Lysophosphatidylcholine uptake and metabolism in the adult rabbit lung

Tracer quantities of 3H-labeled lysoPC and 32P-labeled natural rabbit surfactant were given intratracheally via a bronchoscope and [14C]palmitate was given intravenously to 25 rabbits with labeled PC and lysoPC measured in the alveolar wash, lung homogenate, lamellar bodies and microsomes at five times from 10 min to 6 h after tracheal injection. Surprisingly, only 31% of the administered lysoPC remained in its original form in the total lungs (alveolar wash + lung homogenate) by 10 min, of which 77% was in the alveolar wash. Meanwhile, by 10 min an additional 37% was already converted to PC, of which more than 98% was in the lung homogenate. LysoPC continued to be rapidly and efficiently converted to PC, with 62% conversion measured at 3 h. The converted lysoPC initially appeared with high specific activity in microsomes, then in lamellar bodies, and finally in the alveolar wash. The intravascular palmitate labeled lung PC had similar specific activity-time profiles in the subcellular fractions, while intratracheally administered natural rabbit surfactant had a constantly low specific activity in microsomes and much higher specific activities in lamellar bodies and alveolar wash. Another 25 rabbits received intratracheal lysoPC labeled in both the choline and palmitate moieties and then were studied from 1 to 24 h after tracheal injection. The ratio of the palmitate to choline labels indicated uptake and conversion to PC primarily by direct acylation rather than transacylation and by intact reuptake and conversion rather than breakdown and resynthesis. LysoPC is an attractive 'metabolic probe' of surfactant metabolism which undergoes very rapid and efficient intracellular conversion to PC via a subcellular pathway that parallels the remodeling and de novo synthetic pathways.     

Intrapulmonary catabolism of surfactant-saturated phosphatidylcholine in rabbits

Intrapulmonary surfactant catabolism was investigated by use of a phospholipase A1- and A2-resistant analogue of dipalmitoylphosphatidylcholine (DPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPC ether). [14C]DPC ether, made into liposomes with [3H]DPC and associated with 32P-labeled rabbit surfactant, was given intratracheally to 1-kg rabbits, which were killed at preset times to 48 h. Recoveries of radiolabel as saturated phosphatidylcholine (Sat PC) isolated from alveolar wash (AW), postlavage lung homogenate (LH), and alveolar macrophages were measured. All groups had similar AW and LH Sat PC pool sizes, indicating no perturbation of endogenous Sat PC pools. Despite a nearly fivefold accumulation of [14C]DPC ether in the lung by 48 h (P less than 0.01), the three probes had similar alveolar clearance curves. Furthermore, the Sat PC reutilization efficiency (41.6%) and turnover time (5.9 h) calculated for DPC ether were not different from values for the DPC and rabbit surfactant. Of the DPC ether (0.7%) and DPC (9%) labels recovered as PC in organs outside the lung, greater than 85% was unsaturated, indicating de novo synthesis using precursors from degraded PC. DPC ether was a useful probe of intrapulmonary DPC catabolism, and after alveolar uptake there was no direct reentry of intact DPC from the catabolic compartment(s) into the secretory pathway.     

Reutilization of surfactant phosphatidylglycerol and lysophosphatidylcholine by adult rabbits

Adult rabbits reutilize the phosphatidylcholine (PC) of surfactant much less efficiently than developing rabbits (22% vs. 95%). Comparisons of reutilization efficiency of other components of surfactant in adult rabbits have not been determined. We injected adult rabbits intratracheally with [3H]dipalmitoylphosphatidylcholine (DPPG) mixed with [14C]lysophosphatidylcholine (lysoPC) and natural surfactant or [14C]DPPC mixed with [3H]dipalmitoylphosphatidylglycerol (DPPG) and natural surfactant. Recovery in the alveolar wash and lamellar bodies of labelled DPPC, lysoPC and DPPG was determined at different times after injection. By plotting the ratio of [3H]DPPG to [14C]DPPC in the alveolar wash versus time after injection we found that phosphatidylglycerol was reutilized with an efficiency of only 0-7% which was much less than the reutilization of PC in these animals. At early times after injection, adult rabbits injected with [14C]lysoPC had a ratio of [14C]PC in their alveolar wash to lamellar bodies that was larger than 1.0. By comparison, 3-day old rabbits injected intratracheally with [14C]lysoPC had a ratio of [14C]PC in alveolar wash to lamellar bodies less than 1.0 at the earliest times measurable. Thus adult rabbits demonstrate a pathway for accumulation of PC in their alveolar space prior to its appearance in lamellar bodies. This was not detected in developing rabbits. As in developing rabbits, adult rabbits reutilize the phosphatidylglycerol of surfactant less efficiently than the PC of surfactant.     

