Glycerol as a substrate for phospholipid biosynthesis in type II pneumocytes isolated from streptozotocin-diabetic rats

Glycerol and glucose utilization for phospholipid biosynthesis was examined in type II pneumocytes isolated from normal and streptozotocin-diabetic rats. In cells from diabetic rats, incorporation of [1,3-14C]glycerol into total phosphatidylcholine (PC), disaturated phosphatidylcholine (DSPC), phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) occurred to a greater degree by the glycerol 3-phosphate pathway as opposed to the dihydroxyacetone phosphate pathway. Total incorporation of glycerol into each of the major cellular phospholipids was increased up to 6-fold in cells from diabetic rats, while the total incorporation of glucose into the same lipids was decreased 2-fold. While the percentage of both glucose and glycerol carbons incorporated into the backbone of DSPC was increased in cells from diabetic rats, the percentage of carbons from both substrates incorporated into the fatty acid moieties was decreased. As a measure of DSPC synthesis, choline incorporation into DSPC was significantly decreased in type II cells from diabetic animals if the cells were incubated in the presence of glucose, palmitate and choline but not glycerol. Addition of 0.1 or 0.3 mM glycerol to the incubation medium restored choline incorporation to the control value in cells from diabetic rats, but did not affect the rate of choline incorporation into DSPC in cells from normal rats. These results suggest that exogenous glycerol can compensate for reduced glucose metabolism in type II cells of diabetic animals to maintain a constant rate of DSPC synthesis.     

Alterations in rat alveolar surfactant phospholipids and proteins induced by administration of chlorphentermine

Chlorphentermine is a cationic amphiphilic drug which produces a phospholipid storage disorder in rat lungs. Experiments were carried out to characterize changes in the composition of acellular alveolar lavage materials and to study possible mechanisms by which pulmonary surfactant phospholipidosis is produced by administration of the drug. Following ten daily injections of chlorphentermine (25 mg/kg body weight), there are 12.2- and 13.6-fold increases of pulmonary lavage total phospholipids and disaturated phosphatidylcholines (disaturated PC), respectively. In addition, there is a 2.8-fold increase in total protein and a 12.7-fold increase in the surfactant apoprotein group with molecular weights from 28,000 to 32,000. We measured incorporation of labeled palmitate, choline and glycerol into disaturated PC in type II cells and alveolar macrophages isolated from control and chlorphentermine-treated animals. The drug does not affect the incorporation of labeled substrates into disaturated PC in either cell type. However, in alveolar macrophages there is a decrease in the rate of intracellular degradation of recently synthesized disaturated PC in chlorphentermine-treated animals. The drug also inhibits the phospholipase-induced catabolism of rat surfactant disaturated PC which occurs during incubation of alveolar lavage fluid in vitro at 37 degrees C. When the lavage fluid is divided into subfractions by differential centrifugation, a larger percentage of the phospholipid is distributed in the less sedimentable subfractions in chlorphentermine-treated animals relative to controls, suggesting the accumulation of older surfactant materials. These results suggest that chlorphentermine-induced phospholipidosis of pulmonary surfactant materials is due to decreased rates of phospholipid degradation.     

Incorporation of [3H]palmitate into disaturated phosphatidylcholines in alveolar type II cells isolated by centrifugal elutriation

