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We reported previously that metyrapone inhibited the maturational effect of adrenocorticotropin in the fetal sheep lung, even in the presence of exogenous glucocorticoids. To examine the role of beta-adrenergic input in this response we examined lung maturation in fetal sheep treated for 100 hours in vivo with adrenocorticotropin (66 ng/min for 15 minutes every 2 hours, n = 5); adrenocorticotropin plus propranolol (40 micrograms/min, n = 4), or saline solution (n = 8). Pulmonary maturation was assessed by pressure-volume curves, phospholipid content, and morphologic features. The basal cortisol level rose from less than 5 to 32.0 +/- 12.1 and 83.5 +/- 8.0 ng/ml in the adrenocorticotropin and adrenocorticotropin plus propranolol groups, respectively. The adrenal:body weight ratio (X 10(-4)) rose from 1.43 +/- 0.12 in the saline solution group to 2.90 +/- 0.16 and 2.51 +/- 0.14 in the adrenocorticotropin and adrenocorticotropin plus propranolol groups, respectively. Lung distensibility (milliliters of air per gram of lung) rose from 1.10 +/- 0.14 to 1.90 +/- 0.20 in the adrenocorticotropin group but was unchanged (0.98 +/- 0.24) in the adrenocorticotropin plus propranolol group. Phosphatidylcholine (milligrams per gram of lung) in the lung lavage rose from 0.07 +/- 0.02 to 0.23 +/- 0.11 in the adrenocorticotropin group but was not significantly changed (0.12 +/- 0.06) in the adrenocorticotropin plus propranolol group. We conclude that propranolol inhibits the maturational effects of adrenocorticotropin on the fetal lung, which implies that the mechanism of pulmonary maturation is not solely dependent on endogenous cortisol and must be mediated, at least in part, through adrenergic responses.

1. 1.The development profiles of the phosphatidic acid phosphohydrolase activities in developing rat lung were determined using aqueously dispersed phosphatidic acid (PA aq ), membrane-bound phosphatidic acid (PA mb ) and lipid vesicles (PA 1v ) prepared from extracts of PA m as the substrates. 2. 2. The specific activities of the PA aq -dependent phosphohydrolase activities in the homogenates, microsomes or cytosol did not change appreciably prior to birth but the microsomal and to a lesser extent the homogenate activities increased after birth. The PA mb -and PA 1v -dependent activities increased between 18–21 days gestation (term 22) and thereafter declined. 3. 3. When the phosphatidic acid phosphohydrolase activities were expressed as total activity per g lung, a contrasting picture emerged. The increase in the PA aq -dependent activities in the microsomes and cytosol was also noted prior to birth. Although the increases in the PA mb - and PA 1v -dependent activities were still apparent between 18–20 days gestation, the following decline noted with specific activities was less evident. These differences beween the developmental profiles with specific activities and total activities per g lung reflected changes in the protein contents. 4. 4. Fractionation of adult rat lung cytosol on Bio-Gel A5m columns produced PA aq -dependent phosphohydrolase activities in the void volume and a major PA aq -dependent activity with an apparent molecular mass ( M r ) of 130 kdaltons which was followed by a broad shoulder. The 130 kdalton activity peak was not present in fetal and +1 day neonatal cytosols but the shoulder was still present. 5. 5. The PA mb -dependent phosphohydrolase activities eluted from the Bio-Gel A5m in the void volume, as a major activity with an apparent M r = 390 kdaltons and a minor activity peak with an apparent M r = 110 kdaltons . Except for an increase in the PA mb dependent activity eluting in the void volume, there was little change in the profile. 6. 6. Thermal denaturation curves for the phosphatidic acid-dependent activities in fetal, neonatal and adult cytosols were biphasic with a rapidly inactivated component and a more slowly inactivated component. Little developmental change was noted in the thermal denaturation patterns of the PA mb -dependent activities. However, the thermal denaturation patterns of the PA aq -dependent activities revealed an increase in the heat stability of the more rapidly inactivated coponent with the +3 day neonatal and the adult cytosols.

A phospholipase A2 activity associated with the microsomal fraction of rabbit lung homogenates was studied. The enzyme showed specificity for the sn-2 ester bond of phosphatidylcholine, had an alkaline pH optimum and required Ca2+ for activity. Other divalent cations were unable to support hydrolysis. In the absence of detergents, exogenous phosphatidylethanolamine was deacylated at a rate sevenfold higher than phosphatidylcholine. The activity toward both substrates could be enhanced by sodium deoxycholate or, more effectively, by sodium taurodeoxycholate. Phosphatidylethanolamine required higher detergent/phospholipid molar ratios than phosphatidylcholine. Under these conditions, the preference for the former substrate over the latter was nearly abolished. The zwitterionic detergent 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) and the nonionic detergent Triton X-100 were either ineffective (phosphatidylcholine) or inhibitory (phosphatidylethanolamine). Addition of KCl produced opposite effects on the activity depending on the bile salt used to disperse the substrate. The phospholipase A2 activity was inhibited by p-bromophenacyl bromide but remained unaffected after treatment with diisopropylfluorophosphate or NaF. N-Ethylmaleimide, but not other thiol reagents, partially inhibited the activity.

An effective substitute for pulmonary surfactant must be able to reduce water-vapour surface tensions to under 1 mN/m and it must spread rapidly and spontaneously at the air interface from the aqueous phase. Pure dipalmitoylphosphatidylcholine meets the former requirement, but not the latter. A synthetic surfactant is described which meets both of these criteria. The surfactant is prepared as a DPPC monolayer stabilizing an aqueous emulsion of an inert fluorocarbon oil; it spreads rapidly at the air/liquid interface, lowers the surface tension to under 1 mN/m during compression at the air/liquid interface and restores normal pressure-volume characteristics to surfactant-depleted lungs.

Lipid extracts of bovine pulmonary surfactant contain two very hydrophobic surfactant-associated proteins (SP) designated SP-B (15 kDa nonreduced) and SP-C (3.5 kDa). These two low molecular weight apoproteins were delipidated and purified on silica SEP-PAK cartridges using various reagents. Dansylation studies revealed that the 15 kDa apoprotein has three N-termini: Phe, Leu and Ile, while the 3.5 kDa apoprotein has two N-termini: Leu and Ile. In either protein, only a very small amount of N-Ile is present. Quantitative N-terminal dansylation analysis of the 15 kDa protein indicated that Phe and Leu (plus Ile) are present in a 1:1 ratio. Carboxy-terminal analysis showed that the 15 kDa protein contains C-terminal Gly, and the 3.5 kDa protein contains C-terminal Leu. Gas-phase amino terminal sequencing of the 15 kDa protein revealed almost exclusively the Phe-polypeptide (SP-B). These results suggest that the 15 kDa apoprotein is not an oligomer of SP-B and SP-C. The reason that analysis of SP-B reveals N-terminal Leu and Ile by dansylation which cannot be confirmed by amino acid sequencing is not known.