The significance of reutilization of surfactant phosphatidylcholine

To assess the magnitude of reutilization of surfactant phosphatidylcholine, 68 3-day-old rabbits were injected intratracheally with a trace dose of [3H]choline-labeled surfactant mixed with [14C]palmitate-labeled synthetic dipalmitoylphosphatidylcholine. After timed kills we measured the total phosphatidylcholine associated counts/min in whole lung and alveolar wash and the specific activities of phosphatidylcholine in the alveolar wash, lamellar bodies, and microsomes isolated from the lung of each rabbit. Using a modification of the compartment analysis of Skinner et al. (Skinner, S. M., Clark, R. E., Baker, N., and Shipley, R. A. (1959) Am. J. Physiol. 196, 238-244), we found that surfactant phosphatidylcholine was reutilized with greater than 90% efficiency. The turnover time of the alveolar wash phosphatidylcholine was estimated to be 10.1 h and 9.3 h as measured by the 3H and 14C labels, respectively. From the ratios of alveolar wash-associated natural to synthetic phosphatidylcholine specific activities and from similar ratios obtained in 30 additional rabbits using [14C]choline-labeled natural surfactant and [3H]choline-labeled dipalmitoylphosphatidylcholine, we showed that phosphatidylcholine was reutilized intact rather than as component parts. Within 6 h of injection, the synthetic dipalmitoylphosphatidylcholine functioned metabolically as that administered in the form of natural surfactant.     

Reutilization of surfactant phosphatidylcholine in adult rabbits

32P-saturated phosphatidylcholine was added to [3H]choline-labeled natural surfactant and the mixture was injected intratracheally into 87 adult rabbits. The rabbits were also given [14C]palmitate intravenously at the same time. Rabbits were killed in groups from 10 min to 72 h after injection. In each rabbit we measured the total recovered [3H]phosphatidylcholine (PC) in the alveolar wash, the ratio of [3H]PC to [32P]PC in the alveolar wash, and the specific activity of [14C]PC in the alveolar wash and lamellar bodies. Values were averaged for all rabbits killed at the same times and smooth curves were fit to the data by computer. From the intravenous [14C]palmitate data we calculated a turnover time for alveolar PC of 6.0 h. From the intratracheal labeling data, we calculated a turnover time for alveolar PC of 5.7 h and determined that alveolar PC was reutilized at an efficiency of only 23%. We also concluded that this reutilization occurred as intact molecules.     

Molecular species of phosphatidylcholine and phosphatidylglycerol in rat lung surfactant and different pools of pneumocytes type II.

It is not yet completely understood how a cell is able to export specific phospholipids, like dipalmitoylphosphatidylcholine (dipalmitoyl-PC), which is secreted by pneumocytes type II, into pulmonary surfactant. The acyl species composition of [3H]PC which was synthesized in type II cells in the presence of [2-3H]glycerol resembled the species composition of PC localized in intracellular pneumocyte membranes. This species pattern was different from the pattern of PC of lamellar bodies, i.e., intracellularly stored surfactant, by a higher proportion of dipalmitoyl-PC mainly at expense of 1-palmitoyl-2-oleoyl-PC. Lamellar body PC in turn showed the same species distribution as surfactant PC. The data suggest that subcellular compartmentation and/or intracellular transfer of PC destined to storage in lamellar bodies, but not secretion of lamellar bodies, involves an enrichment of dipalmitoyl-PC and a depletion of 1-palmitoyl-2-oleoyl-PC. In contrast, the acyl species pattern of phosphatidylglycerol does not seem to undergo gross changes on the path from synthesis to secretion.     