In order to study synthesis of pulmonary surfactant materials, we measured incorporation of [3H]palmitate into disaturated phosphatidylcholines (PC) in alveolar type II cells isolated by centrifugal elutriation. The time course for this process is not linear and, at high external palmitate levels (1 mM), incorporation is maximal in 4-5 h. Incorporation is dependent on extracellular palmitate with a Vmax (at 1 mM) of 1.66 nmol palmitate incorporated into disaturated PC/4.2 X 10(5) cells per 2 h and a K1/2 of 0.1 mM palmitate. Addition of an optimal amount of extracellular choline (0.05 mM) increases Vmax and decreases K1/2 for palmitate. Incorporation of palmitate is dependent upon cell number, inhibited by extracellular Ca2+ and stimulated by external Mg2+. Cholinergic and beta-adrenergic agonists do not increase incorporation. Pulmonary lavage fluid inhibits incorporation of palmitate into disaturated PC, suggesting there is negative feedback involved. Disaturated PC which has been recently synthesized (i.e., over a 2 h period) is broken down intracellularly by type II cells when they are suspended in palmitate-free medium. These results indicate that (1) several factors, such as substrate levels, cell number, Ca2+, Mg2+ and amount of surfactant present, are involved in the regulation of palmitate incorporation into disaturated PC; (2) disaturated PC which has been recently synthesized may be broken down by type II cells; and (3) surfactant synthesis in freshly isolated cells differs slightly from that reported by other investigators in type II cells maintained in primary cell culture.     

Phospholipid synthesis in isolated alveolar type II cells exposed in vitro to paraquat and hyperoxia.

Isolated alveolar epithelial type II cells were exposed to paraquat and to hyperoxia by gas diffusion through the thin Teflon bottom of culture dishes. After exposure, type II cells were further incubated in the presence of labelled substrates to assess their capacity to synthesize lipids. Hyperoxia alone (90% O2; 5 h) had minor effects on lipid metabolism in the type II cells. At low paraquat concentrations (5 and 10 microM), hyperoxia enhanced the paraquat-induced decrease of [Me-14C]choline incorporation into phosphatidylcholines. The incorporation rates of [Me-14C]choline, [1-14C]palmitate, [1-14C]glucose and [1,3-3H]glycerol into various phospholipid classes and neutral lipids were decreased by paraquat, depending on the concentration and duration of the exposure. The incorporation of [1-14C]acetate into phosphatidylcholines, phosphatidylglycerols and neutral lipids appeared to be very sensitive to inactivation by paraquat. At 5 microM-paraquat the rate of [1-14C]acetate incorporation was decreased to 50% of the control values. The rate of [1-14C]palmitate incorporation into lipids was much less sensitive; it even increased at low paraquat concentrations. At 10 microM-paraquat both NADPH and ATP were significantly decreased. It is concluded that lipid synthesis in isolated alveolar type II cells is extremely sensitive to paraquat. At low concentrations of this herbicide, lipid synthesis, and particularly fatty acid synthesis, is decreased. The effects on lipid metabolism may be partly related to altered NADPH and ATP concentrations.     

Altered lung phospholipid metabolism in mice with targeted deletion of lysosomal-type phospholipase A2

Lung surfactant dipalmitoylphosphatidylcholine (DPPC) is endocytosed by alveolar epithelial cells and degraded by lysosomal-type phospholipase A2 (aiPLA2). This enzyme is identical to peroxiredoxin 6 (Prdx6), a bifunctional protein with PLA2 and GSH peroxidase activities. Lung phospholipid was studied in Prdx6 knockout (Prdx6-/-) mice. The normalized content of total phospholipid, phosphatidylcholine (PC), and disaturated phosphatidylcholine (DSPC) in bronchoalveolar lavage fluid, lung lamellar bodies, and lung homogenate was unchanged with age in wild-type mice but increased progressively in Prdx6-/- animals. Degradation of internalized [3H]DPPC in isolated mouse lungs after endotracheal instillation of unilamellar liposomes labeled with [3H]DPPC was significantly decreased at 2 h in Prdx6-/- mice (13.6 +/- 0.3% vs. 26.8 +/- 0.8% in the wild type), reflected by decreased dpm in the lysophosphatidylcholine and the unsaturated PC fractions. Incorporation of [14C]palmitate into DSPC at 24 h after intravenous injection was decreased by 73% in lamellar bodies and by 54% in alveolar lavage surfactant in Prdx6-/- mice, whereas incorporation of [3H]choline was decreased only slightly. Phospholipid metabolism in Prdx6-/- lungs was similar to that in wild-type lungs treated with MJ33, an inhibitor of aiPLA2 activity. These results confirm an important role for Prdx6 in lung surfactant DPPC degradation and synthesis by the reacylation pathway.     

