Lysosomal-type PLA2 and turnover of alveolar DPPC

Aron B. Fisher and Chandra Dodia

Institute for Environmental Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study evaluated the role of a lysosomal-type phospholipase A2 (aiPLA2) in the degradation of internalized dipalmitoylphosphatidylcholine (DPPC) and in phospholipid synthesis by the rat lung. Uptake and degradation of DPPC were measured in isolated perfused rat lungs over 3 h following endotracheal instillation of [3H]DPPC in mixed unilamellar liposomes plus or minus MJ33, a specific inhibitor of lung aiPLA2. Uptake of DPPC was calculated from total tissue-associated radiolabel, and degradation was calculated from the sum of radiolabel in degradation products. Both uptake and degradation were markedly stimulated by addition of 8-bromo-cAMP to the perfusate. MJ33 had no effect on DPPC uptake but decreased DPPC degradation at 3 h by ~40-50%. The effect of MJ33 on lung synthesis of DPPC was evaluated with intact rats over a 12- to 24-h period following intravenous injection of radiolabeled palmitate and choline. MJ33 treatment decreased palmitate incorporation into disaturated phosphatidylcholine of lamellar bodies and surfactant by ~65% at 24 h but had no effect on choline incorporation. This result is compatible with inhibition of the deacylation/reacylation pathway for DPPC synthesis. These results obtained with intact rat lungs indicate that aiPLA2 is a major enzyme for degradation of internalized DPPC and also has an important role in DPPC synthesis.

alveolar type II cells; dipalmitoylphosphatidylcholine; phospholipase A2 inhibitors; phospholipid synthesis; endocytosis


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

DIPALMITOYLPHOSPHATIDYLCHOLINE (DPPC), the major phospholipid component of lung surfactant, is synthesized in the lung by type II alveolar epithelial cells (granular pneumocytes) and secreted into the alveolar lumen, where it functions in concert with proteins and other lipid components of surfactant to maintain low surface tension and promote alveolar stability (36). DPPC is removed from the alveolar space by endocytosis, predominantly by type II cells under normal conditions, with a minor contribution from alveolar macrophages (11, 36). Although the mechanisms have not been precisely described, secretion and reuptake of DPPC by the lung alveolar cells appear to be coordinately regulated so as to maintain a relatively constant alveolar phospholipid concentration (11). This fine coordination is necessary since either a deficit or excess of alveolar DPPC would lead to altered lung function. After internalization by type II cells, DPPC is routed either to lamellar bodies for resecretion or to lysosomes for degradation (11). The degradative pathway predominates in the normal adult lung (15, 31), whereas the resecretion pathway appears to predominate in the normal neonate (22). Analysis of degradation products or studies using a nonmetabolizable DPPC analog have demonstrated that phospholipase A2 (PLA2) represents the primary pathway for degradation of internalized DPPC (19, 32, 33). Studies with a transition-state analog competitive PLA2 inhibitor, 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33), have suggested that the specific PLA2 that is responsible for degradation of endocytosed DPPC is a lysosomal-type enzyme that has been called aiPLA2 (1, 12, 19, 26).

While previous studies with primary cultures of isolated type II cells have provided important insights into regulation of DPPC uptake and metabolism (3, 9, 12, 21, 32, 36), this model has the inherent difficulties of poor phenotypic stability and difficulty in extrapolating results to in situ clearance. On the other hand, the isolated perfused lung preparation provides a more physiological model to study clearance of lipid by the intact organ, and clearance can be manipulated by administration of agonists or inhibitors through endotracheal instillation or via the perfusate (14-17, 19). The primary goal of the present study was to evaluate the effect of PLA2 inhibition on the clearance and degradation of DPPC by the intact lung under control conditions and in the presence of a surfactant secretagogue, 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP). Although we have previously reported in similar studies in part (19), the present report represents a new investigation with several key modifications from the previous protocol. First, we decreased the amount of radiolabeled phospholipid instilled into the rat lung to avoid significant augmentation of the endogenous surfactant pool. Second, we determined the reincorporation of labeled choline into the unsaturated phosphatidylcholine (USPC) fraction to give a more accurate representation of DPPC degradation. Results indicate that there is stimulation of uptake and metabolism of DPPC by 8-BrcAMP and that the major fraction of internalized DPPC is degraded by the MJ33-inhibitable PLA2 pathway under both control and secretagogue-stimulated conditions.