1. 1. Administration of estradiol-17β to pregnant rabbits at 25 days gestation (term, 31 days) resulted in a significant increase in the incorporation of [14C]-choline, but not [14C]ethanolamine, into the lipids of fetal lung slices. The incorporation of [35S]methionine was not affected. 2. 2. Enzymatic assays conducted in vitro revealed no significant effect on either the activities of several enzyme markers for subcellular organelles, the activities of the enzymes responsible for the production of phosphatidylglycerol and phosphatidylinositol, membrane-bound or aqueously dispersed phosphatidate-dependent phosphatidic acid phosphohydrolase activities or the activities of the auxiliary enzymes responsible for the synthesis of dipalmitoylphosphatidylcholine. 3. 3. The activity of the enzymes involved in the choline pathway for the de novo biosynthesis of phosphatidylcholine were not significantly altered except for a 66% increase in the CTP:cholinephosphate cytidylyltransferase activity assayed in the cytosol. The addition of phosphatidylglycerol stimulated cholinephosphate cytidylyltransferase activity approx. 3-fold. However, in the presence of this lipid, the activities in cytosol from control and treated fetuses were similar, indicating that the increased activity noted in the absence of phosphatidylglycerol was due to an activation of existing cytidylyltransferase activity rather than an increase in total enzyme units. 4. 4. Estrogen treatment of the does was also associated with a marked decrease in the levels of cholinephosphate in fetal lung and significant increases in the levels of CDPcholine and phosphatidylcholine. These alterations in pool size are consistent with an increase in the activity of cholinephosphate cytidylyltransferase in vivo. The results suggest that cholinephosphate cytidylyltransferase may catalyse an important rate-determining reaction in the synthesis of phosphatidylcholine in fetal lung. The data also support the view that the reaction catalysed by CDPcholine: diacylglycerol cholinephosphotransferase also has a regulatory role during development.

1. 1. The properties of the phosphatidic acid phosphohydrolase activities responsible for the degradation of membrane-bound phosphatidic acid (PA mb ) and aqueously dispersed phosphatidic acid (PA aq ) have been examined in rat lung microsomal and cytosol preparations. 2. 2. The bulk of the PA mb phosphohydrolase activities in the cytosolic and microsomal fractions could be differentiated from the bulk of the PA aq phosphohydrolase activities by their susceptibility to proteolysis and thermal inactivation. 3. 3. EDTA inhibited the PA mb phosphohydrolase activity in the cytosol by 65% but the PA aq phosphohydrolase activity by only 10%. MgCl 2 stimulated the cytosolic PA mb phosphohydrolase activity by 7% and the PA aq phosphohydrolase activity by 80%. Thermal inactivation studies indicated that the PA mb phosphohydrolase activity remaining in the presence of EDTA may be related to the bulk of the PA aq phosphohydrolase activity. Furthermore a portion of the PA mb phosphohydrolase activity may be related to the Mg 2+ -dependent PA aq phosphohydrolase activity. 4. 4. Fractionation of rat lung cytosol on Bio-Gel A-5m revealed the presence of a moderate PA mb phosphohydrolase peak (35% total) in the void volume ( V 0 ), a major PA mb phosphohydrolase activity peak (55% total) with an apparent M r 390 000, and a minor PA mb phosphohydrolase activity peak (12% total) with an apparent M r 110 000. The PA aq phosphohydrolase activities eluted as a moderate peak (25% total) in the void volume, a major peak (50% total) with an apparent M r 130 000 followed by a broad shoulder of PA aq -dependent activity which coeluted with the 110 kdalton PA mb phosphohydrolase peak and accounted for approx. 25% of the total activity. 5. 5. The 390 kdalton PA mb phosphohydrolase activity peak accounted both for most of the EDTA-sensitive PA mb phosphohydrolase activity and for most of the Mg 2+ -dependent PA aq phosphohydrolase activity in the eluates. 6. 6. Treatment of rat lung microsomes with Triton X-100 produced a 5.5-fold increase in the PA mb phosphohydrolase activity but a slight depression of the PA aq phosphohydrolase activity. Filtration of Triton X-100-solubilized microsomes and Triton X-100-treated cytosol on Bio-Gel A-1.5m revealed in both cases PA mb and PA aq phosphohydrolase activity peaks in the V 0 and at 200 ml. With both fractions, PA aq phosphohydrolase activity was also eluted at 295 ml. These results suggest a potential relation between the PA mb phosphohydrolase activities in the microsomes and cytosol. Part of the PA aq phosphohydrolase activities in these fractions may also be related. However, the major PA aq 130 kdalton phosphohydrolase activity peak isolated from untreated cytosol was completely inhibited by Triton X-100 and presumably could not be detected in eluates containing this detergent.

Lipid extracts of bovine pulmonary surfactant containing the 6 kDa apoprotein, but lacking the 35 kDa apoprotein, can mimic the essential characteristics of pulmonary surfactant on a pulsating-bubble surfactometer. Reconstituted surfactant can be produced by combining silicic acid fractions containing 6 kDa apoprotein and phosphatidylglycerol with phosphatidylcholine. Treatment of the protein-containing fraction with proteolytic enzymes abolishes its efficacy. These results indicate that the presence of the 6 kDa apoprotein can account for some of the essential physical and biological characteristics of pulmonary surfactant. Immunodiffusion studies indicate that, contrary to earlier suggestions, the 6 kDa apoprotein is not structurally related to the major surfactant apoprotein that has a molecular mass of 35 kDa.

Natural surfactant possesses the ability to rapidly reduce the surface tension of a bubble pulsating at 20 cycles per min at 37°C to less than 30 dyn/cm at maximum radius and to less than 1 dyn/cm at minimum radius. The preparation of two artificial surfactant systems, containing either dipalmitoylphosphatidylcholine (DPPC) and phosphatidylethanolamine (PE) (5:5) , or DPPC plus PE and phosphatidylglycerol (5:4:1 or 6:3:1) , is described. Formation of artificial surfactants which mimic the essential properties of natural surfactant was correlated with the appearance of particles of aggregated lipids. The effects of lipid composition, calcium ion concentration, pH, temperature and mechanical agitation were determined. It is proposed that these artificial surfactant systems may produce a surface-active monolayer through the involvement of nonbilayer structures with properties similar to hexagonal HII phase.

Lipid extracts of bovine pulmonary surfactant, which retain many of the biophysical characteristics of natural surfactant, contain approx. 98% lipid and 2% protein, as determined by amino acid analysis. Polyacry (amide/urea gel electrophoresis reveals that lipid extract surfactant contained a major apoprotein band with apparent M r 3500 and minor apoprotein bands with apparent M r 15000 and 7000. After reduction, the 15 kDa band disappears and is replaced by a prominent band with apparent M r = 5000. Reduction also results in a relative diminution of the 7 kDa band and a relative increase in the intensity of the 3.5-kDa band. Edman degradation reveals two major peptide sequences which have been designated surfactant-associated peptide (N-terminal Phe) and surfactant-associated peptide (N-terminal Leu) and a minor sequence designated surfactant-associated peptide (N-terminal Ile). The latter surfactant-associated peptide appears to be related to the N-terminal Leu peptide but lacks the terminal Leu. N-Terminal analysis by dansylation demonstrates that the 15 and 5 kDa (reduced) apoprotein species contain N-terminal Phe, Leu and Ile. The 3.5 and 7 kDa bands contain only N-terminal Leu and Ile. Chromatography of lipid extracts on silicic acid columns gives rise to fraction I, which contains protein and phosphatidylglycerol, and fraction II, which contains protein, phosphatidylglycerol and phosphatidylethanolamine. Fraction I was primarily composed of the 15-kDa apoproteins, while fraction II contained mainly the 3.5 and 7 kDa apoproteins. Both fractions exhibited biophysical activity after reconstitution with dipalmitoylphosphatidylcholine. These results indicate that lipid extracts contain an oligomer of 15 kDa containing surfactant-associated peptide (N-terminal Phe) and surfactant-associated peptides (N-terminal Leu or Ile) which interact through sulfhydryl and perhaps other bonds. Lipid extracts also contain 3.5 kDa monomers of surfactant-associated peptides with N-terminal Leu and N-terminal He which can dimerize through sulfhydryl and perhaps hydrophobic interactions.