Culture of fetal alveolar epithelial type II cells in serum-free medium

Summary A serum-free culture medium (defined medium = DM) was elaborated by adding to Eagle’s minimum essential medium (MEM), non-essential amino acids, transferrin, putrescine, tripeptide glycyl-histidyl-lysine, somatostatin, sodium selenite, ethanolamine, phosphoethanolamine, sodium pyruvate, and metal trace elements. This medium was tested for its ability to support sustained surfactant biosynthesis in fetal alveolar epithelial type II cells. For up to 8 days, ultrastructure was maintained with persistance of lamellar inclusion bodies. Thymidine incorporation into DNA was enhanced about 50% in DM as compared with MEM, whereas it was enhanced 300% in 10% fetal bovine serum. With DM, the incorporation of tritiated choline into phosphatidylcholine (PC) of isolated surfactant material was about twice that with MEM. Deletion experiments evidenced the prominent role of pyruvate, transferrin, and selenium in the stimulation of surfactant PC biosynthesis. The addition of biotin to DM enhanced surfactant PC biosynthesis slightly and nonsurfactant PC biosynthesis markedly. The presence of nucleosides seemed unfavorable to the synthesis of surfactant PC. Type II cells responded to the addition of epidermal growth factor and insulinlike growth factor-I both by increased thymidine incorporation into DNA and choline incorporation into PC. It is concluded that DM represents a useful tool for cultivating type II cells without loss of their specialized properties and for studying the regulation of cell proliferation and surfactant biosynthesis in a controlled environment.     

Effects of intratracheal instillation of TNF-alpha on surfactant metabolism.

Tumor necrosis factor-alpha (TNF-alpha) has been shown to play an integral role in the pathogenesis of the acute respiratory distress syndrome. This disorder is characterized by a deficiency of alveolar surfactant, a surface-active material that is composed of key hydrophobic proteins and the major lipid disaturated phosphatidylcholine (DSPC). We investigated how TNF-alpha might alter DSPC content in rat lungs by instilling the cytokine (2.5 microg) intratracheally for 10 min and then assaying parameters of DSPC synthesis and degradation in alveolar type II epithelial cells, which produce surfactant. Cells isolated from rats given TNF-alpha had 26% lower levels of phosphatidylcholine compared with control. TNF-alpha treatment also decreased the ability of these cells to incorporate [(3)H]choline into DSPC by 45% compared with control isolates. There were no significant differences in the levels of choline substrate or choline transport between the groups. However, TNF-alpha produced a 64% decrease in the activity of cytidylyltransferase, the rate-regulatory enzyme required for DSPC synthesis. TNF-alpha administration in vivo also tended to stimulate phospholipase A(2) activity, but it did not alter other parameters for DSPC degradation such as activities for phosphatidylcholine-specific phospholipase C or phospholipase D. These observations indicate that TNF-alpha decreases the levels of surfactant lipid by decreasing the activity of a key enzyme involved in surfactant lipid synthesis. The results do not exclude stimulatory effects of the cytokine on phosphatidylcholine breakdown.     

Altered lipid synthesis in type II pneumonocytes exposed to lung surfactant.

