Prostaglandins Lcukotrienes and Medicine 0 Longman Group UK Ltd 1987
METABOLISM OF PROSTAGLANDIN PG-F2a BY FRESHLY ISOLATED ALVEOLAR TYPE II CELLS FROM LUNGS OF ADULT MALE OR PREGNANT RABBITS T.R. Devereux, J.R. Fouts and T.E. Eling, National Institute of Environmental Health Sciences, National Institutes of Health, P.O. Box 12233; Research Triangle Park, North Carolina 27709 (Reprint requests to T.R. D.). ABSTRACT Prostaglandin F2a (PG-F2a) was metabolized to different products by freshly isolated alveolar type II cells from adult male or pregnant rabbits. The type II cells from the adult male rabbits metabolized PG-F2a to products which co-chromatographed on HPLC with 15-keto PG-F2a and 13,14-dihydro-15-keto PG-F2a, metabolites of cytosolic metabolism. The cells from the pregnant rabbit metabolized the prostaglandin to several polar metabolites. The major peak co-eluted with PO-hydroxy-PG-F2a, a product dependent on cytochrome P-450 metabolism. The other polar metabolites were likely secondary oxidation products, formed by both the cytosolic 15-dehydrogenase and/or the 13,14-reductase and the microsomal ti-hydroxylase. No metabolism of PG-F2a was observed in fractions of alveolar macrophages or tracheal cells of either adult male or pregnant rabbits. Fractions enriched in Clara cells (40-60% purity) showed little and variable Et$;lsrn of PG-F2a, qualitatively similar to that observed with type However, data was inconclusive due to the low Clara cell purity aid low activity. INTRODUCTION The lung is a major site of metabolism and deactivation of circulating prostaglandins (1). For example, prostaglandin F2a (PG-F2a), a bronchial constrictor, is metabolized in lung by oxidation at C-15 (15-dehydrogenase)to 15-keto PG-F2a and reduction of the A 13 double bond (13,14-reductase; 2, 3) to 13,14-dihydro-15-ketoPG-F2a. Other prostaglandins are metabolized by similar reaction sequences. The lungs from pregnant rabbits are much more active than lungs from nonpregnant rabbits in metabolizing prostaglandins (4, 5). Both the
cytosolic enzyme 15-dehydrogenase (4) and the microsomal enzyme PG-whydroxylase (5), dependent on cytochrome P-450, exhibit high activities in the lungs of pregnant rabbits. The cytochrome P-450 isozyme responsible for PG-w-hydroxylase activity has recently been purified from lungs of pregnant rabbits (6). This isozyme is absent or present at very low activity in lungs of nonpregnant females or male rabbits (5, 7). Thus, in the pregnant rabbit, the major pathway for pulmonary detoxication of prostaglandins may be catalyzed by cytochrome P-450. Previously published studies on pulmonary prostaglandin metabolism used isolated perfused lung (8), subcellular fractions (3-5) or purified enzymes (6). However, the lung is composed of many cell types, and the cellular distribution of the 15_dehydrogenase, 13,14-reductase and ohydroxylase is not known. Our previous studies demonstrated that type II cells, Clara cells and tracheal cells of rabbits contain cytochrome P-450 and.can oxidize xenobiotics, although little or no activity was observed with alveolar macrophages (9-12). The purpose of this study was to determine whether prostaglandin metabolism using PG-F2a as a model substrate occurs in these cell types of rabbit lung and which pathways are responsible for this metabolism. Freshyl isolated cells from both adult male and 28-day pregnant rabbits were used for this study. METHODS Chemicals Ci/mmol) was obtained from New England Nuclear PG-F2a (9-3H(N)-12.2 (Boston, MA). sH-15-keto PG-F2a was synthesized from PG-F2a using 100,OOOxg supernatant fraction from guinea pig lung by the method of Parkes and Eling (13). 13,14-dihydro-15-keto-PG-F2a was purchased from Upjohn, Inc. (Kalamazoo, MI). sH-20-hydroxy-PG-F2a was synthesized by a modification of the method of Powell (14). sH-PG-F2a was incubated with microsomes of 28-day pregnant rabbits and NADPH, and samples were Protease I (lot extracted and analyzed as below for PG-F2a metabolism. #13F-8040), Protease XIV (lot #45F-0545), NAD+, NADPH, and unlabeled PG-F2a were obtained from Sigma Chemical Co. (St. Louis, MO). Cell preparation and identification Adult male or 28-day pregnant New Zealand White rabbits (Dutchland Animals, Denver, PA) weighing 2-3kg were used for the cell preparations. The perfusion and cell isolation procedures were described previously Briefly, the alveolar macrophages were isolated from the (9-11, 15). The alveolar type II cells and Clara lavage fluid by centrifugation. cells were isolated from a protease digest (Protease I) of the lung tissue by centrifugal elutriation followed by density gradient centrifugation (10, 15). The tracheal cells were obtained both by enzyme digestion with Protease XIV (11) and by scraping the tracheas.
