Department of Biochemistry, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284-7760
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ABSTRACT |
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Hepatocytes and Kupffer cells in primary culture both secrete plasma-type platelet-activating factor-acetylhydrolase (pPAF-AH) into serum-free culture medium. The rate of secretion of pPAF-AH by Kupffer cells was 20 to 25 times higher than from hepatocytes, and Kupffer cells expressed a higher level of pPAF-AH mRNA than did hepatocytes. Purified liver cell-secreted pPAF-AH exhibited a major protein band of 65-67 kDa on SDS-PAGE; this was the band predominantly labeled when the enzyme catalytic center was reacted with [3H]diisopropylfluorophosphate ([3H]DFP). Rat bile collected from cannulated bile ducts contained significant PAF-AH activity, and bile samples possessed a prominent band at 30-32 kDa, which was the exclusive target for [3H]DFP. Experiments using tunicamycin, an inhibitor of N-linked glycosylation, and endoglycosidase H suggested that pPAF-AH secreted constitutively by cultured hepatocytes and Kupffer cells is glycosylated. The present study supports the notion that hepatic secretion of pPAF-AH into the blood contributes to the regulation of PAF and oxidized phospholipid levels in the circulation, whereas secretion of PAF-AH into the bile may allow hepatic control of these phospholipid signaling molecules in the gastrointestinal tract.
rat liver; platelet-activating factor-acetylhydrolase; secretion; bile; Kupffer cells; hepatocytes
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INTRODUCTION |
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A CALCIUM-INDEPENDENT phospholipase A2, which hydrolyzes the acetyl moiety at the sn-2 position of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (i.e., platelet-activating factor, PAF), was described initially in rat plasma and was shown to inactivate PAF (3, 4, 47). Accordingly, the enzyme responsible for the hydrolysis of PAF was termed PAF-acetylhydrolase (PAF-AH). Subsequently, it was found that the activity of this novel phospholipase was not limited to PAF itself, but the enzyme was able to hydrolyze PAF analogs containing polar and oxidized fatty acid residues at the sn-2 position, suggesting that PAF-AH may be involved in the regulation of lipid peroxidation (32, 39, 40, 46). In fact, a family of unique PAF-AHs has been described in mammalian tissues (18, 19, 34-37). The extracellular, plasma-type PAF-AH (pPAF-AH) is resistant to treatment with proteases or thiol reagents, such as DTNB, whereas the intracellular, predominantly cytosolic PAF-AH is almost completely inactivated by proteolysis or DTNB. Both the pPAF-AH and the intracellular PAF-AH are inhibited by diisopropylfluorophosphate (DFP), suggesting the presence of serine residues in the active center of the enzyme (18, 25, 33, 35). The human pPAF-AH was purified to homogeneity (34, 43), and the cDNAs for human, rat, mouse, dog, bovine, and chicken pPAF-AH have been cloned from spleen libraries (44). Recently, Tew et al. (43) reported that human pPAF-AH associated with low-density lipoproteins (LDL) is N-glycosylated, but the physiological significance of glycosylation of PAF-AH remains to be established.
It has been proposed that the PAF-AH circulating in plasma, predominantly associated with lipoproteins, may play a major role in the prevention of both systemic and local inflammatory effects of PAF and oxidized phospholipids. Elstad et al. (11) have demonstrated that human blood monocytes secrete large amounts of pPAF-AH during maturation to macrophages in culture, implicating macrophages as a major source of this enzyme in the circulation. Later, Tarbet et al. (42) reported that media from rat hepatocytes cultured in the presence of serum, in which the endogenous PAF-AH was inhibited by DFP, contained additional PAF-AH activity. Hattori and co-workers isolated PAF-AH II, a 40-kDa protein, from bovine liver (18) and cloned bovine and human cDNAs for this isoform (17). It was found that PAF-AH II was most abundant in the liver, shared 41% homology with pPAF-AH, and was distributed in a membranous fraction as well as in the cytosol. However, this enzyme was sensitive to treatment with DTNB and thereby differed from pPAF-AH. More recently, we demonstrated that rat hepatocytes in primary culture secrete PAF-AH activity into serum-free medium in a time-dependent fashion and that the release of PAF-AH was not a consequence of cell leakage or damage (41). Moreover, the secretion of PAF-AH by rat hepatocytes was regulated by PAF, its analogs, and PAF-receptor antagonists (41). These results suggested that the liver may be a prominent source of pPAF-AH in the circulation and that this enzyme may be involved in the regulation of oxidative and inflammatory responses in the liver. Recent data from our laboratory indicated that in endotoxin-exposed rat livers, Kupffer cells, but not hepatocytes or endothelial cells, expressed a 20-fold increase in pPAF-AH mRNA levels (20). However, the exact cellular origin and the nature of PAF-AH released in vivo under normal conditions have not been established. An exclusive role for hepatocytes in the formation of bile components implies that PAF-AH may be secreted into bile and may be involved in the mechanism(s) by which the liver controls inflammatory and oxidative events in the gastrointestinal tract. To our knowledge, there has been no previous report of PAF-AH activity in bile, although patients with chronic cholestasis have elevated serum PAF-AH activity (26).
