein, mouse; C546387:P2ry2 protein, rat; D011663:Pulmonary Surfactants; D018048:Receptors, Purinergic P2; D058473:Receptors, Purinergic P2Y2; D000255:Adenosine Triphosphate