Cells were counted with the Electrozone/Celloscope (Particle Data Inc., Elmhurst, IL). Viability of cells was estimated by the trypan blue (0.04%) dye exclusion method (16). Alveolar type II cells were identified and counted by the modified Papanicolaou staining method without acid alcohol (17). Clara cell identification and enumeration were made by nitroblue tetrazolium staining of air-dried smears (18). PG-F2a metabolism Incubations contained whole or sonicated cells, or microsomes or 100,OOOxg supernatant from cells or from whole lung (l-3 mgs protein), 0.1 PM 9-sH PG-F2a (1 ,Ci), 2 mM NAD+, in 0.06 M KH,PO, buffer, pH 8.0 in 1 ml volume. Incubations with microsomes contained 2 mM NADPH instead of NAD+. Following incubation at 37°C for 0, 5, 10, 15 or 20 min, metabolism was stopped by acidifying the samples to pH 3.5 with 1 M HCl and extracting 3 times with 4 ml ethyl acetate. The organic phases were dried with anhydrous MgSO, and evaporated under N . Residues were suspended in 2 ml methanol and evaporated again. Samp 7es were resuspended in 0.45 ml 30% aqueous methanol and stored in the freezer (-20°C). Recovery of radioactivity at this point was greater than 90%. 0.2 ml sample was injected into a Waters HPLC and analyzed by the method of Henke et al. (19) using an Altex Cl8 column with a flow rate of 1.1 ml/minrne elution gradient consisted of 53% solution A (90% water: 10% methanol titrated to pH 5.05 with 2 ml/l acetic acid and ammonium hydroxide) and 47% solution B (100% methanol) for 50 min and 100% solution B for 20 min. RESULTS PG-F2a metabolism
in lungs of adult male rabbits
Metabolism of sH-PG-F2a was examined in isolated alveolar type II cells (>80% purity), Clara cells (40-60% purity with up to 40% macrophages and lo-15% type II cells), alveolar macrophages (from the lavage fluid) and tracheal cells of adult male rabbits. Peaks co-chromatographing with the 15-keto PG-F2a (peak 4) and 13,14-dihydro-15-keto PG-F2a (peak 7) standards were observed in the HPLC profile of metabolites formed when PG-F2a was incubated with freshly isolated alveolar type II cells [;A:; ;I)' Peak 5 was an unidentified contaminant of the substrate . No metabolism of PG-F2a was detected in the tracheal cell fraction or in the alveolar macrophages. Little and variable PG-F2a metabolism occurred in the Clara cell enriched fractions (data not shown). However, because of the low Clara cell purity, detailed studies of PG-F2a metabolism in these isolated cells were not pursued. Metabolism of PG-F2a by the 100,OOOxg supernatant fraction of whole rabbit lung (Fig. 1B) was observed with the major metabolite corresponding to the 15-keto PG-F2a standard (peak 4). Little or no 13,14-dihydro-15keto PG-F2a was observed. Metabolism by the supernatant fraction was dependent on the addition of NAD+ in agreement with a requirement for NAD+ by the 15dehydrogenase (20). No polar metabolites were observed when PG-F2a was incubated with the isolated cells, and no metabolism was observed when PG-F2a and NADPH were incubated with microsomal preparations from the isolated pulmonary cells or from whole lung (Fig. 1C).
50 I I
i3 i 0 RETENTION TIME (mid
Figure 1. Profiles of PG-F~CZ metabolites from lung fractions of adult male rabbits. HPLC profiles of metabolites formed when sH-PG-F2a was incubated at 37'C for 10 min with whole freshly isolated alveolar type II cells (3 mg prot/ml; 1A); 100,OOOxg supernatant of whole lung (1 mg protein/ml; 15); or lung microsomes (1 mg protein/ml; 1C); all from adult male rabbits. Conditions of the assays are given in the "Methods" section. Peaks l-3 were observed only with samples from lungs of pregnant rabbits (see Fig. 2). PG-F2a metabolism
in lungs of pregnant rabbits
The metabolism of PG-F2a was then assessed using isolated pulmonary cells from pregnant rabbits. Alveolar type II cells isolated from 2%day pregnant rabbits metabolized PG-F2a to several polar metabolites (peaks l-3) as observed in the HPLC profile of metabolites (Fig. 2A). No metabolite corresponding to the 15-keto PG-F2a standard (retention
time 35 min) was observed, although 15-dehydrogenase activity is reported to be elevated in rabbit lung during pregnancy (4). No metabolism of PG-F2a was detected in fractions of tracheal cells or in alveolar macrophages of pregnant rabbits. What little metabolism was observed in the enriched fractions of Clara cells (data not shown) was qualitatively similar to that seen with type II cells. This small amount of metabolism may have been due to the lo-15% contamination of the Clara cell fraction by type II cells.