In the present study, we demonstrate that rat hepatocytes and Kupffer cells contain constitutive pPAF-AH, which is secreted by cultured cells. The isolated and characterized enzymes are likely glycosylated and capable of hydrolyzing PAF and PAF analogs containing short-chain length, polar fatty acids at the sn-2 position. Also, we present evidence that rat bile contains significant amounts of PAF-AH exhibiting pPAF-AH characteristics. A novel mechanism for protecting the gastrointestinal tract against an excess of PAF and oxidized phospholipids is proposed.
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EXPERIMENTAL PROCEDURES |
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Materials. Radiolabeled 1-hexadecyl-2-[3H(N)]acetyl-sn-glycero-3-phosphocholine (sp act 7.1 Ci/mmol), 1-[3H]octadecyl-2-lyso-sn-glycero-3-phosphocholine (sp act 150 Ci/mmol), and (1,3-[3H])diisopropylfluorophosphate (sp act 8.4 Ci/mmol) were purchased from NEN (Boston, MA). Unlabeled PAF and lyso-PAF were obtained from Bachem (Bubendorf, Switzerland). Insulin (culture grade), collagenase (type IV), proteinase K, trypsin, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), HEPES, and fatty acid-free BSA (fraction V) were supplied by Sigma Chemical (St. Louis, MO). DTNB and DFP were obtained from Calbiochem (La Jolla, CA). Williams' medium E and RPMI 1640 were purchased from GIBCO (Grand Island, NY). N-Glycosidase F and endoglycosidase H were obtained from Boehringer Mannheim (Indianapolis, IN). Affi-Gel Blue Gel was purchased from Bio-Rad (Hercules, CA). Octyl-Sepharose 4-FF, Q Sepharose HP, concanavalin A Sepharose, HiTrap Q, and HiTrap blue Sepharose were products of Pharmacia (Piscataway, NJ). Other chemicals and solvents were of the highest analytic grade available.
Isolation and culture of rat hepatocytes and Kupffer cells. To isolate hepatocytes, rat livers were perfused and enzymatically digested as described previously (41). The digested liver was passed through a nylon filter, and hepatocytes were obtained after low-speed centrifugation at 4°C and with three successive washing cycles with Krebs-Henseleit buffer. The hepatocytes were resuspended in Williams' medium E supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan, UT), insulin (7 mg/l), penicillin (100 U/ml), streptomycin (100 µg/l), and L-glutamine (2 mM) at a density 2.5 × 105 cells/ml. Hepatocytes (3-4 ml) were plated on 60-mm culture plates (Corning, Houston, TX) and were cultured in an incubator at 37°C in a humidified atmosphere of 95% air-5% CO2. After 4 h of incubation, unattached cells were removed by aspiration, the medium was replaced, and hepatocytes were cultured for an additional 20 h. Medium was aspirated and cells were washed five times with Krebs-Ringer-HEPES (KRH) buffer, pH 7.4, containing 0.1% BSA, and cultured in serum-free media for the times indicated or for 40 h to supply hepatocyte-conditioned media.
Kupffer cells were isolated from enzymatically digested rat livers followed by centrifugal elutriation, as described previously in detail (27). Freshly isolated cells were suspended in RPMI 1640 medium supplemented with 25 mM HEPES, 10% fetal bovine serum, penicillin (100 U/ml), streptomycin (100 µg/ml), and L-glutamine (2 mM). Cell suspensions (4 ml, 8 × 106 cells) were plated on 60-mm culture dishes and maintained in an incubator at 37°C in a humidified atmosphere of 90% air-10% CO2. On the second day in culture, media were changed and incubations were continued for an additional 48 h. Media were aspirated and cells were washed five times with KRH-0.1% BSA buffer and incubated in serum-free RPMI 1640 media for the desired times.Cell fractionation. Freshly isolated hepatocytes were suspended in buffer consisting of 50 mM Tris · HCl (pH 7.4), 0.25 M sucrose, and 2 mM EDTA, and homogenized with 10 to 15 strokes in a Potter-Elvehjem homogenizer. Homogenate was centrifuged at 450 g for 5 min to remove unbroken cells and debris, and the supernatant was centrifuged at 105,000 g for 1 h. The pellet was termed the membrane fraction and the supernatant the cytosol for use in subsequent experiments. Freshly isolated Kupffer cells were suspended in the same buffer and sonicated three times using 20-s pulses at setting 3 in an ice bath, using a microtip probe. The cell sonicates were centrifuged at 450 g for 10 min at 4°C, and supernatant was centrifuged at 105,000 g for 1 h. The activity of PAF-AH in the membrane fraction was referred to as the membrane-associated PAF-AH and in the supernatant as cytosolic PAF-AH.