1. 1. The properties of a stable glycerophosphate acyltransferase preparation obtained by lyophilization of rabbit lung microsomal fractions are described. Phosphatidic acid was the principal radioactive product formed in the presence of either sn-[ 14 C] glycerol -3-P or 3H- or 14C fatty acids plus rac-glycerol-3-P. 2. 2. The subcellular localization of glycerophosphate acyltransferase was examined by comparing the distribution of this enzyme with that of “marker” enzymes. A similarity was observed between the percentage distribution and the relative specific activities of glycerophosphate acyltransferase and NADPH-cytochrome c reductase in the different fractions, which indicates that endoplasmic reticulum is the principal organelle responsible for pulmonary glycerolipid synthesis. The mitochondrial fraction contained glycerophosphate acyltransferase activity which could not be explained through contamination with endoplasmic reticulum. However, under the assay conditions used, the activity associated with the mitochondria could only account for a small proportion of the total lung glycerophosphate acyltransferase activity. 3. 3. The kinetics of the reaction indicated that considerable free fatty acid was either associated with the preparation or produced during the incubations. Treatment of the fresh microsomal preparations with serum albumin removed some of the endogenous fatty acid. 4. 4. The relative incorporation of radioactive fatty acids into phosphatidic acid was in the following order: linoleate > palmitate, oleate and stearate > myristate > laurate > linolenate and arachidonate. A small acylation of endogenous lyso-derivatives was also indicated by the experimental results. 5. 5. Phospholipase A2 degradation of the radioactive phosphatidic acids revealed that laurate, myristate, linolenate and arachidonate were preferentially incorporated to the 2-position. Palmitate, oleate, stearate and linoleate were randomly incorporated.

A preterm rat model has been developed for studies of acute and chronic neonatal lung disease. Premature delivery 24 h before the normal time of delivery is associated with immature pulmonary phospholipid and antioxidant enzyme profiles. The premature lung is more fragile and ruptures at a lower lung vol (172 +/- 8 microL) than the full-term fetal lung (259 +/- 14 microL). Only 7% of premature lungs will float in liquid, after inflation to 85% of the rupture vol, compared with 87% of term fetal lungs. This lung immaturity was reflected in a survival rate of only 6% by 36 h after delivery if the preterm pups were placed in air, which increased to 47% when they were placed in greater than 95% oxygen. Though greater than 95% oxygen enhanced survival of preterm pups during the 1st wk of life, these survivors had a 50% mortality during the 2nd wk of exposure to greater than 95% oxygen. The preterm pup will tolerate intraperitoneal injection of antioxidant enzymes entrapped in liposomes and has a better retention of these liposomes in the lung compared with the term pup. We conclude that the preterm rat is a suitable model for studies of acute and chronic neonatal lung disease.

(1) Pre-type II alveolar cells isolated from the fetal rabbit lung on the 24th gestational day have been maintained in vitro for 14 days in a chemically defined medium supplemented with hormone-stripped serum. These cells replicate in culture. (2) Measurement of the incorporation of [ 14 C]choline into cellular disaturated phospholipid indicated that those cells grown in vitro under standard conditions for 8 days (pre-confluent) incorporate the radioactive precursor at a similar rate to cells maintained for 14 days (post-confluent). (3) Both dexamethasone and serum-free medium conditioned by monolayer cultures of fetal rabbit lung fibroblasts stimulated [ 14 C]choline incorporation into disaturated phosphatidylcholine (PC) by the pre- and post-confluent cultures after 24 or 48 h of exposure; the conditioned medium was more effective than the steroid. (4) These treatments had little effect on choline incorporation into disaturated phosphatidylcholine of preconfluent cells during the first 12 h. A marked response occurred by 24 h after which the labelling of disaturated phosphatidylcholine plateaued. In contrast, with post-confluent cells labelling of disaturated PC increased m a more linear fashion and only plateaued after 72 h. (5) Determination of the ratio of incorporation of [ 14 C]choline into disaturated versus unsaturated phospholipid indicated that serum-free medium conditioned by monolayer cultures of fetal lung fibroblasts specifically increased the level of radioactive precursor in the disaturated phospholipid in both the pre- and post-confluent cell monolayers.

The incorporation of intraperitoneally injected 3H-glycerol and 14C-palmitate into the lipids of lung, liver and brain was examined in premature rabbits (27 days gestation, term 31) both in utero and in control or surfactant-treated neonates. With the lung, there was an increased incorporation after delivery, especially with the surfactant-treated pups. The incorporation of 3H-glycerol into liver lipids was markedly reduced after birth, particularly with the nontreated controls. The liver:lung ratios of the incorporations were decreased after delivery. In all three tissues, the relative incorporations into the individual lipids were not affected to any great extent by birth or surfactant treatment. In lung, the disaturated and monoenoic species, respectively, of phosphatidylcholine accounted for the major proportion of the radioactivity. After birth there was a small increase in the labelling of the monoenoic species at the expense of the disaturated species with both control and surfactant-treated neonates. These observations support the proposal that surfactant treatment of the prematurely delivered neonate could be used prophylactically for the prevention of the respiratory distress syndrome.

The properties of natural bovine surfactant and its lipid extract have been examined with a pulsating bubble surfactometer which assesses the ability of surfactant lipids to adsorb to the air/liquid interface and reduce the surface tension to near 0 dynes/cm during dynamic compression. Studies conducted at 1 mg/ml phospholipid revealed that the surface activity (i.e., the ability to produce low surface tensions) of lipid extracts could be enhanced by incubating the sample at 37 degrees C for 120 min or by addition of CaCl2. In contrast, incubation at 37 degrees C only slightly improved the biophysical activity of natural surfactant and the addition of CaCl2 had a more modest effect than with lipid extracts. With 20 mM CaCl2, the surfactant activity of lipid extract surfactant was similar to that of natural surfactant. Incubation with EDTA reduced the biophysical activity of natural surfactant. Experiments in which increasing amounts of lipid extract were replaced by natural surfactant revealed that small amounts of natural surfactant enhanced the surfactant activity of lipid extract. The biophysical activity of lipid extract surfactant was also increased by the addition of soluble surfactant-associated protein-A (SP-A) (28-36 kDa) purified from natural bovine surfactant. These results indicate that SP-A (28-36 kDa) improves the surfactant activity of lipid extracts by enhancing the rate of adsorption and/or spreading of phospholipid at the air/liquid interface resulting in the formation of a stable lipid monolayer at lower bulk concentrations of either phospholipid or calcium.

1. Pulmonary maturation in the rat is accompanied by a 30% postnatal increase in the pool size of choline, a 4-fold overall prenatal and postnatal decrease in the level of cholinephosphate, a 3-fold decrease in CDPcholine levels and a 2-fold increase in the content of phosphatidylcholine. 2. The level of 1,2-diacyl-sn-glycerol in rat lung increases 5-fold during the fetal and neonatal periods. Only minor alterations were noted in the fatty acid composition. 3. These results are consistent with an increase in the relative rates of the cholinephosphate cytidylyl-transferase and cholinephosphotransferase steps of phosphatidylcholine production during pulmonary maturation. The relative rate of the step catalyzed by phosphatidate phosphohydrolase may also be increased.

The 104,000 × g supernatant fraction from rat lung contains a greater proportion of the phosphatidic acid phosphatase activity toward membrane-bound phosphatidic acid than the microsomal fraction. The microsomal fraction is more effective in hydrolyzing aqueously dispersed phosphatidic acid. The effects of various ions and chelators, particularly Mg 2+ and EDTA, suggest that these two activities are distinct. These results indicate that the supernatant fraction of rat lung contains a phosphatidic acid phosphatase activity which may play an important role in pulmonary glycerolipid synthesis.