10,000 2 I
1 3 2
RETENTION TIME (min)
Figure 2. HPLC profiles of metabolites formed when sH-PG-F2a was incubated at 37°C for 10 min (5 min for 28) with whole freshly isolated alveolar type II cells (2.3 mg protein/ml; 2A); 100,OOOxg supernatant of whole lung (0.2 mg protein/ml; 28); or lung microsomes (1 mg protein/ml; 2C); all from pregnant rabbits. Conditions of the assays are given in the "Methods" section. was also incubated with the 100,OOOxg supernatant obtained from lungs of pregnant rabbits (Fig. 28). We observed extensive metabolism of PG-F2a. to 15-keto PG-F2a in the presence of NAD+. Essentially complete metabolism of PG-F2a was observed in incubations which contained only 20% of the protein used when supernatants from control lungs were PG-F2a
incubated with PG-F2a, suggesting that pregnancy elevated nase activity in lung (4).
The polar metabolites observed in incubations of PG-F2a with type II cells from pregnant animals (Fig. 2A) could be 20-hydroxy-PG-F2a formed by cytochrome P-450 oxidation of PG-F2a as described previously (5) and/or secondary oxidation products of cytosolic PG-F2a metabolites. The major polar metabolite (peak 2) observed with the type II cells appeared to co-elute with the 20-hydroxy-PG-F2a standard (14) formed when a microsomal fraction from whole lung of pregnant rabbit was incubated with PG-F2a in the presence of NADPH (Fig. 2C). In order to further separate the polar PG-F2a metabolites formed by the type II cells (Fig. 2A), incubations of PG-F2a with type II cells or whole lung microsomes were rechromatographed with a shallower elution gradient to separate the peaks (Fig. 3). Under these conditions, several additional peaks were visualized, but the largest peak co-chromatographed with the major peak observed when microsomal preparations of whole lung from pregnant rabbits were incubated with PG-F2a and NADPH. These results suggest that PG-F2a is primarily metabolized by these type II cells to The other smaller peaks observed may represent PO-hydroxy-PG-F2a. secondary oxidation products of 15-keto PG-F2a and/or 13,14-dihydro-15-keto PG-F2a.
Figure 3. HPLC profiles of polar metabolites formed when sH-PG-F2a was incubated at 37°C for 10 min with whole freshly isolated type II cells or lung microsomes (1.0 mg protein/ml) i2.3 mg protein/ml) (-) _ _ - -) from pregnant rabbits. Duplicate samples from Fig. 2A and 2C were rechromatographed using a shallower elution gradient (60% A: 40% B for 20 min) in order to separate the metabolites. Time course of PG-F2a metabolism
in isolated type II cells
A time course for the formation of PG-F2a metabolites by alveolar type II cells isolated from both adult male and pregnant rabbits is shown in Fig. 4. PG-F2a was metabolized more rapidly by the cells isolated from
pregnant rabbits than from the control males. In these experiments, the only metabolite detected with the cells from the control was 15-keto The 13,14-dihydro-15-keto PG-F2a metabolite was below its limit PG-FZa. of detection but was observed in other experiments (e.g., Fig. 1). As observed in Fig. 2, PG-F2a was extensively oxidized to polar metabolites No 15-keto PG-F2a and only minor with cells from the pregnant animals. amounts of 13,14-dihydro-15-keto PG-F2a was observed with these cells. These data indicate the predominance of cytochrome P-450 compared to the dehydrogenase and reductase in the metabolism of PG-F2a in the type II cells from lungs of pregnant rabbits.
