Alveolar type II cell cNOS activity and ATP levels are increased by lung surfactant or DPPC vesicles

P. R. Miles, L. Bowman, A. Rengasamy and L. Huffman Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, West Virginia, USA. In a previous study, we reported that nitric oxide (.NO) affects surfactant synthesis and ATP levels in alveolar type II cells and suggested that there is constitutive nitric oxide synthase (cNOS) activity in the cells. In the present study, we performed experiments to confirm further the presence of cNOS and to determine the effects of lung surfactant on type II cell .NO and ATP levels. The supernatant from freshly isolated cells contains .NO (0.26 +/- 0.08 nmol/10(6) cells). During incubation, the cells produce additional .NO at a rate of approximately 0.3 nmol.10(5) cells-1.h-1. .NO formation is inhibited by 28-46% by three inhibitors of cNOS and inducible NOS (iNOS), NG-monomethyl-L-arginine (L-NMMA), L-N5-(1-iminoethyl)ornithine hydrochloride, and NG-nitro-L-arginine methyl ester, but a specific inhibitor of iNOS, aminoguanidine, has no effect. The production of .NO is reduced in Ca(2+)-free medium, is stimulated by the Ca2+ ionophore A-23187, and is independent of extracellular L-arginine. One known type of cNOS, endothelial NOS (eNOS), can be detected in the cells by using Western blot analysis. Incubation of the cells with lung surfactant leads to a relatively rapid (approximately 15 min), concentration-dependent increase in .NO formation that reaches levels as high as 238 +/- 14% of control. The surfactant effects appear to be caused by its major component, dipalmitoyl phosphatidylcholine (DPPC). Exposure of type II cells to DPPC results in maximal increases in .NO formation, ATP content, and O2 consumption, which are 268 +/- 32, 234 +/- 24, and 131 +/- 6% of control, respectively. The DPPC-induced increases in .NO, ATP, and O2 consumption are inhibited by L-NMMA. These results confirm the presence of type II cell cNOS and suggest that it may have a role in the cellular processing of lung surfactant.