Distribution and characteristics of Ca +2- phosphatidylserine-dependent protein kinase C in subcellular fractions and lamellar bodies of adult rabbit lung

Abstract

Pulmonary surfactant prevents lung collapse at minimal alveolar diameter. Since surfactant acts extracellularly, secretion is vitally important in regulating the alveolar surfactant levels. Studies with phorbol esters which stimulate protein kinase C (PKC) activity suggest PKC is involved in regulating surfactant secretion. This study was done to characterize PKC activity in adult rabbit lung fractions. Lungs were removed, homogenized and subcellular fractions prepared by centrigugation on a discontinuous sucrose gradient. Calcium-phosphatidylserine-dependent PKC activity was assayed in fractions in the presence of 4ωM phorbol 12-myristate 13-acetate, 1mM EDTA, 8 mole% phosphatidylserine and 1mM Ca 2+ by measuring the transfer of 32P from [γ- 32P]ATP to protein. Concurrent assays were done without Ca +2 or PS. Ca +2-dependent PKC activity was defined as the difference between the two. Select fractions were incubated with PKC inhibitors sangivamycin, acridine orange or 9-aminoacridine and activity measured. The results showed the majority of the PKC activity was in the cytosolic fraction (87%, specific activity, 142 pmoles/min/mg) but the lamellar bodies also appeared to contain a small amount of PKC activity (∼4.0%, 151 pmoles/min/mg). PKC inhibitors were used to examine the characteristics of the enzyme in the microsomal and lamellar body fractions. Sangivamycin was the most potent inhibitor. Some differences in the inhibition characteristics between the lamellar body and microsomal fractions were observed. However using an add-back approach with the lamellar body fraction, indicated that the small quantity of activity in this fraction be attributed to contamination by microsomes. These results indicate that PKC is active in adult rabbit lung subcellular compartments but is probably not associated with the intracellular surfactant storage organelles.