1 DNA Unit and
4 Hormonal Laboratory, Novel aspirin (ASA)-triggered 15-epi-lipoxins (ATL) comprise new
potent bioactive eicosanoids that may contribute to the therapeutic effect of this drug. ATL biosynthesis is initiated by ASA acetylation of cyclooxygenase (COX)-2 and was originally identified during the
interaction of leukocytes with either endothelial or epithelial cells.
Here, we examined ATL biosynthesis in rat hepatocytes either alone or
in coincubation with nonparenchymal liver cells (NPC) and in liver
homogenates from ASA-treated rats. Rat hepatocytes and CC-1 cells, a
rat hepatocyte cell line, displayed COX-1 but not COX-2 mRNA expression
and predominantly produced thromboxane A2
(TXA2) and
15-hydroxyeicosatetraenoic acid (15-HETE). In these cells, ASA shifted
the arachidonic acid metabolism from
TXA2 to 15-HETE in a
concentration-dependent manner. In contrast, neither indomethacin,
ibuprofen, valeryl salicylate, nor nimesulide was able to trigger
15-HETE biosynthesis. SKF-525A, a cytochrome
P-450 inhibitor, significantly reduced
the effect of ASA on 15-HETE biosynthesis. Furthermore, phenobarbital,
a potent inducer of cytochrome P-450
activity, further increased ASA-induced 15-HETE production. ASA
treatment of hepatocyte-NPC coincubations resulted in the generation of
significant amounts of ATL. In addition, in vivo experiments
demonstrated augmented hepatic levels of 15-epi-lipoxin A4 in ASA-treated rats. Taken
together and considering that ASA is hydrolyzed on its first pass
through the portal circulation, these data indicate that, during ASA's
consumption, liver tissue generates biologically relevant amounts of
ATL by COX-2-independent mechanisms.
liver cells; acetylsalicylic acid; 15-hydroxyeicosatetraenoic acid; cytochrome P-450
ASPIRIN (ASA) is the nonsteroidal anti-inflammatory
drug (NSAID) most widely employed and the standard against which all
new anti-inflammatory agents are compared. Because of its long history of use and availability without prescription and its low cost and
safety, ASA is the drug of choice for relieving inflammation and mild
to moderate pain and fever (16, 28). ASA also inhibits platelet
aggregation and can reduce the incidence of coronary artery thrombosis
and myocardial infarction by >40% (27). Numerous epidemiological
studies have also shown that the long-term use of low doses of ASA
represents a potentially viable option in the prevention of sporadic
colon cancer (22, 36). The efficacy of ASA as a NSAID is closely
related to its ability to inhibit PG and thromboxane (TX) biosynthesis
by acetylating the cyclooxygenase (COX) enzyme (37). However, not all
of the beneficial effects associated with ASA consumption can be
ascribed to the inhibition of PG and TX biosynthesis, and the precise
mechanism of action of ASA is at present still unsettled (2, 29).
In this regard, recent studies have demonstrated that ASA triggers the
generation by transcellular routes of novel eicosanoids [i.e.,
ASA-triggered 15-epi-lipoxins (ATL)] during the interaction
between lipoxygenase (LO) and COX pathways (12, 13, 32). These novel
ATL effectively may account for the beneficial effects of ASA and are
indeed able to inhibit cell adhesion and proliferation in vitro and are
able to block local inflammation by reducing both leukocyte
adhesion and infiltration in vivo (12, 13, 17, 31, 32, 34, 35).
During ASA intake, the active compound acetylsalicylic acid is absorbed
from the upper small intestine and rapidly presystemically metabolized
by enzymatic hydrolysis on its first pass through the liver (6, 16).
Within the liver, ASA is hydrolyzed by esterases (ASA-esterases)
abundant in parenchymal liver cells and in particular in hepatocyte
mitochondria and endoplasmic reticulum (1, 18). Because all blood
draining the gastrointestinal tract enters the liver via the portal
vein and ~73% of ASA is converted to salicylate within 30 min of its
ingestion (16, 23), the concentration of acetylsalicylic acid in the
liver probably exceeds that in serum and other tissues. Thus it seems reasonable to conceive that the impact of ASA on arachidonic acid metabolism is higher in liver cells than in any other cell type. This
notion prompted us to investigate ATL biosynthesis in rat liver tissue
and specifically in rat hepatocytes. In this report, we demonstrate
that ASA triggers the generation of novel ATL by rat liver cells and
that a similar effect can also be observed in vivo.