When type II pneumonocytes were exposed to purified lung surfactant that contained 1-palmitoyl-2-[3H]palmitoyl-glycero-3-phosphocholine, radiolabelled surfactant was apparently taken up by the cells since it could not be removed by either repeated washing or exchange with non-radiolabelled surfactant, but was released when the cells were lysed. After 4 h of exposure to [3H]surfactant, more than half of the 3H within cells remained in disaturated phosphatidylcholine. Incorporation of [3H]choline, [14C]palmitate and [14C]acetate into glycerophospholipids was decreased in type II cells exposed to surfactant and this inhibition, like surfactant uptake, was half-maximal when the extracellular concentration of surfactant was approx. 0.1 mumol of lipid P/ml. Inhibition of incorporation of radiolabelled precursors by surfactant occurred rapidly and reversibly and was not due solely to dilution of the specific radioactivity of intracellular precursors. Activity of dihydroxyacetone-phosphate acyltransferase, but not glycerol-3-phosphate acyltransferase, was decreased in type II cells exposed to surfactant and this was reflected by a decrease in the 14C/3H ratio of total lipids synthesized when cells incubated with [U-14C]glycerol and [2-3H]glycerol were exposed to surfactant. Phosphatidylcholine, phosphatidylglycerol and cholesterol, either individually or mixed in the molar ratio found in surfactant, did not mimic purified surfactant in the inhibition of glycerophospholipid synthesis. In contrast, an apoprotein fraction isolated from surfactant inhibited greatly the incorporation of [3H]choline into lipids and this inhibitory activity was labile to heat and to trypsin. It is concluded that the apparent uptake of surfactant by type II cells in vitro is accompanied by an inhibition of glycerophospholipid synthesis via a mechanism that involves a surfactant apoprotein.     

Degradation of pulmonary surfactant disaturated phosphatidylcholines by alveolar macrophages

Experiments were performed to determine whether rat pulmonary surfactant disaturated phosphatidylcholines (DSPC) are degraded by alveolar macrophages in vitro. When [3H]choline-labeled surfactant materials are incubated with unlabeled alveolar macrophages, approximately 40% of the labeled DSPC is broken down in 6 h. There is just a slight decrease in the specific activity of DSPC, which suggests that most products of degradation are not reincorporated into DSPC, at least during the 6-h incubation period. There is a time- and temperature-dependent association of surfactant DSPC with alveolar macrophages, and some of the cell-associated materials are released from the cell fragments after sonication. Association of surfactant with the cells precedes degradation. The breakdown of surfactant DSPC by intact alveolar macrophages lags behind that produced by sonicated cell preparations with disrupted cell membranes. These data and other information suggest that the surfactant materials are internalized by the cells, before the breakdown. The products of degradation probably include free choline and fatty acids, most of which appear in the extracellular fluid. The breakdown processes do not seem to depend on the physical form of the surfactant or on the presence of surfactant apoproteins. Incubation of the cells alone also results in disappearance of intracellular DSPC, some of which may be surfactant phospholipid taken up by the cells in vivo. These results indicate that alveolar macrophages can degrade surfactant DSPC and suggest that these cells may be involved in catabolism of pulmonary surfactant materials.     

Altered phospholipid biosynthesis in type II pneumocytes isolated from streptozotocin-diabetic rats

To determine whether type II pneumocytes isolated from diabetic animals could serve as a useful model for the study of surfactant phospholipid biosynthesis and its regulation, type II pneumocytes were isolated from adult streptozotocin-diabetic rats and placed in short-term primary culture. On a DNA basis, total cellular disaturated phosphatidylcholine (disaturated PC) and phosphatidylglycerol (PG) were decreased 36 and 66%, respectively, in type II cells from diabetic animals. 7 days of insulin treatment of diabetic rats returned the cellular disaturated PC and PG content to control values and increased the total cellular phosphatidylethanolamine (PE) content by 51%. The rates of glucose and acetate incorporation into disaturated PC per unit DNA were reduced 32 and 38%, respectively, in cells isolated from diabetic rats, while glycerol incorporation was increased by 143%. Insulin treatment of diabetic rats returned the glucose and glycerol incorporation rates to control values and increased acetate incorporation into disaturated PC by 66%. These data suggest that the biosynthesis of surfactant is altered by both diabetes mellitus and in vivo insulin treatment.     

Subcellular site and mechanism of synthesis of disaturated phosphatidylcholine in alveolar type II cell adenomas.