Bile collection. Rat bile ducts were cannulated under general anesthesia using a polytetrafluoroethylene catheter (ID 0.3 mm, OD 0.76 mm). The initial portions of bile (20-25 µl) were discarded to avoid blood contamination. Bile was collected for the desired times up to 6 h with periodic irrigation of the operative field with warm Krebs-Ringer solution, pH 7.4.
Synthesis of phospholipids.
PAF analogs containing short-chain-length polar residues at the
sn-2 position were synthesized using
the coupling method of Gupta et al. (15) with minor modifications.
Briefly, 10 µmol of unlabeled lyso-PAF, 25 µCi of labeled lyso-PAF,
5 mg of
4-(N,N'-dimethylamino)pyridine, and 100 µmol each of succinic or glutaric anhydride were suspended in
1 ml of anhydrous chloroform in a 15-ml tube with a ground glass cap.
Tubes were closed tightly under a stream of nitrogen and stirred on a
magnetic stirrer at 60°C for 4-5 h. Phospholipids were
extracted and were separated by TLC using a solvent system consisting
of chloroform-methanol-acetic acid-water (50:30:8:4, vol/vol). The TLC
procedure was repeated using the same solvent system, and the resulting
phospholipid, products,
1-[3H]octadecyl-2-succinoyl-sn-glycero-3-phosphocholine
or
1-[3H]octadecyl-2-glutaroyl-sn-glycero-3-phosphocholine,
were extracted from silica gel and stored in chloroform-methanol (1:1,
vol/vol) at 20°C until use.
Assay of PAF-AH. The activity of PAF-AH was determined as described previously (35) with minor modifications. Briefly, 5-25 µl of the sample were adjusted to 50 µl by diluting with 0.1 M HEPES containing 2 mM EDTA, pH 7.2. The labeled PAF solution was prepared by suspending 400 nmol of C16 PAF and 25 µCi of [3H]acetate-PAF in 0.5 ml 0.1 M HEPES buffer containing 2 mM EDTA and sonicating the mixture three times for 1 min. The enzymatic reaction was initiated by addition of 5 µl substrate and was performed for 30 min at 37°C. Incubations were terminated by adding 50 µl of 10 M acetic acid, and the mixture was applied on to a Sep-Pak C18 cartridge column. The column was washed with 3.0 ml of 0.1 M sodium acetate, and the radioactivity in the eluate was measured in a liquid scintillation counter.
During the purification procedure, the activity of PAF-AH was measured as described by Hattori et al. (19) with minor modifications. The incubation mixture consisted of 100 mM Tris · HCl, pH 7.4, 5 mM EDTA, 1% 2-mercaptoethanol, and the sample (5-25 µl) in a total volume of 100 µl. After addition of 10 µl of [3H]PAF prepared as previously described, incubations were performed for 30 min at 37°C and stopped by adding 2.5 ml of chloroform-methanol (4:1, vol/vol) and 0.4 ml of water. Tubes were mixed vigorously and centrifuged at 1,400 g for 5 min, and the radioactivity of the upper phase was measured in a liquid scintillation counter. The activity of PAF-AH using the synthetic PAF analogs as substrates was determined in 100 µl of 0.1 M HEPES buffer containing 2 mM EDTA. Labeled sn-2-succinoyl or sn-2-glutaroyl-PAF (10 µl, 8 nmol) was added, and incubations were conducted for 30 min at 37°C. Phospholipids were extracted and were separated by TLC on K6 plates using a solvent system consisting of chloroform-methanol-acetic acid-water (50:30:8:4, vol/vol). The spot of the TLC plate corresponding to a lyso-PAF standard was scraped, and radioactivity was measured in a liquid scintillation counter.Ribonuclease protection assays.