A second goal of this study was to evaluate the role of aiPLA2 in DPPC synthesis, since activity of this enzyme can provide lysophosphatidylcholine (lysoPC) or, in concert with other enzymes, choline phosphate for DPPC synthesis via reacylation or the de novo pathway, respectively. Our previous results with type II cells in primary culture indicated that the presence of MJ33 markedly inhibited the incorporation of labeled palmitate into DPPC (13), and this result was confirmed in the present study utilizing intact rats.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Authentic lipids of the highest purity available were obtained from Avanti (Birmingham, AL). 8-BrcAMP, p-bromophenacyl bromide (pBPB), and diethyl p-nitrophenyl phosphate (DENP) were obtained from Sigma Chemicals (St. Louis, MO), fatty acid-free BSA was from Boehringer Mannheim (Indianapolis, IN), and radiochemicals were from New England Nuclear (Boston, MA). MJ33 was synthesized by Dr. Mahendra Jain (University of Delaware, Newark, DE) as previously reported (24). The monoclonal antibody to aiPLA2 (MAb 8H11) was raised against recombinant protein and has been described previously (7, 25). Sprague-Dawley male rats weighing ~200 g were obtained from Charles River Breeding Laboratories (Kingston, NY).

Liposomes were prepared from L-alpha -DPPC, egg phosphatidylcholine (PC), egg phosphatidylglycerol (PG), and cholesterol in molar ratio 10:5:2:3 with or without trace [choline-methyl-3H]DPPC by evaporating the lipids to dryness. In some preparations, MJ33 was added at 3-5 mol/100 ml. The evaporated film was resuspended in PBS and was vigorously mixed, then frozen and thawed three times by alternating liquid N2 and a 50°C water bath, and then extruded at 50°C for 10 cycles through a 100-µm pore size filter. Liposomes were stored overnight at 4°C before use.

To measure DPPC uptake and metabolism, rats were anesthetized with intraperitoneal pentobarbital sodium (50 mg/kg body wt). Radiolabeled phospholipid vesicles (0.1 µmol of DPPC in 0.1 ml buffer) with or without 3 mol/100 ml MJ33 were instilled into the airway through a catheter inserted into the trachea to the level of the carina. The thorax was incised, and lungs were cleared of blood by gravity perfusion through the pulmonary artery and then removed from the rat for isolated organ perfusion in a recirculating system using 40 ml of Krebs-Ringer bicarbonate buffer, pH 7.4, containing 3% fatty acid-free BSA and 10 mM glucose (20). The time between instillation of liposomes and the start of perfusion was 5 min. Perfusate was gassed with 5% CO2 in air, and lungs were continuously ventilated with the same gas mixture at 60 cycles/min, 2-ml tidal volume, and 2 cmH2O end-expiratory pressure. In some experiments, 0.1 mM 8-BrcAMP, 0.5 mM DENP, or 2 mM pBPB was added to the perfusate at the start of recirculating perfusion. 8-BrcAMP is a secretagogue that has been shown previously to stimulate DPPC uptake and metabolism by the perfused lung (14-17); DENP is a serine protease inhibitor that has been shown to inhibit authentic aiPLA2 (1, 7, 25, 26); pBPB is a histidine-active agent that inhibits secreted type PLA2 enzymes (10). Lungs were perfused for varying times up to 3 h. As previously documented for similar experiments (20), none of the lungs showed gross evidence of fluid accumulation during perfusion, and the pressures required for ventilation and for perfusion of the lungs did change significantly.

At the end of the perfusion, lungs were lavaged five times with ice-cold saline (7 ml each lavage), and lung tissue was homogenized in saline. Aliquots of the homogenate and the perfusate were assayed for radioactivity to calculate accumulation of radiolabel. Uptake of DPPC by the lung was expressed as the percent of endotracheally instilled disintegrations per minute (dpm) remaining in the lung after lung lavage plus dpm in the perfusate. Perfusate dpm accounted for ~1-2% of internalized phospholipid. The lung homogenate was extracted using the Bligh and Dyer procedure (4) to derive lipid and aqueous fractions (19). In the extracted perfusate, all dpm recovered were in the aqueous fraction, which, as we have shown previously, is free choline, presumably derived from intracellular degradation of DPPC (16).