The site of synthesis of 1,2-disaturated-(diacyl)-sn-glycero-3-phosphocholine (Sat2PC) in mouse alveolar type II cell adenomas has been studied by conducting pulse-chase experiments. Isolation of microsomal and lamellar body fractions from adenomas after a 20-min pulse with [methyl-3H]choline demonstrates that Sat2PC first appears in the microsomal fraction, and after a short lag subsequently appears in the lamellar body fraction. The kinetics of labeling of Sat2PC are consistent with the microsomal membranes functioning as the subcellular site of synthesis for this pulmonary surfactant phospholipid. Short term labeling experiments with [9,10-3H]palmitate demonstrate that this fatty acid is incorporated into the sn-2 position of Sat2PC at a faster rate than its incorporation into the sn-1 position. This finding indicates that the synthesis of Sat2PC occurs by a deacylation-reacylation mechanism.     

Type II pneumocyte changes during hyperoxic lung injury and recovery

Adult rabbits exposed to 100% O2 for 64 h and then returned to room air for up to 200 h, develop a lung injury characterized by decreased levels of alveolar surfactant followed by a rebound recovery. In the present study we isolated alveolar type II cells from rabbits at various times during hyperoxic exposure and recovery and measured rates of phosphatidylcholine (PC) synthesis, cellular lipid content, and the specific activity of glycerol 3-phosphate (G-3-P) acyltransferase, an enzyme that catalyzes one of the early reactions in phosphoglyceride biosynthesis. These biochemical parameters were compared with measurements of cell size and cell cycle phase by laser flow cytometry. Results showed that alterations in alveolar phospholipid levels in vivo correlated consistently with cellular lipid metabolic changes measured in isolated type II pneumocytes. In particular, alveolar pneumocytes isolated from lungs of rabbits exposed to 100% O2 for 64 h exhibited a 60% decrease in PC synthesis, cell lipid content, and G-3-P acyltransferase activity. All variables then followed a pattern of recovery to normal and ultimately supranormal levels beginning at approximately 3 days postexposure, at which point there was also a measured increase in the number of type II cells in S phase. These findings suggest that O2-induced changes in type II cell surfactant biosynthesis may account, at least in part, for observed changes in lung phospholipid levels in vivo.     

Incorporation of [3H]palmitate and [14C]choline into disaturated phosphatidylcholines in rat alveolar macrophages

We studied the synthesis of disaturated phosphatidylcholines in rat alveolar macrophages and, in some cases, compared it with that which occurs in isolated alveolar type II cells. Alveolar macrophages suspended in phosphate-buffered medium incorporate palmitate, choline and glycerol into disaturated phosphatidylcholines. The time-course for incorporation of palmitate into disaturated phosphatidylcholines is linear for 20-30 min and reaches a maximum in 2-3 h. Incorporation is dependent on extracellular palmitate with a Vmax (at 1 mM) of 1.53 nmol palmitate incorporated into disaturated phosphatidylcholines per 5 X 10(5) cells per 2 h and a K 1/2 of 0.19 mM palmitate. Exposure of the cells to zymosan particles increases incorporation of palmitate disaturated phosphatidylcholines by almost 2-fold, while cholinergic and beta-adrenergic agonists have no effect. On a per cell basis, alveolar macrophages incorporate only one-third to one-half as much palmitate into disaturated phosphatidylcholines as do type II cells isolated by centrifugal elutriation. The following results suggest there is extensive remodeling of disaturated phosphatidylcholines in alveolar macrophages: (1) palmitate- and choline-labeled disaturated phosphatidylcholines are catabolized by the cells; (2) the products of catabolism are palmitate and water-soluble choline products; (3) addition of unlabeled palmitate and choline to the medium enhances catabolism of the labeled phospholipid. Addition of oleate also enhances catabolism, suggesting that modification of phospholipids is not specific for the saturated variety. Some of the recently labeled disaturated phosphatidylcholines is released from alveolar macrophages into the extracellular space. Several possible functions of alveolar macrophage disaturated phosphatidylcholines are discussed.     