A 3' truncated pPAF-AH cDNA was linearized with
Cla I (nucleotide 950), and T3 RNA
polymerase was used to create a 245-bp [-32P]UTP-labeled
antisense RNA probe (MaxiScript; Ambion, Austin, TX). As an internal
control, a 355-bp rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
antisense RNA probe was generated using T3 RNA polymerase from the
pTRI-GAPDH template (Ambion). Forty micrograms of hepatocyte and
Kupffer cell RNA were hybridized in solution with both antisense RNA
probes (RPA II kit; Ambion). After ribonuclease digestion, the samples
were separated on a denaturing 5% polyacrylamide-8 M urea gel.
Differences in the amount of pPAF-AH and GAPDH mRNA were visualized
using a PhosphoImager (Molecular Dynamics, Sunnyvale, CA). Yeast tRNA
was included as a negative control. In addition, one sample did not
receive ribonucleases and demonstrated the absence of probe
degradation.
PAF-AH purification procedure. Kupffer cell conditioned medium was concentrated using Centriprep-10 (Amicon) with a cut-off diameter of 10,000 Da to a volume of ~20 ml. The concentrate was dialyzed overnight against 50 mM sodium phosphate, pH 7.2 (buffer A), using Slide-A-Lyzer cassettes (Pierce, Rockford, IL). Solid CHAPS (0.1%) and ammonium sulfate (1 M) were added, and the mixture was stirred for 1 h. Hepatocyte-conditioned media were subjected to LDL and very low-density lipoprotein (VLDL) precipitation using sodium phosphotungstate and MgCl2 according to the procedure of Burstein and Scholnick (5). The precipitate was washed with cold deionized water and resuspended in buffer A containing 0.1% CHAPS. The mixture was stirred for 1 h to achieve complete solubilization and was then centrifuged at 1,400 g for 20 min. Solid ammonium sulfate (1 M) was added, and the mixture was stirred for 1 h. The preparations were filtered and applied to an octyl-Sepharose column (2.6 × 18 cm) equilibrated with buffer A containing 0.1% CHAPS and 1 M ammonium sulfate at a flow rate of 0.2 ml/min. The column was washed with equilibration buffer and then with buffer A containing 0.1% CHAPS without ammonium sulfate. PAF-AH activity was eluted using an increasing linear gradient of buffer A containing 0.1% CHAPS to buffer A with 3% CHAPS. Enzymatically active fractions were pooled, concentrated, and dialyzed for 24 h against buffer A. The mixture was supplemented with 0.1% CHAPS, stirred, filtered, and loaded on a blue Sepharose column (2.6 × 10 cm) equilibrated with buffer A containing 0.1% CHAPS at a flow rate of 0.4 ml/min. A linear gradient of equilibration buffer to equilibration buffer containing 1 M sodium chloride was applied, and 2.5-ml fractions were collected. Active fractions were pooled, concentrated, and dialyzed 24 h against buffer B consisting of 50 mM Tris · HCl, pH 8.0. CHAPS (0.1%) was added, and the mixture was filtered and applied to a Q-Sepharose column (2.6 × 10 cm) using a flow rate of 0.4 ml/min. The column was washed with loading buffer, and the PAF-AH activity was eluted using a linear gradient of NaCl from 0 to 1 M. Fractions (0.8 ml) were collected; active fractions were concentrated by ultrafiltration using Centricon-10 concentrators (Amicon) and stored on ice until use.
Rat bile (5-8 ml) was subjected to LDL plus VLDL sedimentation using sodium phosphotungstate and MgCl2 as described previously (5). The precipitate was washed with cold, deionized water, resuspended in 50 mM sodium phosphate, pH 7.2, containing 0.1% CHAPS, stirred for 1 h, and centrifuged at 1,400 g for 10 min. The supernatant was subjected to one-step purification on a 5-ml octyl-Sepharose column as previously described. Active fractions were dialyzed overnight against 100 mM Tris · HCl, pH 7.4, concentrated, and stored on ice until use.Treatment with endoglycosidases. Purified PAF-AH or crude hepatocyte media, Kupffer cell media, and bile preparations (10 µl each) were adjusted to 50 µl with either endoglycosidase H buffer (50 mM citrate-phosphate, 0.1% 2-mercaptoethanol, 0.1% SDS, 0.1% CHAPS, pH 5.6) or N-glycosidase F buffer (100 mM Tris · HCl, 0.1% 2-mercaptoethanol, 0.1% SDS, 5 mM EDTA, 0.1% CHAPS, pH 8.0). Endoglycosidase H (0.01 U) or N-glycosidase F (0.5 U) was added, and incubations were conducted at 37°C for 2 h. After incubation, the sample volumes were adjusted to 250 µl with 100 mM Tris · HCl, pH 7.4, and PAF-AH activity was measured as described by Hattori et al. (19).
Labeling of PAF-AH with
[3H]DFP.