The incorporation of radioactive glycerol into phosphatidylcholine by rat lung slices increased approximately 4-fold between 19 days gestation and the first day after birth. Increases were also observed with a number of other pulmonary lipids. The specific activities of a number of enzymes related to the biosynthesis of phosphatidylcholine and the acidic phospholipids, phosphatidylglycerol and phosphatidylinositol, were examined in whole homogenates and in the nuclear, mitochondrial, microsomal and cytosolic fractions. The specific activity profiles for a number of enzymatic markers for mitochondria and endoplasmic reticulum were also examined. The specific activity of phosphatidate cytidylyltransferase in the mitochondrial fraction was high during the perinatal period but fell in the adult. On the other hand, the specific activity of the microsomal fraction was low, compared to the mitochondrial fraction in early gestation, but increased near term and after birth, resulting in a specific activity in the adult which was greater than that observed with the mitochondria. Similar specific activity profiles were observed with the endoplasmic reticulum marker NADPH-cytochrome c reductase and with cholinephosphotransferase and inositol phosphatidyltransferase. Although the specific activity of glycerophosphate phosphatidyltransferase in the mitochondrial fraction also increased in late gestation and declined somewhat after birth, the specific activity of the microsomal fraction showed little alteration during development and remained low, compared to the mitochondria, even in the adult. Plots of total enzymatic activity per g lung were consistent with the view that the endoplasmic reticulum is the primary site for the biosynthesis of phosphatidylcholine, CDPdiacylglycerol and phosphatidylinositol, but the mitochondria possess the bulk of the enzymatic activity required for the formation of phosphatidylglycerophosphate and, therefore, phosphatidylglycerol. The basis for the unanticipated sedimentation of a considerable proportion of the activities of a number of those enzymes normally recognized as endoplasmic reticulum constituents with the mitochondria fraction during early development remains unresolved. It is clear that these changes in sedimentation properties could influence the results of experiments where a single subcellular fraction was examined.

When d -β-[3-14C]hydroxybutyrate was injected via the femoral vein into pregnant Sprague-Dawley rats at 21 days of gestation, d -β-[3-14C]hydroxybutyrate was enzymatically detected in fetal plasma within 5 min. The time course of the incorporation of dl -β-[3-14C]hydroxybutyrate into fetal lipids was studied. Lipid extracts of brown adipose tissue exhibited the greatest relative incorporation followed by pancreas, liver and lung. Less radioactivity was incorporated into brain and placenta. The incorporation into fetal lipids was several-fold greater than into maternal lipids. The labelling of the individual phospholipids was similar in the different tissues with phosphatidylcholine accounting for more than 50%. 75% of the radioactivity in brown adipose tissue was in the triacylglycerol fraction. In brain, liver and placenta, approximately half of the neutral lipid radioactivity was in cholesterol. Experiments in which d -β-[3-14C]hydroxybutyrate was directly injected into fetuses in utero confirmed that this substrate was directly used by the fetuses without maternal intervention. These studies demonstrate that the rat placenta is permeable to β-hydroxybutyrate and suggest that this ketone body is rapidly used by the fetus for the synthesis of fatty acids and cholesterol.

1. 1. Maternal administration of betamethasone (0.2 mg/kg) on day 25 or 26 of gestation produced a significant decrease in the lung/body weight ratio of the rabbit fetuses within 24 h. 2. 2. The incorporation of [14C]choline but not [14C]ethanolamine into the lipids of fetal lung slices was significantly increased, indicating that there was a specific effect on phosphatidylcholine synthesis. 3. 3. The activities of a number of marker enzymes for subcellular organelles were elevated, especially in the lungs of fetuses delivered on day 26. The increases in monoamine oxidase (mitochondrial outer membrane), β-glycerophosphatase and aqueously dispersed phosphatidic acid-dependent phosphatidic acid phosphohydrolase (lysosomal) activities were significant. 4. 4. Although the activity of cholinephosphotransferase was not affected by glucocorticoid treatment, the activities of glycerol-3-phosphate phosphatidyl-transferase and the activities of two enzymes in the auxiliary pathways for the production of disaturated phosphatidylcholine (lysophosphatidylcholine: lysophosphatidylcholine transacylase and lysophosphatidylcholine racyl-CoA acyl-transferase) were significantly increased. 5. 5. Membrane-bound phosphatidic acid-dependent phosphatidic acid phosphohydrolase activity was elevated to a lesser extent than the aqueously dispersed phosphatidate-dependent activity and this increase was not significant. 6. 6. The incorporation of [35S]methionine into protein by slices of fetal lung was significantly reduced after maternal treatment with betamethasone. 7. 7. These results are consistent with the general view that glucocorticoids can induce pulmonary maturation and surfactant production in the rabbit fetus but indicate that some of the former hypotheses regarding the mechanism by which lipid synthesis is accelerated must be reevaluated.

CDPdiacylglycerol pyrophosphatase (E. C. 3.6.1.26) activity has been examined in rat lung mitochondrial and microsomal fractions. While the mitochondrial hydrolase exhibited a broad pH optimum from pH 6–8, the microsomal activity decreased rapidly above pH 6.5. Apparent Km values of 36.2 and 23.6 μM and Vmax values of 311 and 197 pmol∙min−1∙mg protein−1 were observed for the mitochondrial and microsomal preparations, respectively. Addition of parachloromercuriphenylsulphonic acid led to a marked inhibition of the microsomal fraction but slightly stimulated the mitochondrial activity at low concentrations. Mercuric ions were inhibitory with both fractions. Although biosynthetic reactions utilizing CDPdiacylglycerol require divalent cations, addition of Mg2+, Mn2+, Ca2+, Zn2+, Co2+, and Cu2+ all inhibited the catabolic CDPdiacylglycerol hydrolase activity in both fractions. EDTA and EGTA also produced an inhibitory effect, especially with the mitochondrial fraction. Although addition of either adenine or cytidine nucleotides led to a decrease in activity with both fractions, the marked susceptibility to AMP previously reported for this enzyme in Escherichia coli membranes, guinea pig brain lysosomes, and pig liver mitochondria was not observed. These results indicate that rat lung mitochondria and microsomes contain specific CDPdiacylglycerol hydrolase activities, which could influence the rate of formation of phosphatidylinositol and phosphatidylglycerol for pulmonary surfactant.

With a randomized clinical trial, the possibility was assessed that a tracheal instillation of pulmonary surfactant prior to the first breath might prevent the development of some of the signs of neonatal respiratory distress syndrome. Of the 72 infants in the trial, all born at a gestational age of less than 30 weeks, 39 received 3 or 4 mL of surfactant, prepared from the lipids extracted from calf lung lavage. The treatment resulted in a significantly improved gas exchange during the first 72 hours of life. On the average, the arterial/alveolar Po2 ratio was 0.15 higher for the treated infants, and only about half as much extra oxygen had to be supplied. The respiratory support (peak inspiratory pressure x frequency) could be lowered significantly. Pulmonary interstitial emphysema occurred in 13 of the 33 control infants, but in only three of the 39 treated infants. Six of the control infants died in the neonatal period, but only one treated infant died. It is concluded that surfactant supplementation prior to the first breath is feasible and is of value as protection against the respiratory distress syndrome and the negative effects of hypoxia and ventilatory support.

Experiments have been conducted investigating the synthesis of phosphatidyl glycerol and phosphatidyl glycerophosphate in preparations from rat brain cerebral hemispheres. [32P]rac-Glycero-3-phosphoric acid in the presence of CDP-diacylglycerol, was incorporated into a lipid tentatively identified as phosphatidyl glycerophosphate. The addition of HgCl2 stimulated this incorporation. [14C]sn-Glycero-3-phosphoric acid was incorporated into two lipids, one tentatively identified as phosphatidyl glycerophosphate and the other identified as phosphatidyl glycerol. In the presence of an acylating system, small amounts of [32P]rac-glycero-3-phosphoric acid could be incorporated into phosphatidyl glycerol. This latter incorporation was markedly stimulated by CTP and CDP-choline. These results are consistent with the view that these lipids can be synthesized in brain by the reactions described by kiyasu et al.4 in which CDP-diacylglycerol reacts with sn-glycero-3-phosphoric acid to form phosphatidyl glycerophosphate which is subsequently hydrolyzed to phosphatidyl glycerol.