Surfactant-associated protein inhibits phospholipid secretion from type II cells

Secretion of [3H]phosphatidylcholine ([3H]PC) from isolated rat pulmonary type II epithelial cells was inhibited by the surfactant-associated protein of Mr = 35,000 (SAP-35) purified from canine lung surfactant. SAP-35 inhibited [3H]PC secretion in a dose-dependent manner and significantly inhibited basal, phorbol ester, beta-adrenergic, and P2-purinergic agonist-induced [3H]PC secretion. SAP-35 significantly inhibited [3H]PC secretion from 1 to 3 h after treatment. The IC50 for inhibition of [3H]PC secretion by canine SAP-35 was 1-5 X 10(-6) g/ml and was similar for inhibition of both basal and secretagogue-stimulated release. Heat denaturation of SAP-35, addition of monoclonal anti-SAP-35 antibody, reduction and alkylation of SAP-35, or association of SAP-35 with phospholipid vesicles reversed the inhibitory effect on secretagogue-induced secretion. Inhibitory effects of SAP-35 were observed 3 h after cells were washed with buffer that did not contain SAP-35. Although SAP-35 enhanced reassociation of surfactant phospholipid with isolated type II cells, its inhibitory effect on secretion of [3H]PC did not result from stimulation of reuptake of secreted [3H]PC by type II cells. The inhibition of phospholipid secretion by SAP-35 was also not due to inhibition of PC or disaturated PC synthesis by SAP-35. SAP-35, the major phospholipid-associated protein in pulmonary surfactant, is a potent inhibitor of surfactant secretion from type II cells in vitro and may play an important role in homeostasis of surfactant in the alveolar space.     

Stimulation of surfactant phospholipid biosynthesis in the lungs of rats treated with silica.

The effects of intratracheally instilled silica (10 mg/rat) on the biosynthesis of surfactant phospholipids was investigated in the lungs of rats. The sizes of the intracellular and extracellular pools of surfactant phospholipids were measured 7, 14 and 28 days after silica exposure. The ability of lung slices to incorporate [14C]choline and [3H]palmitate into surfactant phosphatidylcholine (PC) and disaturated phosphatidylcholine (DSPC) was also investigated. Both intra- and extra-cellular pools of surfactant phospholipids were increased by silica treatment. The intracellular pool increased linearly over the 28-day time period, ultimately reaching a size 62-fold greater than controls. The extracellular pool also increased, but showed a pattern different from that of the intracellular pool. The extracellular pool increased non-linearly up to 14 days, and then declined. At its maximum, the extracellular pool was increased 16-fold over the control. The ability of lung slices to incorporate phospholipid precursors into surfactant-associated PC and DSPC was elevated at all time periods. The rate of incorporation of [14C]choline into surfactant PC and DSPC was maximal at 14 days and was nearly 3-fold greater than the rate in controls. The rate of incorporation of [3H]palmitate was also maximal at 14 days, approx. 5-fold above controls for PC and 3-fold for DSPC. At this same time point, the microsomal activity of cholinephosphate cytidylyltransferase was increased 4.5-fold above controls, but cytosolic activity was not significantly affected by silica treatment. These data indicate that biosynthesis of surfactant PC is elevated after treatment of lungs with silica and that this increased biosynthesis probably underlies the expansion of the intra- and extra-cellular pools of surfactant phospholipids.     

Compartmentation of phosphorylated precursors of phospholipid biosynthesis in cultured neuroblastoma cells

