Localization of Phosphatidic Acid Phosphatase Activity in Granular Pneumonocytes

Abstract

The alveoli of the mammalian lung are lined by a film of surface-active material (surfactant) which is commonly believed to be a phospholipid (1,5,8,11) . A considerable body of evidence has accumulated which suggests that surfactant is synthesized by the granular pneumonocytes (type II cells) of the alveolar epithelium (1, 9, 10) . Although a number of acid hydrolases have been demonstrated in the inclusion bodies of granular pneumonocytes (4, 6), no attempt has been made to localize enzymes specifically concerned with phospholipid metabolism . Phosphatidic acid phosphatase is of particular interest because of its key role in the biosynthesis of phosphatides . Unfortunately, this enzyme cannot be demonstrated by conventional histochemical methods because of the insolubility of the substrate (phosphatidic acid) in water . Stable emulsions of phosphatidic acid can be prepared but these do not diffuse into cells . One method of overcoming this difficulty is to inflict minor localized damage on cells and expose the organelles to an emulsion of phosphatidic acid in a suitable medium .A method of producing minor trauma by osmotic forces has been developed to enable the localiza-THE JOURNAL OF CELL BIOLOGY . VOLUME 53, 1972 . pages 249-252 tion of phosphatidic acid phosphatase to be studied in granular pneumonocytes . MATERIALS AND METHODSYoung adult hamsters of 150-200 g body weight were killed by cervical dislocation, and small blocks of lung tissue were excised and fixed for 2 hr in ice-cold 3% glutaraldehyde in 0 .1 M cacodylate buffer (pH 7 .2) containing 7 .5% sucrose . After fixation the blocks were washed for 20 min in 0 .1 M cacodylate buffer (pH 7 .2) containing 7 .5% sucrose, and then 0 .5 mm slices were cut freehand. The tissue slices were incubated at 37°C for 15-90 min in a freshly prepared medium containing phosphatidic acid as substrate . The medium consisted of 3 ml of 0.1 M acetate buffer (pH 5 .1) containing 3% sucrose, 5 ml of distilled water, 1 ml of 5% lead nitrate solution, and 1 ml of 2% phosphatidic acid (Sigma London Chemical Co ., London, England) emulsion prepared by the method of Clark and Hiibscher (2) . The medium was preheated to 37°C before use . Control sections were incubated in substrate-free media or in complete media containing 0.01 M sodium fluoride . After incubation the tissues were washed briefly in cacodylate buffer, postfixed in cacodylate bufferOs04 (1%), dehydrated in a series of graded alcohols, and processed through propylene oxide to Araldite CY212 . Ultrathin sections were examined

Fetal Lung Maturation

“…An increase in concentration of the latter compound in human amniotic fluid has been utilized to evaluate fetal lung maturation [4]. Since PAPase has been shown to be present in the type II cells [6], which are the site of synthesis of most of the surfactant [7], it is conceivable that parallel to the discharge of the surfactant there also occurs a release of the enzymes associated with phos pholipid biosynthesis. The purpose of the present study was to determine whether or not phosphatidic acid phosphohydrolase could be detected in human amniotic fluid and if so to examine any possible changes in its specific activity at various stages of gestation.…”

Further ultrastructural observations on the alveolar epithelium of the newborn rat

“…17 mannosidase, p-D-galactosidase, and pnitrophenylacetate esterase. Meban (1972) demonstrated phosphatidic acid phosphatase activity in the lamellated bodies; this is of particular interest, as it provides evidence that the catabolic activity of the bodies can extend to membrane phospholipid substrates.…”

Properties of an acid phosphatase in pulmonary surfactant.

“…Dioleoylphosphatidate (0.3 mM) was suspended in 0.16 ml of reaction mixture of 50 mM sodium maleate (pH 6.5) containing [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] jig of protein. The reaction was carried out at 370C for 30 or 60 min.…”

“…Meban (12), using electron microscopic histochemistry, reported the presence of phosphatidate phosphatase (phosphatidate phosphohydrolase, EC 3.1.3.4) in intracellular lamellar bodies. Spitzer et al (13) also found the enzyme in their preparations of isolated lamellar bodies, whereas Garcia et al (14) found no phosphatidate phosphatase in their lamellar body preparations that they could not attribute to microsomal contamination.…”