Lipid fractions were separated further into individual classes by TLC on silica gel using a solvent system of chloroform-methanol-NH3-H2O (65:35:2.5:2.5) (29). The lipid spots were visualized by brief exposure to iodine vapors and then scraped for determination of dpm. Authentic standards were cochromatographed for identification of individual lipids. The disaturated PC (DSPC) fraction1 was separated from total PC on a neutral alumina column after treatment of lipids with OsO4 (28). All fractions were analyzed for dpm. The difference between total PC and DSPC was termed USPC. Recovery of authentic [3H]DPPC applied to the neutral alumina column exceeded 95%. We have shown previously that recovery of authentic sn-1-palmitoyl,[2-14C]oleoyl PC on the column was only 0.2% (13). Degradation of internalized DPPC was calculated from the sum of dpm in lysoPC, aqueous, and USPC fractions plus dpm in the perfusate. This estimate of degradation does not account for dpm that have been reincorporated into DSPC after degradation of the original radiolabeled DPPC substrate.

To measure DPPC synthesis, rats were anesthetized with ketamine-xylazine (50 mg/kg-10 mg/kg). Nonradioactive liposomes (0.1 µmol) with 5 mol/100 ml MJ33 were instilled into the trachea. After 1 h, 100 µCi of [3H-methyl]choline and 5 µCi of 9,10-[14C]palmitate complexed with BSA were injected into the tail vein. Rats were allowed to recover from anesthesia and were maintained under standard conditions with access to food and water. After 12 or 24 h, rats were anesthetized with pentobarbital sodium and lungs were cleared of blood and lavaged as described above. Lamellar bodies were isolated from postlavaged lungs by upward flotation using a sucrose gradient; we have shown previously that this method yields a lamellar body fraction with a phospholipid-to-protein ratio that is ~8-10 and is essentially free of contamination with mitochondrial (succinic dehydrogenase) and microsomal (NADPH cytochrome c reductase) marker enzymes (5, 17). Surfactant was isolated from the lavage material by a NaCl gradient (17); this method yields surfactant consisting of the large-aggregate fraction but does not include small aggregates. Lipids were extracted from the surfactant and lamellar bodies, separated into DSPC and USPC fractions as described above, and quantitated by measurement of dpm, protein, and phospholipid phosphorus (27). Protein was measured with protein binding dye reagent (Bio-Rad, Richmond, CA) using bovine gamma -globulin as standard.

PLA2 activity in lung homogenates was measured by release of [3H]palmitate at pH 4.0 (40 mM sodium acetate and 5 mm EDTA) with [3H]DPPC as substrate in mixed unilamellar liposomes (12). [3H]palmitate was separated by TLC, and dpm were measured (12).

Results are expressed as means ± SE. Statistical comparisons were evaluated by ANOVA with Boneferroni correction using SigmaStat (Jandal Scientific, San Rafael, CA); the level of statistical significance was taken as P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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REFERENCES

DPPC uptake. Previous studies using endotracheally instilled radiolabeled liposomes have demonstrated a rapid increase of lung-associated radiolabel that cannot be removed by treatment of lungs with detergent or mild trypsinization and presumably represents lipid exchange between the alveolar space and epithelial cell membranes (14-16). To obtain a baseline value for this initial rapid phase, lungs were analyzed 5 min after the endotracheal instillation of [3H]DPPC. This initial association with the lung represented ~10% of instilled dpm (Fig. 1) and was similar in the presence and absence of 8-BrcAMP or PLA2 inhibitor (MJ33). Subsequent time-dependent increase in lung-associated radiolabel occurs by endocytosis (11). Uptake of DPPC by the lung was measured at 0.5, 1, 2, and 3 h of perfusion and demonstrated a linear increase of lung-associated dpm under all four conditions, i.e., with and without 8-BrcAMP with and without MJ33 (Fig. 1). The slopes of these plots indicate uptake from the alveolar space as a percentage of instilled dpm per hour (Table 1). In control lungs, uptake of label from the alveolar space was ~5%/h, which extrapolates to a half-life of 10 h. The rate of uptake was increased ~2.5-fold in the presence of 8-BrcAMP. The presence of MJ33, the PLA2 inhibitor, had no effect on uptake of DPPC (Fig. 1 and Table 1).


