The aims of the research proposed are to study the intracellular mechanisms by which surfactant secretion is regulated. The secretion-inhibiting properties of the surfactant protein group of MW 28-36,000 (SP-A) will be used as a tool to characterize better the coupling of stimulus to secretion. To that end, the mechanism(s) by which SP-A inhibits both basal type II cell secretion and secretion stimulated by all agonists tested, we will focus on the very early or very late exocytotic events that are most likely to be common to secretion initiated by different stimuli. How SP-A interacts with the surface of type II cells will be examined through characterization of the binding and possible internalization of 125-I- or fluorescently labelled-SP-A by type II cells. The effect of preventing internalization of SP-A coupled to Sepharose will also be tested. The intracellular second messengers, and study of cells tested with AlCl3/NaF in order to evaluate the potential role of guanine regulatory (G) proteins. The potential role of G proteins in the regulation of later secretory events, and the possible involvement of SP-A will be tested in permeabilized type II cells. The effect of SP-A on the electrophysiological properties of type II cells will be tested using microelectrode techniques. SDS/PAGE and autoradiography will be used to determine whether treatment of stimulated cells with SP-A alters patterns of protein phosphorylation in type II cells. The role of SP-A in altering lamellar body translocation will be studied using electron microscopy to localize lamellar bodies relative to the plasma membrane of stimulated cells. SP-A mediated effects on cytoskeletal organization will be examined in fixed cells with indirect immunofluorescene and in living cells with fluorescent analogue cytochemistry following microinjection. Various models will be used to study the effect of SP-A on lamellar body/plasma membrane fusion.
| Pian, M S; Dobbs, L G (1995) Evidence for G beta gamma-mediated cross-talk in primary cultures of lung alveolar cells. Pertussis toxin-sensitive production of cAMP. J Biol Chem 270:7427-30 |
| Pian, M S; Dobbs, L G (1994) Activation of G proteins may inhibit or stimulate surfactant secretion in rat alveolar type II cells. Am J Physiol 266:L375-81 |