Characterization of rabbit lung lysosomes and their role in surfactant dipalmitoylphosphatidylcholine catabolism

Although alveolar surfactant is rapidly catabolized in adult rabbit lungs, the pathways have not been characterized. Pathways of surfactant secretion and recycling involve lamellar bodies and multivesicular bodies, organelles shown to be related to lysosomes by cytochemistry and autoradiography. Since lysosomes are central to intracellular catabolic events, it is possible that lysosomes are involved in intrapulmonary surfactant catabolism. Lysosomes relatively free of contaminating organelles (as determined morphologically and by marker enzymes for mitochondria, endoplasmic reticulum, peroxisomes, and plasma membranes) were obtained from post-lavage lung homogenates of 1-kg rabbits by differential centrifugation in buffered sucrose and gradient separation in percoll (density, 1.075-1.165). The role of lung lysosomes in catabolism of dipalmitoylphosphatidylcholine (DPC) was then studied in rabbits killed 4, 12, and 24 h following intratracheal injection of [3H]DPC and [14C] dihexadecyl phosphatidylcholine (DPC-ether). While equal amounts of label were in the lamellar body containing fractions at 4 h, nearly 6-fold more DPC-ether label than DPC label was recovered in the lysosomal fractions. By 24 h, there was 15-fold more DPC-ether in the lysosomes. This is the first report of successful isolation of lysosomes relatively free of other organelles from rabbit lungs. The tracer studies indicate DPC and DPC-ether follow similar intracellular processing after alveolar uptake. The subsequent accumulation of the ether analog in the lysosomal fractions supports a role for these organelles in surfactant DPC catabolism.     

The phosphatidylcholine-transfer protein catalyzed import of phosphatidylcholine into isolated rat liver mitochondria

In order to study the individual steps involved in the import of phosphatidylcholine (PC) into rat liver mitochondria, a number of PC analogues were introduced into the outer membrane of isolated mitochondria. Two fluorescent PC species, i.e. 1-palmitoyl-2-(16-bimanylthio)hexadecanoyl-PC (bimane-PC) and 1-palmitoyl-2-(10-pyrene)decanoyl-PC (pyrene-PC), and one radiolabeled PC species, i.e. 1-palmitoyl-2-[1-14C]oleoyl-PC (14C-POPC), were studied. The PC analogues were introduced from small unilamellar vesicles with the use of PC-specific transfer protein. The amount of PC imported was quantified by reisolation of the mitochondria. Import of the fluorescent PC species was monitored by on-line fluorescence spectroscopy. The distribution of the newly inserted PC between the outer and the inner membrane was assessed by separation of the two membranes using digitonin treatment. All analogues tested remained exclusively localized in the outer membrane thereby suggesting that additional (extramitochondrial) factors are required to initiate transfer of PC to the inner membrane.     

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.     

Role of clathrin- and actin-dependent endocytotic pathways in lung phospholipid uptake

We evaluated the contribution of endocytotic pathways to pulmonary uptake of surfactant lipids from the alveolar space. Resting and stimulated 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) uptake of unilamellar liposomes labeled with either [(3)H]dipalmitoylphosphatidylcholine ([(3)H]DPPC) or 1-palmitoyl-2-[12-(7-nitro-2-1,3-benzoxadiazol-4-yl) amino] dodecanoyl-phosphatidylcholine (NBD-PC) was studied in isolated perfused rat lungs and isolated type II cells. Amantadine and phenylarsine oxide, inhibitors of clathrin-mediated endocytosis, each decreased [(3)H]DPPC uptake under resting conditions by approximately 40%; their combination had no additional effect. Cytochalasin D, an inhibitor of actin-dependent processes, reduced liposome uptake by 55% and potentiated the effect of either clathrin inhibitor alone. Relative inhibition for all agents was higher in the presence of 8-Br-cAMP. The effect of inhibitors was similar for liposomes labeled with [(3)H]DPPC or NBD-PC. By fluorescence microscopy, NBD-PC taken up by lungs was localized primarily to alveolar type II cells and was localized to lamellar bodies in both lungs and isolated cells. These studies indicate that both clathrin-mediated and actin-mediated pathways are responsible for endocytosis of DPPC-labeled liposomes by alveolar type II cells in the intact lung.     