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Fig. 1.   Uptake of [3H]dipalmitoylphosphatidylcholine (DPPC) by the isolated perfused lung plotted vs. time of perfusion in the presence and absence of 0.1 mM 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) added to the lung perfusate. Mixed unilamellar liposomes containing 0.1 µmol of [3H]DPPC ± 3 mol/100 ml MJ33 were instilled into the trachea at zero time. Recovery of dpm in postlavage lungs plus perfusate was used to calculate uptake as percent instilled 3H. The initial time point was 5 min after instillation of liposomes. Each of the 3-h time points represents the mean ± SE for 3 individual experiments. Each of the remaining points represents a single experiment. The effect of 8-BrcAMP on uptake at 3 h was statistically significant (P < 0.05 vs. control) in either the presence or absence of MJ33.


                              
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Table 1.   Uptake and degradation of DPPC by isolated perfused rat lung following endotracheal instillation of [3H]DPPC-labeled mixed unilamellar liposomes

The DSPC content of the lung lavage was measured for each experiment and varied from 620 to 680 µg per lung. Values were similar for the four conditions, and there was no change during the 3-h perfusion period (data not shown). The value for lavage DSPC presumably includes the endogenous DSPC pool plus 0.1 µmol (75 µg) of instilled [3H]DPPC; thus the instilled lipid represents ~12% of the total pool.

DPPC degradation. Degradation of internalized DPPC at 3 h after instillation of liposomes was calculated based on the sum of dpm recovered in three classes of metabolites: the lysoPC and unsaturated PC fractions of the lung homogenate and the aqueous soluble fraction of the homogenate plus perfusate. The contribution of each of these fractions to total DPPC degradation in control lungs was 10% in lysoPC, 48% in the aqueous fraction, and 42% in the USPC fraction (Fig. 2). Approximately half of the aqueous counts was found in the lung homogenate and the other half in the perfusate. The distribution of metabolites in the presence of 8-BrcAMP was similar to control, whereas in the presence of MJ33, the aqueous fraction was increased to ~65% of total and the USPC fraction was decreased to ~25% (Fig. 2).


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Fig. 2.   Effect of various agents on the degradation of [3H]DPPC by the isolated perfused lung at 3 h of perfusion. The conditions were control (Cont; no additions), 3 mol/100 ml MJ33 instilled with liposomes, or 0.5 mM diethyl p-nitrophenyl phosphate (DENP), 2 mM p-bromophenacyl bromide (pBPB), or 0.1 mM 8-BrcAMP added to the perfusate. Degradation was calculated as the sum of radiolabel recovered in lysophosphatidylcholine (lysoPC), unsaturated phosphatidylcholine (unsat.PC), and the aqueous fractions in the postlavaged lung plus radiolabel recovered in the perfusate. Degradation of DPPC is expressed as percent total lung dpm (i.e., lung uptake of DPPC). P < 0.05 for MJ33 and DENP vs. control and for 8-BrcAMP + MJ33 vs. 8-BrcAMP. P > 0.05 for pBPB vs. control.

Under control conditions, 38% of the internalized DPPC at 3 h had been degraded, and this increased 1.9-fold to 72% in the presence of 8-BrcAMP (Table 1). The presence of MJ33 significantly inhibited the degradation of internalized lipid by ~40-50% (Table 1). This effect was similar for unstimulated and secretagogue-stimulated lungs. The effect of the serine protease inhibitor DENP also significantly decreased degradation of DPPC, whereas pBPB had no effect (Fig. 2). In a separate series of experiments (data not shown), lungs perfused for 2 h in the presence of 8-BrcAMP showed a nonsignificant 11% decrease of uptake in the presence of 2 mM pBPB (n = 4, P > 0.05).

DPPC synthesis. Synthesis of DPPC was evaluated by measuring incorporation of radiolabeled substrates ([3H]choline, [14C]palmitate) into DSPC of lamellar bodies (LB) and surfactant (Surf). Lungs were harvested for isolation of LB and Surf fractions at 12 or 24 h after intravenous injection of radiolabeled substrates. The pool sizes for total phospholipid, DSPC, and USPC in both LB and Surf fractions were similar at 12 and 24 h, and values for these time points were combined. The method of surfactant isolation does not recover the small lipid vesicles so that the recovered surfactant DSPC is only ~60% of total lavage DSPC. In LB, DSPC represented 45% and USPC 28% of total phospholipid, whereas the corresponding values in Surf were 52 and 27% (Table 2). The ratio of total phospholipid (µg) to total protein (µg) was 8.7 for LB and 3.7 for Surf (data not shown). Phospholipid pool sizes were unchanged with MJ33 (Table 2).

