Alveolar surfactant is known to exist in at least 3 subtypes that are in metabolic relation with each other: a lamellar body form that evolves in sequence into tubular myelin and vesicular form. A unique way of mimicking this metabolic sequence in vitro has been developed in this laboratory, based on repeated cyclic expansion and contraction of the air-fluid interface of surfactant at 38 C (""""""""cycling""""""""). Manipulating nascent surfactant in this way generates products that exactly resemble the subtypes found in alveolar lavage fluid with respect to phospholipid and protein content, buoyant density, ultrastructure, and surface properties. This technique makes it possible for the first time to study the extra-cellular metabolism of surfactant in detail. Using this methodology, the applicant found that any of large number of serine protease inhibitors inhibited the conversion of tubular myelin to the vesicular form on cycling in vitro, suggesting that a serine protease (called """"""""convertase"""""""" for convenience) was required for this conversion. Alpha-1 antitrypsin was found to be the most potent inhibitor, resulting in 50% inhibition at in vivo alveolar concentrations suggesting physiologic relevance. The enzyme, which appears to be concentrated in a lamellar body-rich preparation and secreted with its substrate, has a number of unique biochemical properties including the absolute requirement for a surface that is cyclically expanded and contracted. The present project seeks to purify the convertase, identify its cell source, and explore some of its actions on the physiology and turnover of alveolar surfactant. Using an assay of the convertase developed for this purpose, the enzyme will be purified from a lamellar body-rich preparation of rat lungs using affinity chromatography. Purity will be checked by 2D-SDS-PAGE and Western transfer of the 3H-DFP reacted yield. Its cellular source will be determined using HRP-linked antibody raised in rabbits against the purified enzyme. The applicants propose to investigate the in vivo physiologic role of convertase by examining the effect of a1-antitrypsin inhibition on the surface tension properties of nascent surfactant, and determining the effect of administering to mice a 5-10 fold excess of a1-AT on surfactant subtype proportions, turnover rates, and lung pressure-volume behavior.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL045782-04
Application #
2222479
Study Section
Respiratory and Applied Physiology Study Section (RAP)
Project Start
1991-01-01
Project End
1995-12-31
Budget Start
1994-01-01
Budget End
1995-12-31
Support Year
4
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Edward Hines Jr VA Hospital
Department
Type
DUNS #
City
Hines
State
IL
Country
United States
Zip Code
60141
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Krishnasamy, S; Gross, N J; Teng, A L et al. (1997) Lung ""surfactant convertase"" is a member of the carboxylesterase family. Biochem Biophys Res Commun 235:180-4
Gross, N J; Veldhuizen, R; Possmayer, F et al. (1997) Surfactant convertase action is not essential for surfactant film formation. Am J Physiol 273:L907-12
Gross, N J; Bublys, V; D'Anza, J et al. (1995) The role of alpha 1-antitrypsin in the control of extracellular surfactant metabolism. Am J Physiol 268:L438-45
Gross, N J (1995) Extracellular metabolism of pulmonary surfactant: the role of a new serine protease. Annu Rev Physiol 57:135-50
Gross, N J; Schultz, R M (1992) Requirements for extracellular metabolism of pulmonary surfactant: tentative identification of serine protease. Am J Physiol 262:L446-53
Gross, N J (1991) Inhibition of surfactant subtype convertase in radiation model of adult respiratory distress syndrome. Am J Physiol 260:L311-7
Gross, N J (1991) Surfactant subtypes in experimental lung damage: radiation pneumonitis. Am J Physiol 260:L302-10
Gross, N J; Holloway, N O; Narine, K R (1991) Effects of some nonsteroidal anti-inflammatory agents on experimental radiation pneumonitis. Radiat Res 127:317-24