An apolipoprotein preferentially enriched in cholesteryl ester-rich very low density lipoproteins

Phosphatidyl glycerol is present in lamellar bodies and in the material obtained by alveolar wash representing 12.3 and 11.5%, respectively, of the total phospholipid phosphorus. Lung microsomes catalyze the formation of phosphatidyl glycerol from the known precursors, L-glycerol 3-phosphate and CDP-diglyceride. The rate of [¹⁴C]L-glycerol 3-phosphate incorporation into phosphatidyl glycerol was 30% higher in microsomes as compared to mitochondria. The addition of mercuric chloride inhibited the synthesis of phosphatidyl glycerol, and stimulated the incorporation into another as yet incompletely identified lipid. After pulse labeling of microsomal phosphatidyl glycerol $\text{in vitro}$, further incubation of microsomes with lamellar bodies or alveolar wash resulted in nearly quantitative appearance of label in surfactant.     

Phospholipid-protein interactions in rat lung lamellar bodies

We investigated the specificity of the cytosol-mediated phosphatidylcholine transfer between isolated rat lung microsomes and rat lung lamellar bodies. For that purpose we labeled the microsomes with 1-acyl-2-[1-¹⁴C]palmitoyl- and 1-acyl-2-[9,10-³H]oleoylphosphatidylcholine through protein-catalyzed phosphatidylcholine exchange. Incubation in buffer resulted in 3–5% transfer of label from microsomes to lamellar bodies. Lung cytosol stimulated this transfer about 2-fold and the presence of 12 μg/ml phosphatidylcholine-transfer protein from bovine liver resulted in a 30 to 35% recovery of radioactivity in the lamellar bodies. When microsomal donor membranes with a ³H/¹⁴C ratio of 2.6 were used, the ³H/¹⁴C ratios of the lamellar bodies were 3.9, 3.7 and 3.7, after incubation in buffer, with cytosol and with bovine liver exchange protein, respectively. Doubling the amount of lamellar body acceptor membranes resulted in ³H/¹⁴C ratios in the lamellar bodies of 4.6 and 4.1, after incubation in buffer and with cytosol, respectively. Furthermore, we isolated the protein component from rat lung lamellar bodies and performed reconstitution experiments with phospholipids. Reconstituted and non-reconstituted phospholipid and protein were separated by either Sepharose 4B gel filtration or discontinuous sucrose gradient centrifugation. The presence of lamellar body protein in the reconstitution mixture resulted in the formation of larger structures with higher density than those formed in control experiments without protein. When 1-acyl-2-[1-¹⁴14C]palmitoyl- and 1-acyl-2-[9,10-³H]oleoylphosphatidylcholine were included in the reconstitution mixture, the structures containing lamellar body protein had 2- to 4-fold lower ³H/¹⁴C ratios than initially present in the incubation. These results suggest that lamellar body proteins associate preferentially with disaturated phosphatidylcholine species.     

Phospholipid composition and acyltransferase activity of lamellar bodies isolated from rat lung.

The role of the lamellar body of the type II pneumocyte in the synthesis and storage of the phospholipids of the surfactant lipoprotein lining the alveolar surface has been investigated. Electron microscopy has been used to establish the purity of the isolated lamellar body, microsomal, and mitochondrial fractions. Additional proof of lamellar body purity was obtained by enzyme marker studies. The phospholipid:protein ratio of each of the above fractions was determined as well as that of surfactant lipoprotein isolated from rat lung. Lamellar body phospholipid:protein ratio was highest, 3.7 μmol of lipid phosphorus/mg of lung protein. The phospholipid composition of the lamellar body fraction was found to be similar to that of the isolated surfactant lipoprotein. Lamellar body phosphatidylcholine and phosphatidylglycerol each contained over 90% saturated fatty acids. The lamellar body fraction was found to possess significant acyltransferase activity between [1-¹⁴C]palmitoyl-CoA and phosphatidylcholine. This activity was somewhat higher than in the microsomal fraction and much greater than in the mitochondrial fraction. The activity in all fractions was stimulated by Ca²⁺ and Mg²⁺. [1-¹⁴C]oleoyl-CoA did not serve as an effective acyl donor. When 1-palmitoyl-2-lysophosphatidylcholine was used as the acceptor molecule and [1-¹⁴C]palmitoyl-CoA the donor, acyltransferase activity was increased over that found with phosphatidylcholine as donor in all fractions. The microsomal fraction had the greatest activity and the lamellar body fraction the least. The data obtained support the hypothesis that the lamellar body is involved in the synthesis and storage of the phospholipids of the surfactant lipoprotein complex.     