                              
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Table 2.   Pool sizes of phospholipids in lamellar body and surfactant fractions of rat lungs

Incorporation of substrates into DSPC in LB and Surf was expressed as specific activity (dpm/nmol DSPC). Specific activity in LB and Surf in control lungs was similar at 12 and 24 h for both choline and palmitate labels (Fig. 3). To evaluate the effect of MJ33, lungs were instilled with MJ33 before intravenous injection of radiolabeled substrates. aiPLA2 activity (activity at pH 4 inhibited by MJ33) in control lungs was 12.9 nmol · h-1 · mg protein-1 and was decreased to 0.8 and 2.2 nmol · h-1 · mg protein-1 at 2 and 24 h, respectively, after endotracheal instillation of 5 mol/100 ml MJ33 in unilamellar liposomes. MJ33 pretreatment had no effect on the specific activity of choline in LB or Surf at either 12 or 24 h (Fig. 3). However, [14C]palmitate incorporation into DSPC in both LB and Surf fractions was decreased in the presence of MJ33 by ~50% at 12 h and 65% at 24 h (Fig. 3).


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Fig. 3.   Effect of MJ33 on incorporation of [3H]choline (A) or [14C]palmitate (B) into disaturated PC (DSPC) of lung lamellar body and surfactant fractions. Lungs were analyzed at 12 and 24 h after intravenous bolus injection of radiolabeled substrates. MJ33 (5 mol/100 ml) was administered intratracheally in liposomes 1 h before substrate infusion. Values are means ± SE for n = 3 (n = 7 for choline at 24 h). For [14C]palmitate, P < 0.05 for all comparisons of control vs. MJ33. For [3H]choline, none of the comparisons between control and MJ33 was statistically significant (P > 0.05).

In vitro aiPLA2 activity. We have shown previously in rat lung homogenate and isolated type II alveolar epithelial cells that MJ33 inhibits ~70-80% of acidic (pH 4) PLA2 activity (12, 19, 25, 26). We have also shown that monoclonal antibody (MAb) 8H11 markedly inhibits activity of recombinant aiPLA2 (7), but we have not previously reported its effect on lung homogenate. In the present study, MJ33 inhibited PLA2 activity in the rat lung homogenate by 73%, whereas MAb 8H11 inhibited PLA2 activity by 85% (Fig. 4). Inhibition by MAb 8H11 was similar in the presence of MJ33, i.e., there was no additive effect of the two inhibitors (Fig. 4). The activity remaining after treatment with antibody plus MJ33 may represent another enzyme of considerably lower abundance in the lung homogenate.


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Fig. 4.   Effect of inhibitors on lysosomal-type phospholipase A2 (aiPLA2) activity of rat lung homogenates. Homogenates were centrifuged at 10,000 g for 10 min, and the supernatant was used for assay. The assay contained 200 µg supernatant protein in buffer (40 mM sodium acetate and 5 mM EDTA) at pH 4. Incubation was for 1 h at 37°C. Substrate was [3H]DPPC (sn-2 fatty acid label) in unilamellar liposomes. MJ33 was added at 3 mol/100 ml at the start of the assay. Monoclonal antibody (mAb) 8H11 at 1:50 dilution was preincubated for 2 h with the homogenate supernatant. Values are means ± range for n = 2.


    DISCUSSION
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ABSTRACT
INTRODUCTION
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Previous studies have established that alveolar DPPC is endocytosed by type II cells followed by intracellular degradation (3, 9, 12, 21, 30, 32), most likely in a lysosomal compartment (33, 34). Complete degradation would generate free choline, palmitic acid, glycerol, and phosphate. Glycerol and fatty acids enter the metabolic pool where they can be reutilized for lipid synthesis but are also metabolized via intermediary metabolism. Choline, on the other hand, is utilized exclusively by the cell for resynthesis of phospholipids, predominantly PC, with a minor contribution from sphingolipids. This differential reutilization of choline presumably accounts for its significantly prolonged biological half-life compared with fatty acids and glycerol (11, 23, 35). Thus DPPC degradation and resynthesis by the type II cell are linked metabolically through the generation and reutilization of choline as well as of other substrates.