PAF-AH partially purified from various hepatic sources (1-5
nmol/min) was incubated for 1 h at room temperature with 10 µCi [3H]DFP in 50 µl of
labeling buffer consisting of 10 mM Tris · HCl (pH
7.4), 0.2 M NaCl, 5 mM 2-mercaptoethanol, 10% glycerol, and 0.5%
CHAPS. After incubation, 2 ml of labeling buffer were added, and the
mixture was concentrated using Centricon-10 concentrators (cut-off
diameter 10,000 Da). The procedure of washing with labeling buffer and
concentrating was repeated twice and the final concentrate was diluted
to a specific radioactivity of 50,000 dpm/25 µl. Samples were
subjected to SDS-PAGE in 10% polyacrylamide gels and impregnated with
Enhance (DuPont). Gels were dried and exposed to Hyperfilm (Amersham)
for at least 72 h at 80°C.
Protein assay. The reagent kits used for protein measurement were provided by Pierce Chemical (Rockford, IL). Protein was determined using bicinchoninic acid or Coomassie G-250 for bile samples according to the manufacturer's instructions.
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RESULTS |
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PAF-AH activity in Kupffer cells, hepatocytes, and bile.
The sensitivity of crude PAF-AH preparations to proteinase K treatment,
the most potent nonselective endopeptidase known, and to DTNB was
employed as the major functional criterion for detecting pPAF-AH.
Hepatocytes and Kupffer cells isolated from rat livers exhibit
relatively low and comparable levels of the proteinase K-resistant
PAF-AH activity (Fig. 1). In Kupffer cells, 46.9% (0.33 ± 0.043 nmol · min1 · mg
protein
1) of total PAF-AH
activity in the membrane fraction (0.703 ± 0.059 nmol · min
1 · mg
protein
1) and 23.3%
(0.25 ± 0.06 nmol · min
1 · mg
protein
1) of total PAF-AH
activity in the cytosol were resistant to proteinase K digestion. In
hepatocytes, 39.1% (0.095 ± 0.011 nmol · min
1 · mg
protein
1) of total
membrane-associated PAF-AH activity (0.24 ± 0.04 nmol · min
1 · mg
protein
1) and 16.1% (0.1 ± 0.019 nmol · min
1 · mg
protein
1) of total
activity in the cytosol (0.62 ± 0.09 nmol · min
1 · mg
protein
1) were not
inhibited by extensive proteinase K treatment. In addition, the
relative amount of PAF-AH activity resistant to treatment with DTNB was
comparable with that resistant to proteinase K digestion (Fig. 1) in
these cell preparations.
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pPAF-AH mRNA in liver cells. The antisense RNA used for RNase protection assays in the present study was synthesized from a rat pPAF-AH cDNA (ICOS, Bothell, WA). A 245-bp antisense PAF-AH RNA was hybridized in solution with 40 µg of total RNA isolated from cultured rat hepatocytes and Kupffer cells. After RNase digestion, the protected fragments were detected by their change in mobility. In both freshly isolated Kupffer cells and hepatocytes, a 175-bp PAF-AH protected fragment was barely detectable (data not shown), which is consistent with an occurrence of relatively low levels of constitutive PAF-AH activity in both types of cells. Kupffer cells in primary culture for 24 h contained high levels of a 175-bp PAF-AH protected fragment compared with 24-h cultured hepatocytes, which exhibited very low amounts of this transcript (Fig. 3). Expression of high PAF-AH mRNA levels at 24 h was accompanied by enhanced PAF-AH secretion from Kupffer cells (Fig. 2).
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Partial characterization of PAF-AH secreted by liver cells and PAF-AH in bile. The hepatocyte- and Kupffer cell-secreted PAF-AHs were completely resistant to treatment with proteinase K or DTNB. In fact, proteinase K treatment increased slightly the activity of liver cell-secreted PAF-AH(s). Also, PAF-AH in bile was proteinase K-insensitive but was inhibited 15-20% by DTNB. The hepatic-derived PAF-AH activities were partially decreased by p-bromophenacyl bromide (by ~40%) and were nearly completely inhibited by DFP or phenylmethylsulfonyl fluoride (5 mM), indicating the presence of serine residues in the active center of the PAF-AH(s). These properties of the secreted activities are consistent with a pPAF-AH.
