Avinash Chander

Isolation of lamellar bodies from rat granular pneumocytes in primary culture

A lamellar body fraction was isolated from rat alveolar granular pneumocytes in primary culture by upward flotation on a discontinuous sucrose gradient and compared with a similar fraction isolated from lung homogenates. Lamellar bodies from granular pneumocytes were free of detectable contamination with either succinate dehydrogenase or NADPH-cytochrome c reductase. There was an enrichment of acid phosphatase activity, which, based on distribution of enzyme activity on the gradient, did not appear to be a contamination from other fractions. The lamellar body fraction of granular pneumocytes yielded approx. 1 microgram protein/10(6) cells with a phospholipid-to-protein ratio (mg/mg) of 9.6 +/- 0.4 (n = 7). Composition with respect to total phospholipids was 71.0% phosphatidylcholine (disaturated phosphatidylcholine, 45.2%), 8.4% phosphatidylglycerol and 12.8% phosphatidylethanolamine. Palmitic acid comprised 66% of the fatty acids in phosphatidylcholine and 34% of those in phosphatidylglycerol. The lamellar body fraction from granular pneumocytes was similar to that from whole lung with respect to phospholipid-to-protein ratio and phospholipid composition and showed only minor differences in fatty acid composition. Ultrastructurally, lamellar bodies showed generally intact limiting membranes and lamellated structure. Lamellar bodies from granular pneumocytes showed occasional multinucleated whorls which were not seen in those isolated from lung homogenates. This study describes a method for preparing a homogeneous fraction of intact lamellar bodies from small amounts of material (6 X 10(7) granular pneumocytes). The yield on a per cell basis was higher when compared with a similar preparation from whole lung, although overall yield is small, due to loss of cells during the cell isolation procedure. This preparation may be useful to evaluate the role of lamellar bodies in the synthesis and secretion of lung surfactant by isolated granular pneumocytes.

In vitro fusion of lung lamellar bodies and plasma membrane is augmented by lung synexin.

Lamellar bodies of lung epithelial type II cells undergo fusion with plasma membrane prior to exocytosis of surfactant into the alveolar lumen. Since synexin from adrenal glands promotes aggregation and fusion of chromaffin granules, we purified synexin-like proteins from bovine lung cytosolic fraction, and evaluated their effect on the fusion of isolated lamellar bodies and plasma membrane fractions. Synexin activity, which co-purified with an approx. 47 kDa protein (pI 6.8), was assessed by following calcium-dependent aggregation of liposomes prepared from a mixture of phosphatidylcholine:phosphatidylserine (PC:PS, 3:1, mol/mol). Lung synexin caused aggregation of liposomes approximating lung surfactant lipid-like composition, isolated lamellar bodies, or isolated plasma membrane fraction. Lung synexin promoted fusion only in the presence of calcium. It augmented fusion between lamellar bodies and plasma membranes, lamellar bodies and liposomes, or between two populations of liposomes. However, selectivity with regard to synexin-mediated fusion was observed as synexin did not promote fusion between plasma membrane and liposomes, or between liposomes of surfactant lipid-like composition and other liposomes. These observations support a role for lung synexin in membrane fusion between the plasma membrane and lamellar bodies during exocytosis of lung surfactant, and suggest that such fusion is dependent on composition of interacting membranes.

Meconium Increases Surfactant Secretion in Isolated Rat Alveolar Type II Cells

One of the underlying causes of pathophysiology of meconium aspiration syndrome is access of meconium to the alveolar space and inhibition of activity of lung surfactant. This study examines the effects of meconium on type II cell function by following surfactant secretion. Isolated rat alveolar type II cells were labeled with [methyl-3H]choline during the initial 21-22 h of incubation. During the subsequent 150 min of incubation, phosphatidylcholine (PC) secretion in the presence of 1% meconium was increased 250 ± 11% (mean ± SE, n = 23) over controls. The secretagogue effect was concentration-dependent and reached a maximum at 0.5% meconium. The meconium effect was not due to cellular toxicity as evaluated by vital dye exclusion, lactate dehydrogenase release, and PC synthesis. The secretagogue effect of meconium was associated with the particulate fraction pelleted by centrifugation of the suspension for 1 h at 100,000 × g. Heat treatment of meconium decreased the effect, suggesting the active component to be a protein. The effect of meconium was additive with that of 0.1 mM terbutaline, or 1 mM ATP, suggesting different pathways of action of each agent. The effect of meconium was reduced in the presence of 0.1 mM 4,4′-diisothiocyanato-2,2′-disulfonic acid, or 100 ng/mL surfactant protein A. These agents were previously shown to inhibit surfactant secretion in a stimulus-independent manner. Our results suggest that meconium at low concentrations is not toxic to type II cells, and a component of meconium, possibly a protein, increases PC secretion.

Choline-phosphate cytidyltransferase activity and phosphatidylcholine synthesis in rat granular pneumocytes are increased with exogenous fatty acids

We investigated the effect of exogenous fatty acids on phosphatidylcholine (PC) and disaturated phosphatidylcholine (DSPC) synthesis by rat granular pneumocytes in primary culture. Synthesis of PC and DSPC from [3H-methyl]choline, as evaluated by increasing specific activity (pmol choline incorporated/microgram phosphorus), was linear for 3 h. Exogenous palmitic, oleic, linoleic, or linolenic acid (100 microM each) increased the synthesis of PC by approx. 50% during incubation for 3 h. In contrast, synthesis of DSPC was increased only by palmitic acid. The increase in DSPC synthesis was approx. 150% after 3 h. Conversion of choline phosphate to PC was increased in the presence of palmitic or oleic acid as indicated by pulse-chase studies with [3H-methyl]choline in the intact cells. Cells incubated for 3 h with either oleic or palmitic acid showed increased choline-phosphate cytidyltransferase activity in the cells and the microsomal fraction. In addition, oleic acid increased the activity of this enzyme in the cytosolic fraction. The distribution of this enzyme in cytosolic and microsomal fraction was 24 and 76% in the cells incubated with palmitic acid and 32 and 68% in control cells. These results suggest that exogenous fatty acids stimulate the de novo pathway of PC synthesis in granular pneumocytes by increasing the microsomal choline-phosphate cytidyltransferase activity.