Previous studies have demonstrated that the initial step in degradation of internalized DPPC by the type II cell occurs predominantly via PLA2 activity. First, using the isolated lung model instilled with DPPC labeled in the sn-2 palmitate moiety, 94% of dpm were recovered in free fatty acids, the product of PLA2 activity (19). Second, there was no appreciable degradation of a DPPC analog in which a nonmetabolizable alkyl linkage replaced the acyl group in the sn-2 position (31, 33, 34). Finally, MJ33, a phospholipid transition state analog PLA2 inhibitor, significantly decreased degradation of DPPC in the lung and type II cells (12, 19).

PLA2 represents a relatively large family of enzymes, each with the same enzymatic activity but with different cofactor requirements and subcellular localization (10). These have been classified as secreted PLA2, cytosolic PLA2, and Ca2+-independent PLA2 (10). Our results suggest that, in the lung, MJ33 inhibits aiPLA2, a Ca2+-independent PLA2 that we have called a lysosomal-type enzyme because of its acidic pH optimum. First, assay of rat lung homogenate or type II cells showed that MJ33 inhibited Ca2+-independent PLA2 activity at pH 4; it did not inhibit Ca2+-dependent activity at pH 8.5 or phospholipase C activity (12, 19, 26). Note that we did not detect significant Ca2+-independent PLA2 activity at pH 8 or Ca2+-dependent activity at pH 4 in the lung homogenate (26). Second, MAb 8H11 inhibited activity in the lung homogenate by 85%, indicating that aiPLA2 is responsible for the bulk of activity at pH 4 (Fig. 4). The combination of MJ33 plus the MAb inhibited similarly to the MAb alone, indicating that MJ33 was not acting on another protein. Third, both an inhibitor of secreted PLA2 (pBPB) or of cytosolic PLA2 (arachidonoyl methylketone) had little to no effect on DPPC metabolism by type II cells (12) or (for pBPB) by the perfused lung (present study). These agents do not inhibit activity of recombinant aiPLA2 (7, 26). Fourth, DENP, a serine protease inhibitor that inhibits aiPLA2 activity (17, 25, 26), inhibited DPPC degradation in the isolated lung (present study); this agent does not inhibit secreted PLA2 that are histidine-based enzymes (10).

The presumption that MJ33 inhibits a lysosomal enzyme is compatible with previous studies, which indicate that the lysosome is the subcellular site of DPPC degradation in the lung (33, 34). Subcellular fractionation of rat lungs has localized aiPLA2 to the lysosomal fraction where the low pH would ensure maximal activity (1, 12). While aiPLA2 is also present in lamellar bodies (1, 12), its possible function in that location is not known; the somewhat higher pH in lamellar bodies compared with lysosomes (6) and the presence of surfactant protein A (SP-A), an inhibitor of aiPLA2 activity (18), raise the possibility that the protein may not be enzymatically active in that organelle.

The present study further investigated the role of aiPLA2 in DPPC degradation and synthesis using MJ33 as an inhibitor of enzymatic activity. When studied over a period of 3 h, the isolated perfused lung demonstrated linear uptake of labeled DPPC at ~5%/h, indicating a DPPC biological half-life of ~10 h. This may be an underestimation of the true rate of uptake since the specific activity of alveolar DPPC was probably decreasing during perfusion due to removal of the labeled compound and secretion of unlabeled DPPC. Uptake and degradation were both markedly stimulated in the presence of 8-BrcAMP. We have provided evidence previously that the stimulation of DPPC uptake by secretagogues is associated with recruitment of SP-A receptors to the plasma membrane of type II cells (8). The mechanism for cAMP stimulation of degradation has not been elucidated but does not appear to be a direct effect on PLA2 (15). MJ33 in this model inhibited degradation by ~50% under both control and stimulated conditions. However, it is important to note that it is not possible to measure the relevant pool sizes, so the actual rates of degradation in the intact lung cannot be calculated. Several factors could influence the degree of inhibition by MJ33. First, the relative distribution of MJ33 and DPPC at either the cellular or subcellular level may not have been identical. Second, MJ33 is a competitive inhibitor, and its intracellular concentration may have limited its effectiveness. Finally, type II cells contain other phospholipases that are not MJ33 sensitive and may participate in DPPC degradation. In a previous study with isolated cells, we found that ~20% of DPPC degradation is sensitive to p-bromophenacyl bromide, suggesting participation of a Ca2+-dependent secreted type PLA2 (12); a similar decrease was seen with the perfused lung in the present study, but it was not statistically significant. In the MJ33-inhibited state, phospholipase C (or D) may show a relative increase in activity; that would be compatible with a relative increase in radiolabel recovery in the aqueous fraction of DPPC metabolites with MJ33-treated lungs as described above. However, under normal conditions, the lysosomal type PLA2 appears to account for the major fraction of internalized DPPC degradation by the lung.