Lung phospholipid metabolism in transgenic mice overexpressing peroxiredoxin 6

Previous studies with peroxiredoxin 6 (Prdx6) null mice demonstrated that the phospholipase A(2) activity of this enzyme plays a major role in lung phospholipid metabolism. This study evaluated lung phospholipid metabolism in transgenic mice that over-express Prdx6. Lung lysosomal type PLA(2) activity in transgenic mice was 222% of wild type in lung homogenate and 280% in isolated lamellar bodies. Total phospholipid, phosphatidylcholine (PC) and disaturated PC were decreased approximately 20-35% in bronchoalveolar lung fluid, lung homogenate, and lung lamellar bodies in transgenic mice although lung compliance and type 2 cell ultrastructure were unaltered. To study metabolism, unilamellar liposomes ((3)H-DPPC: PC: cholesterol: PG, 10: 5: 3: 2 mol fraction) were instilled endotracheally in anesthetized mice and lungs were removed for perfusion. Compared to wild type, transgenic mice showed similar net uptake of liposomes in 2 h, but significantly increased (3)H-DPPC degradation (38.9+/-1.1 vs. 29.0+/-1.3% of recovered dpm). The PLA(2) competitive inhibitor MJ33 decreased degradation to 15% of recovered dpm in both transgenic and wild type lungs. Incorporation of [(14)C] palmitate into DSPC at 24 h after its intravenous injection was markedly increased in both the lung surfactant (+100%) and lamellar bodies (+188%) while incorporation of [(3)H] choline was increased by only 10-20%. These results indicate increased DPPC degradation and synthesis by the reacylation pathway with Prdx6 overexpression and provide additional evidence that the PLA(2) activity of Prdx6 has an important role in lung surfactant turnover.     

Phosphatidylcholine metabolism for energy production in sea urchin spermatozoa

Sea urchin spermatozoa use endogenous phosphatidylcholine (PC) to produce energy for swimming. The catabolism of PC was studied in Hemicentrotus pulcherrimus spermatozoa. Following incubation in sea water, the content of PC decreased and that of choline increased gradually, whereas phosphocholine maintained a constant level. Measurement of the radioactivity in metabolites converted from 1-palmitoyl-2-[1-14C]linoleoyl-PC, [choline-methyl-14C]dipalmitoyl-PC and 1-[1-14C]palmitoyl-lysophosphatidylcholine (LysoPC) showed that the major degradative pathway is PC----LysoPC----glycerophosphocholine----choline. 1-Palmitoyl-2-[1-14C]linoleoyl-PC and [1-14C]oleic acid were oxidized to 14CO2 in a cell-free system of spermatozoa. Sea urchin spermatozoa thus appear to quite likely obtain energy through the oxidation of fatty acid(s) from PC.     

Altered surfactant function and metabolism in rabbits with acute lung injury

Lung injury was induced in rabbits with N-nitroso-N-methylurethane (NNNMU), and saturated phosphatidylcholine (Sat PC) pool sizes and phospholipid compositions were measured in alveolar wash subfractions isolated by differential centrifugation (large and small surfactant aggregates). Surfactant metabolism also was studied using intravascular and intratracheal radiolabels. Protein permeability, gas exchange, and compliance were significantly abnormal as lung injury progressed. At peak injury, there was a decrease in the large aggregate Sat PC pool size in alveolar wash accompanied by increased uptake of Sat PC from the air space and increased specific activity of both intravascular and intratracheal radiolabels in lamellar bodies. This was followed by a marked rise in the small aggregate pool size in the alveolar wash and increased secretion of Sat PC into the air spaces. Phospholipid compositions, total phospholipid-to-protein ratios, and in vivo functional studies using a preterm ventilated rabbit model were abnormal for both large and small aggregate surfactant fractions from the lung-injured rabbits. These studies characterize quantitative, qualitative, and functional changes of alveolar wash surfactant subfractions in NNNMU-injured lungs.     