*_-.__*___ --c INCUBATION
Figure 4. Time course of PG-F2a metabolism in whole freshly isolated alveolar type II cells from adult male (- - - -) and pregnant rabbits @---a, PG-F2a (adult male); ?? --15-keto PG-F2a (adult (-). male); 04, PG-F2a (pregnant); A+, po ir metabolites (pregnant); o--_-O, 13,14-dihydro-15-keto PG-FPa (pregnant). The protein concentrations were 2.3 mg/ml and 2.0 mg/ml for the cells from pregnant and adult male rabbits, respectively. Other conditions of the assay are given in the "Methods" section. The PG-F2a did not start at 100% because of high background radioactivity and some contaminants (e.g., peak 5) in the labeled PG-F2a.
DISCUSSION Our results demonstrate that PG-F2a is metabolized by alveolar type II cells of rabbit, although different pathways appear to be involved in its degradation in lungs of adult male vs. pregnant rabbits. The major metabolic pathways for prostaglandin inactivation in the lungs of adult 49
non-pregnant female rabbits are the 15-dehydrogenase and the 13,14reductase (4). 15-keto PG-F&Z was the major metabolite formed by the alveolar type II cells of adult male rabbits. Small amounts of a metabolite co-chromatographing with the 13,14-dihydro-15-keto PG-F& were observed with about one-half of the cell preparations, but the conditions for optimization of the reductase enzyme activity were not studied. Cytochrome P-450-dependent w-hydroxylation of PG-F2a does not appear to function in the degradation of PG-F2a in lungs of the adult non-pregnant rabbit (both male and female; 7). In our study, this pathway was not detected in microsomal preparations of whole lung, or in type II or Clara cells isolated from adult male rabbits. Cytochrome P-450-dependent PG-w-hydroxylase is induced in lungs of pregnant rabbits compared to either adult male or non-pregnant female rabbits (Z), and metabolites identified after incubation of PG-F2a with lung microsomes from pregnant rabbits appear to be exclusively products of w-hydroxylation (5). One major polar metabolite was observed in the HPLC profile of metabolites formed when whole lung microsomes from pregnant rabbits were incubated with PG-F2a and NADPH, and this metabolite has been identified by Powell (7) as the 20-hydroxy-PG-F2a. Freshly isolated alveolar type II cells from pregnant rabbits metabolized PG-F2a to several polar products. The largest metabolite peak cochromatographed with the major product formed by lung microsomes and NADPH, providing evidence for the oxidation of PG-F2a to 20-hydroxyPG-F2a by alveolar type II cells of pregnant rabbits. The fact that no 15-keto PG-F2a peak was observed in these cells, and yet the 15-dehydrogenase enzyme is induced during pregnancy (4), suggests that this metabolite may also be a substrate for the w-hydroxylase. Indeed, Powell and Solomon (5) identified the 13,14-dihydro-20-hydroxy-15-keto PG-F2a as a product formed when PG-F2a was incubated with lung homogenate from pregnant rabbits. The minor polar metabolites formed by the type II cells were not identified, but are likely to be secondary metabolites formed by both the cytosolic dehydrogenase or reductase and the microsoMoreover, the data suggest that cytochrome P-450ma1 w-hydroxylase. dependent oxidation of PG-F2a in alveolar type II cells may be an important pathway for the metabolism of deactivation of prostaglandins in lungs of pregnant rabbits. We were unable to detect PG-F2a metabolism catalyzed by either cytochrome P-450 or the cytosolic dehydrogenase or reductase enzymes in tracheal cells or alveolar macrophages isolated from adult male or Enriched fractions containing Clara cells from adult pregnant animals. male or pregnant rabbits metabolized PG-F2a to products qualitatively similar to that seen with the type II cells. However, the data are inconclusive due to low Clara cell purity and contamination of the preparations by up to 15% type II cells. Because of localization studies of cytochrome P-450 and cytochrome P-450 monooxygenase activities in Clara cells of various species, we expected to find high w-hydroxylase activity in the isolated Clara cells relative to the type II cells. However, our results indicate that the alveolar type II cell of pregnant rabbit may be a more important contributor than the Clara cell to pulmonary cytochrome P-450-dependent w-hydroxylation of prostaglandins.
Extensive metabolism was observed using type II cells with increased metabolism occurring in cells from pregnant rabbits. The type II cells are a major cell type of lung and may be a major cellular site in lung for the inactivation of prostaglandins particularly in pregnant rabbits. Endothelial cells are also a major cell type in the lung, but studied with cultured endothelial cells (22) suggest that these cells are not rich in prostaglandin metabolizing enzyme activity. Other studies with freshly isolated cells are necessary to fully determine the cellular site(s) of pulmonary deactivation of prostaglandins. ACKNOWLEDGMENTS The authors thank Roberta Danilowicz and Janet Diliberto for their excellent technical assistance. REFERENCES 1.
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