Reported associations between air pollution and pulmonary and cardiovascular diseases prompted studies on the effects of gold nanoparticles (Au NP) on pulmonary surfactant function. Low levels (3.7 mol % Au/lipid, 0.98% wt/wt) markedly inhibited adsorption of a semisynthetic pulmonary surfactant (dipalmitoyl-phosphatidylcholine (DPPC)/palmitoyl-oleoyl-phosphatidylglycerol/surfactant protein B (SP-B); 70:30:1 wt %). Au NP also impeded the surfactant's ability to reduce surface tension (gamma) to low levels during film compression and to respread during film expansion. Transmission electron microscopy showed that Au NP generated by a seed-growth method were spherical with diameters of approximately 15 nm. Including palmitoyl-oleoyl-phosphatidylglycerol appeared to coat the NP with at least one lipid bilayer but did not affect NP shape or size. Similar overall observations occurred with dimyristoyl phosphatidylglycerol. Dipalmitoyl-phosphatidylglycerol was less effective in NP capping, although similar sized NP were formed. Including SP-B (1% wt/wt) appears to induce the formation of elongated strands of interacting threads with the fluid phosphatidylglycerols (PG). Including DPPC resulted in formation of aggregated, less spherical NP with a larger size distribution. With DPPC, strand formation due to SP-B was not observed. Agarose gel electrophoresis studies demonstrated that the aggregation induced by SP-B blocked migration of PG-coated NP. Migration was also influenced by the fluidity of the PGs. It is concluded that Au NP can interact with and sequester pulmonary surfactant phospholipids and, if inhaled from the atmosphere, could impede pulmonary surfactant function in the lung.

The physical properties of organized system (bilayers and monolayers at the air water interface) composed of bovine lipid extract surfactant (BLES) were studied using correlated experimental techniques. 6-Dodecanoyl-2-dimethylamino-naphthalene (LAURDAN)-labeled giant unilamelar vesicles (mean diameter approximately 30 microm) composed of BLES were observed at different temperatures using two-photon fluorescence microscopy. As the temperature was decreased, dark domains (gel-like) appeared at physiological temperature (37 degrees C) on the surface of BLES giant unilamelar vesicles. The LAURDAN two-photon fluorescent images show that the gel-like domains span the lipid bilayer. Quantitative analysis of the LAURDAN generalized polarization function suggests the presence of a gel/fluid phase coexistence between 37 degrees C to 20 degrees C with low compositional and energetic differences between the coexisting phases. Interestingly, the microscopic scenario of the phase coexistence observed below 20 degrees C shows different domain's shape compared with that observed between 37 degrees C to 20 degrees C, suggesting the coexistence of two ordered but differently organized lipid phases on the bilayer. Epifluorescence microscopy studies of BLES monomolecular films doped with small amounts of fluorescent lipids showed the appearance and growth of dark domains (liquid condensed) dispersed in a fluorescent phase (liquid expanded) with shapes and sizes similar to those observed in BLES giant unilamelar vesicles. Our study suggests that bovine surfactant lipids can organize into discrete phases in monolayers or bilayers with equivalent temperature dependencies and may occur at physiological temperatures and surface pressures equivalent to those at the lung interface.

1. The membrane-bound phosphatidate-dependent phosphatidic acid phosphatase activity of rat lung has been investigated in cytosol and microsomal fractions using as a substrate [32P]phosphatidate bound to heat inactivated rat liver microsomes. Both activities demonstrated broad pH optima with a maximum of 7.4--8 for the cytosol and a maximum of 6.5--7.5 with microsomal preparations. 2. At low concentrations (0--5 mM) Mg2+ produced a slight stimulation of the cytosol activity but at higher concentrations an inhibition was observed. Low concentrations (1.0--2.0 mM) of EDTA abolished the cytosol activity and reduced the microsomal activity to half. In both cases, the addition of Mg2+ in the presence of EDTA resulted in an activity which was more than 2-fold greater than that observed in the absence of chelator or divalent cation. 3. The cytosol activity was relatively resistant to the addition of ionic and nonionic detergents. In general, the addition of a number of phosphate esters increased rather than decreased the release of 32Pi, indicating a relative specificity for phosphate groups associated with a hydrophobic environment. The addition of aqueous dispersions of phosphatidate, lysophosphatidic acid or phosphatidylglycerophosphate markedly reduced the hydrolysis of membrane-bound [32P]phosphatidate. The cytosol activity was slightly inhibited by the addition of phosphatidylcholine. 4. In an attempt to estimate the relative contributions of the cytosol and microsomal activities in vivo, these activities were assayed using [32P]phosphatidate endogenously generated on rat lung microsomes. With the 32P-labelled microsomes, the hydrolysis remained linear over the 45 min of the experiment. Addition of high speed supernatant produced a rapid release of 32Pi during the first 10 min followed by a more gradual release similar to that oberved with the microsomes alone. The cytosol activity remained greater than the microsomal activity at all times studied. 5. When [14C]phosphatidate-labelled microsomes were incubated in the presence of nonradioactive CDPcholine, the addition of cytosol markedly stimulated the incorporation of radioactivity into phosphatidylcholine. This observation suggests that the phosphatidic acid phosphatase activity associated with the cytosol has a role in phosphatidylcholine (and presumably surfactant) biosynthesis in rat lung.

Pulmonary surfactant obtained from rabbit lung lavage was evaluated with the pulsating bubble surfactometer. Because surfactant forms a monomolecular film at the air-liquid interface consisting mainly of phospholipids, solvent vapors, which might be inhaled, could have a destructive influence on the surfactant monolayer. To assess the risk of such an inhalation, vapors from five solvents--halothane, chloroform, enflurane, acetone, and diethyl ether--were made to flow into the bubble of the surfactometer as it pulsated for 30 sec, i.e., during 10 pulsations. The vapors from halothane and chloroform, excellent solvents of dipalmitoyl-phosphatidylcholine (DPPC), had a destabilizing effect evidenced by the fact that surface tension at minimal bubble size increased from 0 to as high as 20 mN/m. When the vapors were replaced with a flow of room air, the pressure tracing promptly returned to normal. The concentration of halothane vapor, however, had to be at least 20%, a concentration much higher than that used for anesthesia, to have a destabilizing effect on pulmonary surfactant. Twenty-five percent enflurane vapor had a less pronounced yet conspicuous impact. With 25% acetone and diethyl ether vapors, poor solvents of DPPC, surface tension at minimal bubble size remained unaffected. We conclude that vapors of halothane and chloroform, if inhaled in high concentration, might instantaneously obliterate the stabilizing effect of pulmonary surfactant but that anesthetic concentrations of halothane have no effect.