Inhibition of Trimeresurus flavoviridis phospholipase A2 by lung surfactant protein A (SP-A).

A marked sequence homology has been noted between lung surfactant protein A (SP-A) and an inhibitor of phospholipase A2 (PLA2) isolated from the serum of Trimeresurus flavoviridis (Habu snake). This study evaluated the effect of SP-A on PLA2 activity from several sources. SP-A was isolated from bovine or rat lung surfactant by extraction with 1-butanol and octyl beta-D-glucopyranoside. The addition of SP-A produced a concentration-dependent inhibition of T. flavoviridis PLA2 that indicated non-competitive kinetics with Ki 5 micrograms/ml. Inhibition was reversed by heat inactivation, disulfide bond reduction or alkylation of SP-A, or by the presence of anti-SP-A antibody. Treatment of SP-A with endoglycosidase F or the presence of variation monosaccharides or lectins did not alter SP-A inhibition. Binding of PLA2 to SP-A was shown by ultrafiltration and was abolished by SP-A alkylation or the presence of SDS. The SP-A/PLA2 complex recovered from the ultrafilter had essentially no enzymatic activity, but activity was restored by treatment with mercaptoethanol. SP-A had no effect on activity of PLA2 from Naja naja, Crotalus atrox, or bovine pancreas. These results indicate that surfactant protein A selectively inhibits Trimeresurus phospholipase A2 activity and suggest that binding to the enzyme is the mechanism for inhibition.

“Hydrophobic” Surfactant Apoproteins and Augmentation of Phospholipid Recycling

A 10,000-11,000 molecular weight apoprotein was isolated from an ethanol-ether extract of rat lung surfactant and purified by silicic acid chromatography. The protein (Apo Et) significantly augmented the uptake of phospholipids in liposomal form by cultured rat granular pneumocytes by a time-dependent process that varied with protein concentration and liposome composition. The protein had no effect on cell viability and showed no phospholipase activity. The mechanism for this augmented phospholipid uptake is not known but could be due to an alteration of physical form of the phospholipids by the protein or to a receptor-mediated uptake of phospholipids. This protein may prove to be a physiologically important regulator of the recycling of lung surfactant phospholipids.

Introduction: Lung Surfactant- Phospholipids and Apoproteins

Evidence accumulated over the past 50 years has indicated that the alveolar surface of the lung is lined by a material that when tested in vitro has marked surface activity [1, 2]. This material, called the lung surfactant, can be isolated in relatively pure form through centrifugation procedures such as those described by King and Clements [3]. Surfactant as isolated by this method is a mixture of lipids, proteins, and carbohydrate [4, 5], with phospholipid accounting for 75-80% of the total weight (Table 1). Dipalmitoyl phosphatidylcholine accounts for nearly half and other phosphatidylcholines account for approximately one-third of the total phospholipid. Recently, considerable attention has focussed on phosphatidylglycerol, which comprises only 6-12% of the phospholipid [6], but even this amount is uniquely high compared with other tissues. Cholesterol, the major neutral lipid present in surfactant, accounts for 8% or more by weight of the total surfactant. These lipid components are responsible for s...

Glycerol kinase activity and glycerol metabolism of rat granular pneumocytes in primary culture

Glycerol kinase activity and glycerol utilization by rat granular pneumocytes were determined in order to investigate the rate-limiting step for glycerol incorporation into lung lipids. Granular pneumocytes were isolated in primary culture following trypsinization of rat lungs. Glycerol kinase activity was 8.2 nmol/h per 10(6) cells. Incorporation of [1,3-14C]glycerol into total cell lipids was 0.29 nmol/h per 10(6) cells. In the presence of saturating glycerol concentration, production of 3H2O from [2-3H]glycerol was 13 times greater than incorporation of [14C]glycerol into lipids. Glycerol phosphate dehydrogenase activity in isolated cells was approximately 10 times glycerol kinase activity. In the presence of 5.6 mM glucose, glycerol incorporation into lipids was decreased 79% and detritiation of glycerol was decreased 34%. This effect of glucose was due to a 25% increase in cell glycerol 3-phosphate content, resulting in dilution of the precursor pool and possible inhibition of glycerol phosphorylation. These results indicate that the relatively limited incorporation of glycerol into surfactant phospholipids by lung epithelial cells reflects the relatively high rate of glycerol 3-phosphate oxidation.

Lung Mechanics and Inflammatory Response in Meconium Injured Rats following Lung Lavage with Perfluorochemical or KL4 Surfactant † 1757

Lung Mechanics and Inflammatory Response in Meconium Injured Rats following Lung Lavage with Perfluorochemical or KL4 Surfactant † 1757

Stimulation of the methylation pathway for phosphatidylcholine synthesis in rat lungs by choline deficiency

The methylation of phosphatidylethanolamine (PE) to form phosphatidylcholine (PC) was investigated using the isolated rat lung perfused with radiolabeled ethanolamine. Lungs from choline-deficient rats showed increased incorporation of radiolabel into PC at 2 h of perfusion. Increased PC synthesis from PE was also observed with lungs from rats fed a lipotrophic (choline plus methionine deficient) diet when methionine was added to the lung perfusate. These results indicate increased activity of the methylation pathway for lung PC synthesis during choline deficiency.