The continuous turnover of membrane phospholipids requires a steady supply of biosynthetic precursors. We evaluated the effects of decreasing extracellular Na+ concentration on phospholipid metabolism in cultured neuroblastoma (N1E 115) cells. Incubating cultures with 145 to 0 mM NaCl caused a concentration-dependent inhibition of [32P]phosphate uptake into the water-soluble intracellular pool and incorporation into phospholipid. Phospholipid classes were differentially affected; [32P]phosphate incorporated into phosphati-dylethanolamine (PE) and phosphatidylcholine (PC) was consistently less than into phosphatidylinositol (PI) and phosphatidylserine (PS). This could not be attributed to decreased phospholipid synthesis since under identical conditions, there was no effect on arachidonic acid or ethanolamine incorporation, and choline utilization for PC synthesis was increased. The effect of Na+ was highly specific since reducing phosphate uptake to a similar extent by incubating cultures in a phosphate-deficient medium containing Na+ did not alter the relative distribution of [32P]phosphate in phospholipid. Of several cations tested only Li+ could partially (50%) replace Na+. Incubation in the presence of ouabain or amiloride had no effect on [32P]phosphate incorporation into phospholipid. The differential effects of low Na+ on [32P]phosphate incorporation into PI relative to PC and PE suggests preferential compartmentation of [32P]phosphate into ATP in pools used for phosphatidic acid synthesis and relatively less in ATP pools used for synthesis of phosphocholine and phosphoethanolamine, precursors of PC and PE, respectively. This suggestion of heterogeneous and distinct pools of ATP for phospholipid biosynthesis, and of potential modulation by Na+ ion, has important implications for understanding intracellular regulation of metabolism.     

Impaired pulmonary defense against Pseudomonas aeruginosa in VEGF gene inactivated mouse lung

Repeated bacterial and viral infections are known to contribute to worsening lung function in several respiratory diseases, including asthma, cystic fibrosis, and chronic obstructive pulmonary disease (COPD). Previous studies have reported alveolar wall cell apoptosis and parenchymal damage in adult pulmonary VEGF gene ablated mice. We hypothesized that VEGF expressed by type II cells is also necessary to provide an effective host defense against bacteria in part by maintaining surfactant homeostasis. Therefore, Pseudomonas aeruginosa (PAO1) levels were evaluated in mice following lung‐targeted VEGF gene inactivation, and alterations in VEGF‐dependent type II cell function were evaluated by measuring surfactant homeostasis in mouse lungs and isolated type II cells. In VEGF‐deficient lungs increased PAO1 levels and pro‐inflammatory cytokines, TNFα and IL‐6, were detected 24 h after bacterial instillation compared to control lungs. In vivo lung‐targeted VEGF gene deletion (57% decrease in total pulmonary VEGF) did not alter alveolar surfactant or tissue disaturated phosphatidylcholine (DSPC) levels. However, sphingomyelin content, choline phosphate cytidylyltransferase (CCT) mRNA, and SP‐D expression were decreased. In isolated type II cells an 80% reduction of VEGF protein resulted in decreases in total phospholipids (PL), DSPC, DSPC synthesis, surfactant associated proteins (SP)‐B and ‐D, and the lipid transporters, ABCA1 and Rab3D. TPA‐induced DSPC secretion and apoptosis were elevated in VEGF‐deficient type II cells. These results suggest a potential protective role for type II cell‐expressed VEGF against bacterial initiated infection. J. Cell. Physiol. 228: 371–379, 2013. © 2012 Wiley Periodicals, Inc.     

Comparison of effects of epidermal and insulin-like growth factors,gastrin releasing peptide and retinoic acid on fetal lung cell growth and maturation in vitro

The role in cell multiplication and maturation of several factors present in the late fetal lung was explored on isolated fetal rat pulmonary fibroblasts and alveolar epithelial type II cells cultivated in serum-free medium. The low degree of reciprocal contamination of each cell population was assessed by immunocytochemistry. Epidermal Growth Factor (EGF) stimulated thymidine incorporation and DNA accumulation in both cell types. In type II cells, it increased labeled-choline incorporation into surfactant phosphatidylcholine (PC), consistently with previous data obtained with lung explant cultures, but not into non-surfactant PC. Insulin-like growth factor (IGF)-I slightly stimulated DNA accumulation in fibroblasts although it did not significantly stimulate thymidine incorporation, contrary to IGF-II which presented a dose-dependent stimulating activity of thymidine incorporation. Neither IGF-I nor IGF-II stimulated type II cell growth. IGFs thus appear to primarily control the growth of lung mesenchyme. In type II cells, they stimulated the most non-surfactant PC biosynthesis. Gastrin releasing peptide (GRP) which was recently reported to promote fetal lung growth in vivo and to stimulate surfactant biosynthesis in lung organ culture revealed as a growth factor for type II cells only, at concentrations below 10 ⁻⁹ M. At concentration 10 ⁻⁸ M, although it did not affect DNA synthesis, GRP tended to increase surfactant and non-surfactant-PC biosynthesis. Retinoic acid inhibited thymidine incorporation into type II cells on a dose-dependent manner but nevertheless enhanced surfactant-PC biosynthesis to a similar extent as EGF. It is suggested that retinoic acid may represent a differentiation or maturation factor for the alveolar epithelium.     