Two low-molecular-weight hydrophobic proteins with nominal molecular weights Mr = 15,000 and Mr = 3,500 have been isolated from the lipid extracts of bovine pulmonary surfactant by several methods, including (a) dialysis plus silicic acid chromatography, (b) elution from Waters SEP-PAK silica cartridges with a variety of solvent mixtures, and (c) ultrafiltration. As detailed in the text, these proteins have been designated surfactant-associated protein-BC (SP-BC) (15 kDa: nonreduced), and SP-C (3.5 kDa). The biophysical activities of reconstituted surfactant containing these proteins and the phospholipids present in lung surfactant have been compared with the biophysical activities of bovine lipid extract surfactant on a pulsating bubble surfactometer using a phospholipid concentration of 10 mg/ml. At this concentration, unmodified lipid extract surfactant reduces the surface tension of the pulsating bubble to near 0 within 10 pulsations at 20 cycles per min. Similar biophysical properties were observed with modified lipid extract surfactant in which the relative concentration of hydrophobic protein had been reduced from 1 to 0.4% (W/W) of the phospholipids by addition of dipalmitoylphosphatidylcholine (DPPC) or DPPC plus phosphatidylglycerol. Reconstituted surfactants, which contained partially delipidated SP-BC (15 kDa: nonreduced) obtained by method (a) at a relative concentration of 0.1%, were also capable of reducing the surface tension to near 0 mN/m. Preparations of SP-BC (15 kDa: nonreduced) obtained by method (b), which had been subjected to very low pH levels during isolation and were extensively delipidated, exhibited full biophysical activity only at higher protein concentrations and with prolonged pulsation. Extensively delipidated samples of SP-BC obtained by method (c) exhibited impaired biophysical activities, even when prepared with neutral organic solvents. Reconstituted surfactant samples containing SP-C (3.5 kDa) obtained by any of the methods listed above were only able to reduce the surface tension at minimum bubble radius to approx. 20 mN/m. The biophysical activity of SP-C (3.5 kDa) was not significantly affected by low pH or extensive delipidation. Reconstituted samples containing mixtures of SP-BC (15 kDa: nonreduced) and SP-C (3.5 kDa) were more effective than samples containing either protein alone. Furthermore, with samples containing both hydrophobic proteins the final surface tensions at maximum bubble radius were attained within a few bubble pulsations.(ABSTRACT TRUNCATED AT 400 WORDS)

Rat lung cytosol and microsomal fractions both contain phosphohydrolase activity towards membrane-bound phosphatidic acid (PAmb) and aqueously dispersed phosphatidic acid (PAaq) which cannot be explained through contamination with the other fraction. The phosphohydrolase activities with PAaq demonstrated Km and Vmax values which were more than an order of magnitude greater than those observed with PAmb and with vesicles prepared from the lipids extracted from [32P]PA-labelled microsomes. The PAaq-dependent activities in both fractions were stimulated by preparing mixed liposomes with phosphatidylcholine. The PAmb-dependent activities in rat lung microsomes and cytosol were markedly stimulated by high concentrations of Triton X-100 and Nonidet P-40. The PAmb- and PAaq-dependent activities in the microsomes were stimulated by deoxycholate. Although no difference was observed in the inhibition profiles of the PAmb- and PAaq-dependent activities of the cytosol in the presence of various mercurials, the PAmb-dependent activity in the microsomes was somewhat more susceptible than the PAaq-dependent activity. The PAmb-dependent activities in both fractions were more susceptible to inhibition by iodoacetamide. These results support the view that separate rat lung enzymes were involved in the hydrolysis of PAmb and PAaq. The relative abilities of rat lung cytosol and microsomes to hydrolyse PA endogenously generated on the microsomes were compared using relative concentrations of cytosol corresponding to the levels in intact rat lung. During the initial period (5–10 min) the cytosol phosphohydrolase activity was more effective than the microsomal activity. At later stages (10–20 min), the rates were comparable.

Rat lung microsomes washed with increasing concentrations of NaCl show a displacement of protein from microsomes to the wash supernatant. Among the proteins removed from the microsomal surface was the Mg2+-dependent phosphatidate phosphohydrolase, while the Mg2+-independent activity remained associated with the microsomes. The Mg2+-dependent activity could be quantitatively assayed in the wash supernatant. Microsomes washed with increasing concentrations of NaCl showed a progressive impairment in the synthesis of labelled neutral lipid and phosphatidylcholine from [14C]glycerol 3-phosphate with a concomitant increase in the labelling of phosphatidic acid. The impairment was sigmoidal and correlated highly with the decrease in Mg2+-dependent phosphatidate phosphohydrolase activity. When Mg2+-dependent phosphatidate phosphohydrolase from wash supernatant was incubated with microsomes previously washed with high salt concentrations, the labelling of neutral lipid and phosphatidylcholine was returned to control levels. Labelling of neutral lipids and phosphatidylcholine could be restored upon addition of a cytosolic Mg2+-dependent phosphatidate phosphohydrolase isolated by gel filtration. Mg2+-independent phosphatidate phosphohydrolase isolated from cytosol was incapable of restoring the labelling of neutral lipids and phosphatidylcholine. These findings confirm that the Mg2+-dependent phosphatidate phosphohydrolase of rat lung is involved in pulmonary glycerolipid biosynthesis. The role of the Mg2+-independent phosphatidate phosphohydrolase activity remains unknown.

The mammalian alveolus is stabilized by the presence of a specialized material, the pulmonary surfactant, which reduces the surface tension at the air-liquid interface. The disaturated lecithin, 1,2-dipalmitoyl-sn-phosphatidylcholine, is a major constituent of the surfactant. Unsaturated lecithins, other phospholipids, neutral lipid and protein are also present. The properties of compressed films of natural surfactant strongly resemble those of a monolayer of dipalmitoylphosphatidylcholine. However, below the transition temperature of 41°C, liposomes of dipalmitoylphosphatidylcholine are in the condensed gel state from which monolayers form very slowly. The mechanism by which dipalmitoylphosphatidylcholine is transferred from bilayers to monolayers has been investigated with a pulsating bubble technique. Natural surfactant reduces the surface tension of the pulsating bubble to 25–30 dynes/cm at maximal radius (0.55 mm) and to 0 dynes/cm at minimal radius (0.4 mm). Evidence is presented which strongly indicates that the proteins associated with natural surfactant are not required for the rate or the extent of the reduction of surface tension. Divalent cations appear to be important for surfactant behaviour. Using combinations of pure lipids, artificial surfactant preparations have been obtained which possess the basic physical and biological properties of natural surfactant.

Pulmonary surfactant contains phospholipids including dipalmitoyl-phosphatidylcholine and three surfactant-associated proteins designated SP-A, SP-B and SP-C. A cDNA for rabbit SP-B has been isolated from a fetal (30 days gestation) rabbit lung cDNA library constructed in lambda gt11. The cDNA and deduced amino acid sequences show strong homology with the cDNAs and predicted 40 kDa proproteins for human and canine SP-B. Strong homology is also observed with the amino acid sequences directly determined for the mature 8 kDa bovine and porcine SP-B isolated from lung lavage. SP-B is remarkable for its high cysteine and proline content and for the hydrophobic nature of the organic solvent-soluble, mature protein. In vitro translation of sense but not antisense RNA transcribed from the cDNA led to the production of 40 kDa and 32 kDa proteins. These proteins were immunoprecipitated by an antibody raised against bovine SP-B. Northern blot analysis revealed the mRNA for rabbit SP-B appears in fetal rabbit lung late in gestation and falls slightly in the neonate.

— A comprehensive study has been undertaken on the subcellular and subsynaptosomal distribution of a number of markers for subcellular organelles in preparations from rat brain. Although the activity of most enzymatic markers was decreased by freezing and storage at - 70oC, no significant changes were noted in the distribution of these activities. This demonstrates that contamination of brain fractions by subcellular organelles can be accurately assessed after freezing and thawing. A marked discrepancy was noted between the distribution of three putative markers for endoplasmic reticulum. CDP-choline-diacylglycerol cholinephosphotransferase (EC 2.7.8.1) activity was mainly limited to the microsomal fraction and was present to a lesser extent in the synaptosomal fraction than the other putative markers for endoplasmic reticulum. Estrone sulfate sulfohydrolase (EC 3.1.6.2) activity demonstrated a bimodal distribution between the crude nuclear and microsomal fractions. However, considerable activity was associated with the synaptosomal fraction. NADPH-cytochrome c reductase (EC 2.3.1.15) activity sedimented in the microsomal and the synaptosomal fractions. Calculations based on the relative specific activities of the microsomal and synaptic plasma membrane fraction indicated that the contamination of the synaptic plasma membranes by endoplasmic reticulum was 44.5% (NADPH-cytochrome c reductase), 38.0% (estrone sulfatase) and 9.0% (cholinephosphotransferase). Since it is believed that virtually all of the synthesis of phosphatidylcholine by cholinephosphotransferase occurs in the neuronal and glial cell bodies, it was concluded that cholinephosphotransferase is a satisfactory marker for the endoplasmic reticulum derived from these sources. The results suggest that NADPH-cytochrome c reductase and estrone sulfatase may be present in the smooth endoplasmic reticulum system responsible for the fast transport of macromolecules along the axon to the nerve endings as well as in the endoplasmic reticulum of the cell bodies. The possible relation between that portion of the smooth endoplasmic reticulum involved in fast axonal transport and the GERL (Golgi, Endoplasmic Reticulum, Lysosomes) complex discovered by Novikoff and his coworkers (Novikoff, 1976) is discussed.