The incorporation of choline into disaturated phosphatidylcholine in the microsomal, lamellar body, and surfactant fractions of hamster lung tissue

The specific activity of disaturated phosphatidylcholine in microsomes and lamellar bodies prepared from hamster lung tissue and in surfactant obtained by lung lavage was determined at various times following the intraperitoneal administration of [Me-3H]choline. The highest specific activity of disaturated phosphatidylcholine in the lung microsomes was attained 1 h after the administration of [3H]choline; thereafter, the specific activity declined. The specific activity of disaturated phosphatidylcholine in lamellar bodies increased steadily for 12 h after [3H]choline administration. The specific activity in lamellar bodies ater 12 h exceeded the maximum specific activity achieved in the microsomal fraction (p less than 0.005). The specific activity of the disaturated phosphatidylcholine in the alveolar lavage increased after an initial lag period of approximately 3 h, attaining the same specific activity as that of the lamellar bodies at the 12-h time point. The reported results are discussed in relation to the biosynthesis, storage, and secretion of the disaturated phosphatidylcholine associated with the lipoprotein, surfactant.     

Flip-flop rates of individual molecular species of phosphatidylcholine in the human red cell membrane

Trace amounts of four different, well-defined species of phosphatidyl[N-methyl-14C]choline ([14C]PC), differing in their fatty acyl constituents, were introduced exclusively into the outer membrane leaflet of the intact erythrocyte by using a PC-specific phospholipid transfer protein. The rate of transbilayer equilibration of these probe molecules was calculated from the time-dependent decay in specific radioactivity of the PC pool in the outer monolayer, which was discriminated from that in the inner leaflet by treating the intact cells with phospholipase A2 in the presence of sphingomyelinase C. At 37 degrees C, 1,2-dipalmitoyl-, 1,2-dioleoyl-, 1-palmitoyl-2-linoleoyl- and 1-palmitoyl-2-arachidonoyl-PC revealed halftime values for the rate of their transbilayer equilibration of 26.3 +/- 4.4, 14.4 +/- 3.5, 2.9 +/- 1.7 and 9.7 +/- 1.6 h, respectively.     

Effect of hematocrit on vascular pressure profile in dog lungs

Retention of radiolabeled phospholipid vesicles instilled into the alveolar space was studied with the isolated perfused rat lung and quantitated by measuring the percent of instilled radioactivity remaining in lung after five lavages. With synthetic [14C]dipalmitoyl phosphatidylcholine (PC):egg PC: phosphatidylglycerol:cholesterol (10:5:2:3) vesicles, there was a rapid (within 5 min) base-line retention of 10.3 +/- 0.25% (n = 11) followed by a slower phase of linear retention over the next 4 h. Retention at 2 h with perfused lungs was 18.6 +/- 0.60% (n = 9) and was similar to values obtained with lungs in vivo. Net retention (total minus base line) was stimulated 93% by isoproterenol, 173% by 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP), and 39% by 8-bromoguanosine 3',5'-cyclic monophosphate; propranolol blocked the effect of isoproterenol. The retention of natural (biosynthesized) surfactant [14C]PC was stimulated 92% by 8-Br-cAMP. The results suggest that the retention of exogenous phospholipid by the isolated perfused lung represents phospholipid uptake and that this process is under beta-adrenergic control. Secretion and uptake may be physiologically linked to regulate the concentration of surfactant on the alveolar surface.     

Alveolar pre-type II cells from the fetal rabbit lung: effect of confluence on the production of disaturated phosphatidylcholine

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. Measurement of the incorporation of [14C]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). Both dexamethasone and serum-free medium conditioned by monolayer cultures of fetal rabbit lung fibroblasts stimulated [14C]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. 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 in a more linear fashion and only plateaued after 72 h. Determination of the ratio of incorporation of [14C]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.