The synthesis of phosphatidylcholine by adult rat lung alveolar type II epithelial cells in primary culture

1. The formation of phosphatidylcholine from radioactive precursors was studied in adult rat lung alveolar type II epithelial cells in primary culture. 2. The incorporation of [Me-14C]choline into total lipids and phosphatidylcholine was stimulated by addition of palmitate, whereas the incorporation of [U-14C]glucose into phosphatidylcholine and disaturated phosphatidylcholine was stimulated by addition of choline. Addition of glucose decreased the absolute rate of incorporation of [1(3)-3H]glycerol into total lipids, phosphatidylcholine and disaturated phosphatidylcholine, decreased the percentage [1(3)-3H]glycerol recovered in phosphatidylcholine, but increased the percentage phosphatidylcholine label in the disaturated species. 3. At saturating substrate concentrations, the percentages of phosphatidylcholine radioactivity found in disaturated phosphatidylcholine after incubation with [1-(14)C]acetate (in the presence of glucose) [1-(14)C]palmitate (in the presence of glucose), [Me-14C]choline (in the presence of glucose and palmitate) and [U-14C]glucose (in the presence of choline and palmitate) were 78, 75, 74 and 90%, respectively. 4. Fatty acids stimulated the incorporation of [U-14C]glucose into the glycerol moiety of phosphatidylcholine. The degree of unsaturation of the added fatty acids was reflected in the distribution of [U-14C]glucose label among the different molecular species of phosphatidylcholine. It is suggested that the glucose concentration in the blood as related to the amount of available fatty acids and their degree of unsaturation may be factors governing the synthesis of surfactant lipids.     

Comparison of effects of epidermal and insulin-like growth factors, gastrin releasing peptide and retinoic acid on fetal lung cell growth and maturation in vitro.

The role in cell multiplication and maturation of several factors present in the late fetal lung was explored on isolated fetal rat pulmonary fibroblasts and alveolar epithelial type II cells cultivated in serum-free medium. The low degree of reciprocal contamination of each cell population was assessed by immunocytochemistry. Epidermal Growth Factor (EGF) stimulated thymidine incorporation and DNA accumulation in both cell types. In type II cells, it increased labeled-choline incorporation into surfactant phosphatidylcholine (PC), consistently with previous data obtained with lung explant cultures, but not into non-surfactant PC. Insulin-like growth factor (IGF)-I slightly stimulated DNA accumulation in fibroblasts although it did not significantly stimulate thymidine incorporation, contrary to IGF-II which presented a dose-dependent stimulating activity of thymidine incorporation. Neither IGF-I nor IGF-II stimulated type II cell growth. IGFs thus appear to primarily control the growth of lung mesenchyme. In type II cells, they stimulated the most non-surfactant PC biosynthesis. Gastrin releasing peptide (GRP) which was recently reported to promote fetal lung growth in vivo and to stimulate surfactant biosynthesis in lung organ culture revealed as a growth factor for type II cells only, at concentrations below 10(-9) M. At concentration 10(-8) M, although it did not affect DNA synthesis, GRP tended to increase surfactant and non-surfactant-PC biosynthesis. Retinoic acid inhibited thymidine incorporation into type II cells on a dose-dependent manner but nevertheless enhanced surfactant-PC biosynthesis to a similar extent as EGF. It is suggested that retinoic acid may represent a differentiation or maturation factor for the alveolar epithelium.