Synthesis of DSPC from radiolabeled precursors was evaluated in intact rats that had been pretreated with MJ33. Specific activity of DSPC was measured in lamellar bodies, representing the storage site of lung surfactant, and in the secreted material obtained by bronchoalveolar lavage. MJ33 had no effect on the incorporation of [3H]choline, but significantly inhibited incorporation of [14C]palmitate into DSPC. Incorporation of these labels cannot be translated directly into rates of synthesis, since the intracellular specific activities of the relevant precursor substrates are not known and might be changed by MJ33. The results obtained with the in vivo lung confirm the decreased palmitate incorporation into DSPC with MJ33 treatment of isolated type II cells (13). Unlike the present results with intact lung, experiments with isolated cells also showed a modest decrease in choline incorporation. Interpretation of the present metabolic data is based on the known pathways for DSPC synthesis. The de novo pathway requires the condensation of cytidyl diphosphocholine and diacylglycerol (36), and its inhibition would be reflected by decreased incorporation of both fatty acid (i.e., palmitate) and choline into DSPC. A second pathway for DSPC synthesis is via acylation of lysoPC, which in turn is generated by the action of PLA2 (36). Inhibition of this pathway with the conditions used in this study would be reflected by decreased incorporation of palmitate and no change in choline incorporation. Thus the present results are compatible with inhibition by MJ33 of the deacylation/reacylation pathway for DPPC synthesis, with relatively little effect on de novo synthesis. The decreased synthesis of DSPC with aiPLA2 inhibition is ~60% compared with control. Thus aiPLA2, a Ca2+-independent enzyme with maximal activity in the acidic pH range, has a major role in the synthesis of DSPC by providing lysoPC substrate. Interestingly, Balsinde et al. (2) have shown involvement of a Ca2+-independent PLA2 in membrane PC remodeling in a macrophage cell line, although the putative PLA2 in that system is not a lysosomal enzyme.

In summary, we have shown in the normal rat lung that internalized DPPC is degraded predominantly via a lysosomal-type PLA2 and that activity of this enzyme is also required for synthesis of DPPC via the deacylation/reacylation pathway. The interrelationships of these pathways and the products of phospholipase activity are shown in Fig. 5.


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Fig. 5.   Metabolic pathways for following the choline label in DPPC. DPPC is degraded by phospholipases (PL) A2, C, or D to generate lysoPC or aqueous soluble metabolites glycerophosphorylcholine (GPC), choline, and choline phosphate. The primary product of PLA2 activity is lysoPC (fatty acyl group in sn-1 position), which can then be further degraded to aqueous metabolites. Not indicated in the figure is that unsaturated PC (USPC) is also a substrate for these same phospholipases. Also not shown is the action of PLA1, which would generate lysoPC but with the fatty acyl group in the sn-2 rather than the sn-1 position. USPC or DPPC is synthesized either by reacylation of lysoPC or via the de novo pathway utilizing cytidyl diphosphocholine (CDP choline). The pathway for synthesis via lysoPC is called the deacylation/reacylation pathway. Not shown is the pathway for incorporation of phosphocholine into sphingomyelin.


    ACKNOWLEDGEMENTS

We thank Drs. Mahendra Jain and Avinash Chander for sage advice, Jamie Fisher for technical assistance, and Elaine Primerano for typing the manuscript.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grant HL-19737.

This work was presented in part at the Experimental Biology 2000 Meeting in San Diego, CA, in April, 2000.

Address for reprint requests and other correspondence: A. B. Fisher, Institute for Environmental Medicine, Univ. of Pennsylvania School of Medicine, 1 John Morgan Bldg., 3620 Hamilton Walk, Philadelphia, PA 19104-6068 (E-mail: abf{at}mail.med.upenn.edu).

1 The osmication method measures disaturated PC, which includes DPPC as the major component, with a minor contribution from PC containing other saturated fatty acids. We have used the terms DSPC for PC measured by the osmication method and DPPC for the authentic phospholipid.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 3 May 2000; accepted in final form 9 November 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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