Materials
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Cell Isolation and Culture
Hepatocyte isolation. Male Wistar rats weighing 200-300 g were treated with gadolinium chloride (GdCl3, 10 mg/kg iv in normal saline), a Kupffer cell-specific toxicant (19). Twenty-four hours later, animals were anesthetized with ketamine (50 mg/kg body wt), and the hepatocytes were isolated by in situ collagenase perfusion through the portal vein according to the method of Berry and Friend (4) with minor modifications. Briefly, livers were perfused for 20 min at a flow rate of 14 ml/min at 37°C with HBSS without calcium and magnesium containing 10 mmol/l HEPES (pH 7.4) and 1 mmol/l EGTA, followed by 5 min with HBSS containing 10 mmol/l HEPES (pH 7.4), 1.3 mmol/l CaCl2, and 0.6% BSA and then 10 min with a 0.05% collagenase (A type) solution containing 10 mmol/l HEPES (pH 7.4) and 1.3 mmol/l CaCl2. The resultant digested liver was excised and minced, and the dispersed cells were passed through nylon mesh filters (100 µm). The hepatic cell suspension was centrifuged two times at 50 g for 5 min, and the supernatant containing mainly nonparenchymal cells (NPC) was used as described below. The cell pellet enriched with hepatocytes was then washed two times with cold HBSS containing 10 mmol/l HEPES (pH 7.4), 1.3 mmol/l CaCl2, and 1% BSA, followed by two washes with DPBS. Hepatocytes were purified by layering the cells over a 60% Percoll solution and centrifuging at 400 g for 7 min at 4°C. Cell viability was determined by trypan blue exclusion, and hepatocytes were characterized by a combination of phase-contrast microscopy and immunocytochemical analysis. Purified hepatocytes were either suspended in DPBS with calcium and magnesium (DPBS++) or plated on collagen-coated plastic dishes in Williams' medium E supplemented with 10Nonparenchymal liver cells. The NPC-enriched supernatants obtained during the isolation of hepatocytes (vide supra) were centrifuged at 800 g for 10 min, and the resultant pellet was suspended in DPBS++.
CC-1 cell cultures. CC-1 cells, a rat Wistar hepatocyte cell line (European Collection of Cell Cultures, Salisbury, UK), were plated in EMEM with 2 mM L-glutamine, 1% nonessential amino acids, 20 mM HEPES, and 10% FCS. Cells were maintained in a humidified 5% CO2 incubator at 37°C and were used before the 17th passage.
Cell incubations. Freshly isolated hepatocytes suspended in 1 ml of DPBS++ (5 × 106 cells/ml) were exposed to either vehicle (0.1% ethanol, EtOH) or ASA (1 mM) and were stimulated with calcium ionophore A-23187 (5 µM) for 20 min at 37°C. Confluent CC-1 cells (0.6-0.8 × 106 cells/ml) growing in 75-cm2 culture flasks were incubated for 20 min at 37°C with either vehicle (0.1% EtOH), ASA (1 mM), ibuprofen (50 µM), indomethacin (1 µM), nimesulide (2 µM), or valeryl salicylate (0.5 mM) before stimulation with A-23187 (5 µM) for 20 min at 37°C in 3 ml of DPBS++. For concentration-response curves, CC-1 cells growing in 24-well tissue culture plates were treated with increasing doses of ASA (0.01-10 mM) and then exposed to A-23187 for 20 min at 37°C. For experiments investigating enzymatic induction, CC-1 cells were treated with PMA (1 µM, 6 h), LPS (1 µg/ml, 18 h), or rat recombinant interleukin 6 (1 ng/ml, 18 h) at 37°C. In some experiments, proadifen (SKF-525A, 100 µM) was added 20 min before the addition of ASA (1 mM, 20 min) to either vehicle-, phenobarbital (2 mM)-, or resveratrol (30 µM)-treated (18 h) CC-1 cells.