PAF analogs with sn-2-succinoyl and sn-2-glutaroyl moieties, which represent oxidized fatty acids, were synthesized and used as substrates. The activity of the Kupffer cell-secreted enzyme using sn-2-succinoyl and sn-2-glutaroyl derivatives of PAF was ~37 and 31% of that for acetyl-PAF. The hepatocyte-secreted and bile PAF-AH exhibited ~49 and 51% of the activity toward sn-2-succinoyl-PAF and ~36 and 39% toward sn-2-glutaroyl-PAF, respectively, compared with acetyl-PAF. The activities of the Kupffer cell- and hepatocyte-derived PAF-AH enzymes and that in bile were not detected using 1-alkyl-2-arachidonoyl-sn-glycero-3-phosphocholine or 1-palmitoyl-2-lyso-sn-glycerophosphocholine as substrates. Interestingly, treatment with N-glycosidase F did not affect the activities of the Kupffer cell- and hepatocyte-secreted enzymes or the activity of PAF-AH in bile. In contrast, endoglycosidase H increased by 50-60% the activity of Kupffer cell- and hepatocyte-secreted enzymes as well as the activity in bile (Fig. 4A), whereas endoglycosidase H exhibited no intrinsic PAF-AH activity. Assuming the secreted PAF-AH(s) may be glycosylated, we investigated the effects of tunicamycin, an established inhibitor of N-linked glycosylation. Incubation with tunicamycin (1-25 µM) for 6 h increased the activity of PAF-AH in Kupffer cell- and hepatocyte-conditioned media (Fig. 4B). However, incubation of cells with tunicamycin for 42 h decreased significantly the activity of PAF-AH in the media (data not shown).
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Partial purification and characterization of liver cell-secreted PAF-AH. The PAF-AH enzymatic activity of Kupffer cell- and hepatocyte-conditioned media and bile was retained on octyl-Sepharose at low ionic strength and a high CHAPS concentration, indicating the high hydrophobicity of the enzyme. PAF-AH was eluted from the column at 2.0-2.3% of CHAPS. As a first step, the octyl-Sepharose chromatography was very effective in the purification of PAF-AH from Kupffer cell-conditioned media. The second step included anion exchange chromatography on Q Sepharose. PAF-AH activity was eluted as a single peak with high activity from a 5-ml HiTrap Q column using a sodium chloride gradient (Fig. 5A). The purification factor and overall yield were routinely ca. 2,600 and 11%, respectively. For purification of the enzyme from the hepatocyte-conditioned media, an additional chromatographic step on blue Sepharose was required after octyl-Sepharose chromatography to remove excess albumin. Finally, PAF-AH activity was eluted from the Q-Sepharose column as a peak containing a shoulder that could not be further resolved (Fig. 5B). In a typical experiment, the purification factor and overall yield were found to be 1,800 and 5%, respectively.
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DISCUSSION |
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Initially, PAF-AH was found in mammalian plasma and was determined to be the enzyme that inactivated PAF (see Ref. 37 for review). Later, it was discovered that oxidized phospholipids containing short-chain- length polar residues at the sn-2 position also serve as substrates for both the secreted pPAF-AH and for the intracellular PAF-AH (19, 39, 40). Because of the unique enzymatic characteristics of the various PAF-AHs, an important role has been postulated for these enzymes in the inactivation of bioactive oxidized phospholipids, which are generated as a consequence of pathophysiological episodes (1, 28, 31, 38, 48). The plasma-type and intracellular PAF-AHs exhibit quite distinct activity profiles in response to inhibitors. Whereas the plasma-type enzyme is resistant to proteolysis and treatment with thiol reagents such as DTNB, the activity of intracellular PAF-AH is abolished by the same treatment. In fact, the intracellular and secreted PAF-AHs are the products of different genes (17, 44).
In the present study, we observed that freshly isolated rat liver hepatocytes and Kupffer cells exhibited low PAF-AH activity that localized in both membrane and cytosolic fractions (Fig. 1). In both cases, a significant fraction of the cellular PAF-AH activity was resistant to extensive proteolysis with proteinase K and to treatment with DTNB, characteristic of pPAF-AH. With use of a ribonuclease protection assay, low levels of pPAF-AH mRNA were detected in hepatocytes and Kupffer cells isolated from untreated rats (data not shown), which is in agreement with our data obtained previously using whole liver total mRNA (20). Assuming that the protein content of a single hepatocyte is three to five times higher than that of a Kupffer cell and that the number of hepatocytes in the liver is nearly 10 times greater than the number of Kupffer cells, it can be estimated that under normal conditions in vivo most of the hepatic PAF-AH originates from hepatocytes, not from Kupffer cells. The absence of pPAF-AH mRNA in human liver samples observed by Tjoelker et al. (45) can be explained by the much lower sensitivity of the Northern analyses used in their study.