The enzymes responsible for the biosynthesis of phosphatidylglycerol, CTP:phosphatidate cytidylyltransferase, CDP-diacylglycerol: glycerophosphate phosphatidyltransferase and phosphatidylglycerophosphate phosphatase demonstrated a coordinate increase in activity in fetal rat lung at term when the demand for pulmonary surfactant increases. The activity of CTP:cholinephosphate cytidylyltransferase, the enzyme responsible for CDP-choline production also increased in the perinatal period. The activity of cholinephosphate cytidylyltransferase in fetal and neonatal cytosol was stimulated by the addition of phosphatidylglycerol but no effect was noted with cytosol from adult lung. These results are consistent with the suggestion that the activity of cholinephosphate cytidylyltransferase, a potential rate-determining enzyme in pulmonary phosphatidylcholine synthesis, may be regulated in the perinatal period both through an activation by phosphatidylglycerol and by an increase in total enzyme units.

The surfactant properties of natural bovine pulmonary surfactant, its lipid extracts and acetone precipitates of lipid extracts have been examined with an artificial alveolus model, the pulsating-bubble surfactometer. At bulk concentrations of 0.4% (wt/vol.) phospholipid in saline, all three preparations exhibited surfactant activity i.e., were capable of reducing the surface tension of the pulsating bubble to approx. 27 dynes/cm at maximum bubble radius and to near zero at minimum bubble radius. At a concentration of 0.1% (wt./vol.) in saline, only natural surfactant was effective. Acetone-precipitated surfactant at 0.1% (wt./ vol.) achieved these criteria in the presence of 5 mM calcium, but 15–20 mM calcium was required to restore the surfactant activity of lipid extract surfactant. Chemical analysis revealed that lipid extraction decreases the protein content but does not alter the endogenous calcium levels. A calcium requirement for natural surfactant could only be demonstrated after repeated treatment with chelators for divalent cations. Surfactant activity was restored by low levels of calcium or high levels of magnesium. Paradoxically, a calcium requirement could not be demonstrated by treating acetone-precipitated lipid extract with chelators. The subtle differences noted between natural, lipid extract and acetone-precipitated lipid extract surfactant with the pulsating-bubble assay show that the latter preparations do not represent simplified model systems for the natural product.

Bovine pulmonary surfactant was obtained by endotracheal lavage of lungs from newly slaughtered cows followed by differential centrifugation. Lipid extracts of bovine surfactant contained 3% neutral lipid, mainly as cholesterol and diacylglycerol and 97% phospholipid. Phosphatidylcholine (79%) and phosphatidylglycerol (11%) accounted for most of the phospholipids with smaller amounts of phosphatidylethanolamine, phosphatidylinositol, lyso-bis-phosphatidic acid and sphingomyelin. Fatty acid analysis revealed high levels of palmitate in phosphatidylcholine and to a lesser extent phosphatidylglycerol, but not in the other diacylphospholipids. Phosphatidylcholine was 53% disaturated and phosphatidylglycerol was 23% disaturated. Monoenoic species accounted for the major proportion of the remaining lipid. The protein content was 10% as estimated by the Lowry procedure and 5% when determined by amino acid analysis. Extraction with chloroform/methanol removed ca. 90% of the protein but had no effect on the surfactant properties as evaluated by a pulsating bubble technique.

In the sheep tracheal lobe model of silicosis, we have recently reported that total phospholipid, lecithin, and phosphatidylglycerol levels were elevated in lung lavage. To investigate further this observation, we obtained complete phospholipid profiles of lung lavage in 10 sheep exposed to saline only (Sa group), 10 sheep exposed to aluminum lactate inhalation only (Al group), 10 sheep exposed to 100 mg Minusil-5 in saline followed by monthly saline inhalation (Si group), and 10 sheep exposed to 100 mg Minusil-5 in saline followed by monthly aluminum lactate inhalation (Si-Al group). The following phospholipid components were measured: total phospholipids, phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylcholine, disaturated phosphatidylcholine, sphingomyelin, and lyso-phosphatidylcholine. All values were comparable in the Sa group, Al group, and Si-Al group. In the Si group, there was a significant increase in total phospholipid to approximately 200% of the control values. The phospholipid profile of this group demonstrated an increase in all of the phospholipid components with some enrichment of the fraction of PG, PE, and PI. We concluded that lung exposure to silica dust significantly increases the concentration of phospholipids in the alveoli. This increase is of a large spectrum of alveolar phospholipids and is completely suppressed by aluminum lactate inhalation.

Summary The presence of CMP, CDP and CTP markedly reduced the incorporation of [14C] sn -glycerol-3-phosphate into the total lipids of rat brain prepartions. CDP-choline produced a slight stimulation but this was not observed in all experiments. The addition of various amounts of inorganic pyrophosphate up to 1 mM produced a decrease in the incorporation of [14C] sn -glycerol-3-phosphate, but this effect was small compared to the effect of similar concentrations of CTP. Increasing the energy charge of the cytidylate pool produced a decrease in the incorporation of glycerol-3-phosphate in the region of low energy charge. The inhibition of [14C] sn -glycerol-3-phosphate incorporation observed in the presence of CMP or CTP was almost completely reversed by the addition of similar amounts of CDP-choline. Addition of phosphoryl choline also reversed the inhibition due to CTP. This latter effect was partially dependent upon the presence of rat brain supernatant. These results indicate that one of the initial reactions in phospholipid metabolism may be regulated by the relative concentration of the cytidine nucleotides, particularly CTP and CDP-choline.

The possibility that pulmonary surfactant, characterized by a phosphatidylglycerol deficiency, as in early fetal life, might have inferior surface properties was evaluated. We obtained this specific surfactant from adult rabbits by withholding glucose and giving them an excess of myoinositol by mouth and intravenously. Controls were given a similar quantity of glucose. The myoinositol resulted in a drastic reduction of surfactant phosphatidylglycerol, from 7.2 to 0.3% of phospholipids, and a corresponding increase in phosphatidylinositol from 4.8 to 11.3%. In addition, the myoinositol treatment increased the myoinositol that was disaturated from 18.5 to 27.3% (p less than 0.05). The corresponding figures for disaturated phosphatidyl-choline were 56.0 and 60.5%, respectively (NS). The myoinositol treatment for 4 days increased the pool size of alveolar surfactant by 32% (p less than 0.01). The surface activity was studied with modified Wilhelmy balance and the pulsating bubble surfactometer. Surfactant containing phosphatidylinositol rather than phosphatidylglycerol was not inferior, as compared to surfactant that contained phosphatidylglycerol (minimum surface tension: 2.0 versus 2.2 mN X m-1; collapse rate at 10 nM X m-1: 1.85 versus 1.95 min-1; rate of adsorption from subphase to surface: 32 versus 35 mN X m-1 X 30 s-1), nor was there a difference in the ability of the two surfactants to improve lung stability of 27-day-old rabbit fetuses (air retention at 35 cm H2O: 1.8 versus 1.8 ml/30 g; air retention at 0 cm H20: 0.8 versus 0.9 ml/30 g). We conclude that phosphatidylinositol surfactant does not have inferior surface properties.(ABSTRACT TRUNCATED AT 250 WORDS)

