ABSTRACT
Lung surfactant is secreted from epithelial type II cells into alveolar airspace in response to airborne and circulating stimuli. Nitric oxide (NO) can be generated by constitutive and inducible nitric oxide synthases (cNOS and iNOS) in pulmonary endothelial and epithelial cells. The authors therefore examined the effects of NO on lung surfactant secretion using an isolated perfused rat lung model and primary culture of type II cells. Infusion of L-N(G)-nitroarginine methyl ester (L-NAME) (100 micro M), an inhibitor of cNOS and iNOS, via pulmonary circulation for 90 minutes resulted in a decrease of lung surfactant secretion (1.55%+/-0.15% in control versus 0.79%+/-0.16% in L-NAME-treated lungs, P <.05). However, aminoguanide, an inhibitor of iNOS, had no effect, indicating that the decline of lung surfactant secretion is due to the specific blockage of cNOS rather than iNOS activity in perfused lungs. A reduction of cGMP level by 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (25 micro M), a specific inhibitor of guanylyl cyclase, inhibited surfactant secretion by 64%. Furthermore, KT5823 (1 micro M), an inhibitor of protein kinase G, depressed surfactant secretion by 40%. These results suggest that physiological concentrations of NO are required for lung surfactant secretion and NO-mediated secretion is at least partly via a rise of cGMP level and activation of protein kinase G. In primary culture of alveolar type II cells, spermine NONOate (SPER/NO), a NO donor, increased basal phosphatidylcholine (PC) secretion in a dose-dependent manner. Maximal stimulation was observed at 1 micro M. However, in the ATP-stimulated type II cells, SPER/NO displayed a biphasic effect on PC secretion. At low concentrations (0.1 to 1 micro M), SPER/NO increased ATP-stimulated PC secretion, whereas at a high concentration (100 micro M), SPER/NO inhibited the secretion. The results suggest that NO may play an important role in lung surfactant secretion.
Subject(s)
Epithelial Cells/metabolism , Nitric Oxide/metabolism , Pulmonary Alveoli/metabolism , Pulmonary Surfactants/metabolism , Spermine/analogs & derivatives , Animals , Carbazoles/pharmacology , Cells, Cultured , Cyclic GMP/metabolism , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Guanidines/pharmacology , Indoles/pharmacology , Male , NG-Nitroarginine Methyl Ester/pharmacology , Nitric Oxide/antagonists & inhibitors , Nitrogen Oxides , Oxadiazoles/pharmacology , Perfusion , Phosphatidylcholines/metabolism , Pulmonary Alveoli/drug effects , Quinoxalines/pharmacology , Rats , Rats, Sprague-Dawley , Spermine/pharmacologyABSTRACT
We investigated the effect of nitric oxide (NO) donors on the activities of annexin II tetramer (AIIt), a member of the Ca2+- dependent phospholipid-binding protein family. Incubation of purified AIIt with S-nitrosoglutathione (GSNO) led to the inhibition of AIIt-mediated liposome aggregation. This effect was dose-dependent with an IC50 of approximately 100 micro m. Sodium nitroprusside, another NO donor also inhibited AIIt-mediated liposome aggregation, whereas reduced glutathione, nitrate, or nitrite had no effects. GSNO also inhibited AIIt-mediated membrane fusion, but not the binding of AIIt to the membrane. GSNO only has a modest effect on liposome aggregation mediated by annexins I, III or IV. The binding of AIIt to the membrane protected the reactive sites of GSNO on AIIt. GSNO did not inhibit AIIt-mediated liposome aggregation in the presence of dithiothreitol. Taken together, our results suggest that GSNO inactivates AIIt possibly via S-nitrosylation and/or the formation of disulfide bonds.