Next, we asked whether the pPAF-AH can be secreted by liver cells. Both hepatocytes and Kupffer cells were established in primary culture and were observed to secrete pPAF-AH into the culture media as a function of time. Hepatocytes and Kupffer cells exhibited a similar time course of secretion (Fig. 2), whereas the amount of pPAF-AH secreted was dramatically different. Kupffer cells developed 20 to 25 times higher levels of PAF-AH in the medium compared with hepatocytes, which secreted amounts of PAF-AH roughly comparable to those found in freshly isolated cells (see Figs. 1 and 2). Moreover, Kupffer cells cultured for 24 h expressed very high levels of pPAF-AH mRNA compared with cultured hepatocytes, which possessed very low levels of this transcript (Fig. 3). Our laboratory previously demonstrated that pPAF-AH mRNA expression is upregulated during the process of plating and culturing Kupffer cells (20). The high levels of pPAF-AH mRNA result in elevated rates of synthesis and secretion of PAF-AH into the culture medium (Fig. 3). On the other hand, hepatocytes in primary culture maintained a low level of PAF-AH mRNA and subsequent enzyme secretion, most probably from a constitutive, preformed cellular pool. These data are supported by our previous results, which demonstrated that in cultured hepatocytes at least a portion of PAF-AH was located in the outer leaflet of the plasma membrane and can be secreted into the medium (41).
The role of the hepatocyte in the secretion of the major constituents of bile prompted us to investigate whether PAF-AH is secreted into bile. Indeed, rat bile collected from cannulated bile ducts contained significant amounts of PAF-AH (Fig. 2). The specific enzymatic activity varied slightly in bile samples collected during a 6-h cannulation period, and the variations in the specific activity may reflect differences in the amount of total protein secreted as a result of operative invasion. The average amount of the PAF-AH activity in bile is in the range of the activity secreted by cultured hepatocytes (Fig. 2). To our knowledge, the present study is the first demonstration of the constitutive presence of PAF-AH in bile. Because rats do not possess gallbladders and the cannulation of the bile duct in our study was accomplished in the upper (liver) third of the duct, the origin of PAF-AH in bile is most likely the hepatocytes lining canalicular spaces. However, the secretion of PAF-AH into the bile may occur, at least in part, by means of transcytosis from the plasma. In any case, in rats, PAF-AH secreted into the bile reaches the intestine directly, which may have an important impact during the pathophysiological episodes discussed below. The hepatic cell-derived enzymes hydrolyzed PAF analogs with sn-2 polar succinoyl and glutaroyl residues but were not able to hydrolyze either sn-2 arachidonoyl-PAF or 1-palmitoyl-lyso PC, indicating that the PAF-AH activities secreted by cultured hepatocytes and Kupffer cells and the bile enzyme are specific; this activity cannot be attributed to long-chain-length phospholipase A2 or lysophospholipase. Moreover, these PAF-AH activities satisfy all of the pharmacological characteristics attributed to pPAF-AH.
Recently, it was reported that PAF-AH in human and guinea pig plasma may be N-glycosylated (24, 43). However, the physiological significance of glycosylation was not determined. For these reasons, an established inhibitor of N-linked glycosylation, tunicamycin, was incubated with cultured hepatocytes and Kupffer cells to assess changes in the activity of PAF-AH in the media. Incubation with tunicamycin for 6 h in serum-free medium increased the PAF-AH activity of the culture media by nearly 3-fold in cultured Kupffer cells and 1.5-fold in hepatocytes (Fig. 4B). Unexpectedly, treatment of PAF-AHs secreted by untreated cells and bile PAF-AH with N-glycosidase F either did not change or slightly decreased the activity of the enzymes (Fig. 4A). Under the same conditions, treatment of the glycosylated standard protein fetuin resulted in complete deglycosylation as indicated by protein band shifts on SDS-PAGE (data not shown). In contrast, deglycosylation with endoglycosidase H led to a significant increase in PAF-AH activity from all sources (Fig. 4A). Further studies will be required to elucidate the precise roles for glycosylation of PAF-AH in the processing, secretion, and catalytic function of pPAF-AH derived from liver cells.
The PAF-AH purified first by Stafforini et al. (34) from human plasma LDL and VLDL was shown to be a single 43-kDa protein. The cDNA for the plasma type PAF-AH was cloned, and the predicted molecular mass of the protein was confirmed as a 441-amino acid, 43-kDa protein (45). However, Tew et al. (43) subsequently isolated PAF-AH from human plasma LDL as a 65- to 66-kDa protein, which was extensively glycosylated. Cloning of the cDNA confirmed the nucleotide and predicted amino acid structure reported previously. Moreover, it has been reported recently that PAF-AH isolated from guinea pig plasma is a 58- to 63-kDa protein and that its cDNA contains an open reading frame encoding a 436-amino acid protein with a predicted molecular mass of 49 kDa, suggesting that guinea pig pPAF-AH is modified posttranslationally, probably by glycosylation (24).