The incorporation of radioactive choline into phosphatidylcholine and disaturated phosphatidylcholine in rat lung slices increased markedly before term and peaked after birth. The specific activity of cholinephosphate cytidylyltransferase in the microsomal fraction increased before birth but fell after delivery. The specific activity of this enzyme in the cytosol showed a marked increase at birth. The developmental profile for the total cytosolic activity per gram lung was similar to the pattern observed with choline incorporation. Although the specific activity of cholinephosphotransferase in the whole homogenate remained relatively constant throughout pulmonary maturation, there was a marked increase in the specific activity of this enzyme in the microsomal fraction at term. Similar findings were obtained with the microsomal marker NADPH-cytochrome c reductase. The basis of this disparity in specific activity profiles is being investigated further. The specific activity of lysophosphatidylcholine: lysophosphatidylcholine transacylase in rat lung homogenates increased during gestation but rose a further 10-fold between day 3 after birth and the adult. The specific activity of lysophosphatidylcholine: palmitoyl-CoA acyltransferase remained relatively constant throughout development. At term, the specific activity of the acylation enzyme was 10- to 15-fold greater than the specific activity of the transacylation enzyme. These observations are consistent with previous studies indicating that the accumulation of phosphatidylcholine and dipalmitoyl phosphatidylcholine during the perinatal period may be due to alterations in the activity of cholinephosphate cytidylyltransferase. Cholinephosphotransferase could also play a regulatory role. The formation of dipalmitoyl phosphatidylcholine appears to occur via the acylation, rather than the transacylation pathway.

We tested a new captive bubble surface tensiometer with films adsorbed from aqueous suspensions of rabbit lung surfactant and a bovine lung surfactant lipid extract and with films of dipalmitoyl-sn-3-glycerophosphorylcholine (DPPC) spread from solvents. The lack of tubes penetrating the bubble surface eliminated potential leakage pathways for the surface film, which was compressed by increasing external pressure. Surface tensions and areas were calculated directly from bubble shapes without the need of pressure measurements. After only one to two compressions, the rabbit surfactant films exhibited the low surface tension, collapse rates, and compressibilities characteristic of the alveolar surface in situ and approached the behavior of spread DPPC films. The bubble “clicking” phenomenon described earlier by Pattle (Proc. R. Soc. Lond. B Biol. Sci. 148: 217-240, 1958) was also reproduced, but only with the bovine extract, which did not perform as well as the rabbit surfactant in surface tests. These findings suggest that surfactant apoprotein SP-A, which was probably present in the rabbit but not the bovine preparations, enhances both adsorption and stability of pulmonary surfactant monolayers.

An assay of pulmonary phosphatidate phosphohydrolase activity has been developed that employs a chemically defined liposome substrate of equimolar phosphatidate and phosphatidylcholine. Enzyme assays employing this substrate resolved two distinct activities based upon their requirements for Mg2+. Assays were performed in the presence and absence of 2 m m MgCl2 and the Mg2+-dependent phosphatidate phosphohydrolase activity calculated by difference. The Mg2+-independent phosphatase activity resembled that found using aqueous dispersions of phosphatidate (PAaq). Approximately 90% of the Mg2+-dependent phosphatidate phosphohydrolase activity was recovered in the cytosol and the remainder was associated with the microsomal fraction. The Mg2+-dependent phosphatidate phosphohydrolase activity has kinetic parameters of Km = 55 μ m , Vmax = 1.6 nmol/min/mg protein for the microsomal fraction, and Km = 215 μ m , Vmax = 6.8 nmol/min/mg protein for the cytosolic fraction. These parameters resembled those found using the microsomal membrane-bound (PAmb) substrate. In addition, the pH optima and sensitivity to detergents and thermal inactivation are equal to those for the PAmb-dependent phosphatidate phosphohydrolase activity. In the course of these studies the microsomal and cytosolic activities were qualitatively equal, indicative of a single enzyme in two subcellular locations. In conclusion, the assay of Mg2+-dependent phosphatidate phosphohydrolase activity measured using equimolar phosphatidate and phosphatidylcholine liposomes is equivalent to that activity previously described using microsomal membrane-bound substrate. However, the chemically-defined system provides a more simplified starting point for further studies on this important enzyme.

Differentiation-arrested lung cell cultures were developed from fetal rats of various gestational ages. In contrast to previously published observations with cultures in a pO2 of approximately 142 mm Hg, cultures developed in a pO2 of approximately 30 mm Hg, close to the normal fetal arterial pO2, have improved plating efficiency and a slightly increased growth rate. They did not, however, show gestation-dependent increases of choline incorporation into phospholipids, nor did immature lung cell cultures respond to dexamethasone or triiodothyronine, singly or in combination, by increased choline incorporation into saturated lecithin. The incorporation of choline and glycerol into lipids suggested a mature rate of lipid synthesis by immature cultures at a pO2 approximately 30 mm Hg, despite preservation of an immature morphology. Electron microscope observations revealed no gross differences between immature cultures developed at either pO2. The cellular mechanisms underlying these differences are unclear but suggest that oxygen tension may significantly influence results obtained with in vitro studies of lipid synthesis by immature lung.

Lipid extracts of bovine pulmonary surfactant, which exhibit biophysical and biological activity, contain two hydrophobic proteins which have been designated surfactant protein-B (SP-B) and SP-C. Amino terminal amino acid sequence analysis of whole lipid extracts and partially purified protein fractions gave rise to three sequences, two major and one minor. The first sequence, identified as a member of the SP-B family, extended for 60 amino acids beginning with an amino terminal phe. The second polypeptide, identified as a member of the SP-C family, sequenced for 35 amino acids and had a leu amino terminus. The third minor sequence corresponded to amino acids 2–9 of SP-C (N-leu) and was designated SP-C (N-ile). Sequence analysis of cyanogen bromide peptides derived from methyl isocyanate-blocked lipid extract material produced two peptides which extended the amino acid sequence of SP-B to residue 79, which appears to be a glycine.

Pregnant guinea pigs of 50 to 53 days' gestation (term 63 days) were anesthetized with ether, and their fetuses were injected intramuscularly with 30 mg of dexamethasone or sterile saline. One week later, the fetuses were injected with 3 H-thymidine intramuscularly under direct vision at laparotomy; after one hour, the incorporation of thymidine into deoxyribonucleic acid (DNA) was analyzed in various fetal tissues. The relative labeling of DNA was significantly depressed in the cerebral hemispheres, cerebellum, medulla oblongata, and midbrain of the treated fetuses compared to their littermate controls. The relative labeling of the DNA of lungs, kidneys, heart, and adrenals was also significantly reduced. Increasing the dose of dexamethasone produced a progressive inhibition of the incorporation of 3 H-thymidine into DNA. A variable recovery from the inhibition became apparent by 14 days following exposure to dexamethasone. The evidence suggests that exposure of the fetus to dexamethasone may exert a potentially deleterious effect on fetal tissues.

The potential involvement of the glycerophosphorylcholine (GPC) pathway for the synthesis of phosphatidylcholine (PC) has been examined in rat liver and lung and in a human line, the A549 cell which possesses characteristics representative of mature alveolar type II epithelial cells. Although mitochondrial and microsomal fractions from the above sources readily incorporated radioactive glycerophosphate into lipids, the only incorporation observed with radioactive GPC was a small variable labelling with the mitochondrial and microsomal fractions from rat lung. Even with these fractions, no radioactivity from GPC was incorporated into PC or lysoPC. Attempts to increase the incorporation of GPC into lipids by manipulating the incubation conditions were unsuccessful. It was concluded that the occurrence of the GPC pathway in liver and lung is unlikely.