Our present data demonstrate that rat hepatocytes and Kupffer cells, potential sources of PAF-AH in plasma, secrete two PAF-AH entities, a 65- to 67-kDa and a 44- to 45-kDa protein, which likely correspond to the glycosylated and the nonglycosylated forms of the enzyme, respectively (Fig. 6). Active site labeling of purified Kupffer cell- and hepatocyte-secreted PAF-AH with [3H]DFP indicated incorporation of the label predominantly into the 65- to 67-kDa protein band, confirming the nature of this protein as a serine esterase (Fig. 7). Moreover, the enzyme isolated from rat plasma exhibited a major protein band at 65-67 kDa and a minor band at 44-45 kDa, which disappeared after an additional purification step on concanavalin A Sepharose, whereas the material eluted from the column retained the enzymatic activity and a protein band at 65-67 kDa (S. I. Svetlov and M. S. Olson, unpublished data). In addition, the hepatocyte-secreted PAF-AH contained a minor 30- to 32-kDa protein band (Fig. 6), and this band also was labeled with [3H]DFP. Partially purified bile PAF-AH incorporated [3H]DFP exclusively into the 30- to 32-kDa protein, similar to that observed for the hepatocyte-secreted enzyme. This indicates that the biliary processing of the PAF-AH, either by transcytosis from plasma and/or via secretion by hepatocytes per se, is different from the secretion into the circulation and may involve lysosomal degradation or truncation of PAF-AH similar to that shown for several other bile proteins (see Ref. 10 for review).
The unique role of the hepatobiliary system as a "biochemical bridge" between the gastrointestinal tract and the circulation makes it possible for the liver to participate in signaling mechanisms, which regulate metabolic events on both sides of this axis and which influence pathophysiological episodes either in the circulation, the gastrointestinal tract, or both. Recent studies have afforded the perception of a dual function for the liver as a major target and scavenger for bioactive lipids, including lipid peroxides and PAF under normal and pathological conditions such as endotoxemia, sepsis, or hypoxia (2, 16, 29, 30). It has been demonstrated that intravascular PAF can induce an ischemic bowel necrosis similar to that observed in neonatal necrotizing enterocolitis and that PAF is crucially involved in pathogenesis of endotoxin- and hypoxia-induced intestinal necrosis in rats (6, 7, 9, 14, 21-23). Moreover, recombinant PAF-AH prevents the development of ischemia-evoked necrosis of the duodenum, jejunum, and ileum after injection of PAF into the descending aorta of the rat (12). In addition, it has been shown that PAF and PAF-AH are involved in the pathogenesis of liver diseases in human patients (8, 26). Also, it was reported that human breast milk contains PAF-AH, probably secreted by milk macrophages, which may be an important factor in defending the gastrointestinal tract of newborns against lipoperoxides and PAF (13). Our present data demonstrate that rat bile contains a constitutive pPAF-AH, which may be an important component in the ability of the liver to protect the gastrointestinal system under normal conditions. In fact, we contend that bile plays both a metabolic role in helping to digest and absorb lipids and also functions as an anti-inflammatory element in the hepatobiliary-gastrointestinal system. The nature of and the mechanism for the processing of rat bile PAF-AH, as well as its role in models of liver and intestinal pathology, still remain to be elucidated.
In conclusion, this study demonstrates that normal rat hepatocytes and Kupffer cells possess constitutive pPAF-AHs, which are secreted by these cells in culture. It is our current view that the pPAF-AH secreted by Kupffer cells plays a role in vivo in pathophysiological situations involving PAF and oxidized phospholipids, e.g., endotoxic shock. Moreover, we suggest that the hepatocyte-derived pPAF-AH is secreted into the bile and represents a novel mechanism by which the liver participates in the elimination of excess PAF and oxidized phospholipids in the gastrointestinal tract.
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ACKNOWLEDGEMENTS |
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The authors thank Dr. S. A. K. Harvey for critical reading of the manuscript.
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FOOTNOTES |
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This study was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-33538.
Present address of S. I. Svetkov: Dept. of Pathology, Yale Univ. Medical School, 310 Cedar St., New Haven, CT 06520.
Address for reprint requests: M. S. Olson, Dept. of Biochemistry, Univ. of Texas Health Science Center, 7703 Floyd Curl Dr., San Antonio, TX 78284-7760.
Received 2 September 1997; accepted in final form 21 January 1998.
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