For coincubations, hepatocytes were treated with vehicle (0.1% EtOH) or ASA (1 mM) for 20 min at 37°C. Coincubations were performed by adding the NPC to hepatocytes (1:10 ratio) followed by costimulation with ionophore A-23187 (5 µM) in DPBS++ for 20 min at 37°C.
All incubations were terminated by the addition of MeOH (2 vol), and
products were stored at 20°C until further analysis.
Liver Tissue Samples
Rats were fed ad libitum with standard chow and distilled water. Animals were randomly divided to receive ASA (30 mg/kg body wt in 1 ml of 0.5% carboxymethylcellulose) or vehicle (0.5% carboxymethylcellulose) by gavage. Six hours later, animals were killed, and the liver was excised and rinsed with ice-cold DPBS++. Samples of ~1-2 g were individually homogenized with a Polytron homogenizer in 4 ml of DPBS++, precipitated with 2 vol of ice-cold methanol (MeOH), and extracted with Sep-Pak C18 columns.Analysis of Eicosanoids by Reverse-Phase-HPLC
Materials that eluted in the methyl formate fractions from Sep-Pak extractions were concentrated under a stream of N2 and were scanned for ultraviolet (UV)-absorbing material (in MeOH) with a Kontron spectrophotometer (Milan, Italy) before injection into the reverse-phase (RP)-HPLC system. This system consisted of a Waters integrated system controller (model 600E) equipped with a 996 Photodiode Array Detector and Millennium HPLC analysis software (Waters). For analysis of 5-, 12-, and 15-HETE, a Spherisorb ODS-2 (5 µm, 4.6 × 250 mm) column eluted with MeOH-H2O-acetic acid (65:35:0.01 vol/vol/vol) as phase one (time 0 to 20 min) and a linear gradient with MeOH-acetic acid (99.9:0.1 vol/vol) as phase two (20-45 min) at a flow rate of 1.0 ml/min was used. For analysis of LXA4 and ATL, methyl formate fractions from Sep-Pak-extracted samples were resolved with a Kromasil C18 (5 µm, 4.6 × 250 mm) column eluted with MeOH-H2O-acetic acid (60:40:0.01 vol/vol/vol) at a flow rate of 1 ml/min.Analysis of Eicosanoids by Enzyme-Linked Immunosorbent Assay
15-HETE and TXB2, the stable metabolite for TXA2, levels were measured in unextracted samples by highly specific enzyme-linked immunosorbent assays (ELISAs) from PerSeptive Diagnostics (Cambridge, MA) and Cayman Chemical, respectively. Immunoreactive 15-epi-LXA4 (i15-epi-LXA4) levels were measured in Sep-Pak C18-extracted samples by a recently developed ELISA (9).RNA Isolation and Reverse Transcriptase-PCR
Total RNA was obtained by the guanidinium isothiocyanate-cesium chloride method (10). Samples were homogenized in guanidinium isothiocyanate (4 M) and were placed on 5.7 M of cesium chloride and centrifuged at 40,000 rpm for 20 h at 20°C. The integrity of all samples was documented by visualization of 18S and 28S ribosomal bands after electrophoresis through an 0.8% formaldehyde-agarose gel stained with ethidium bromide. Reverse transcription (RT) was performed with random primers using a cDNA synthesis kit. PCR was performed with oligonucleotides constructed from published sequences of rat COX-1 and COX-2 (15): COX-1, 5'-GTCATTCCCTGTTGTTACTATCC-3' (sense) and 5'- CTCCCTTCTCAGCAGCAATCG-3' (antisense) and COX-2, 5'-ACTTGCTCACTTTGTTGAGTCATTC-3' (sense) and 5'-TTTGATTAGTACTGTAGGGTTAATG-3' (antisense). Amplification of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was performed with 5'-TCCCTCAAGATTGTCAGCAA-3' (sense) and 5'-AGATCCACAACGGATACATT-3' (antisense). COX-1, COX-2, and GAPDH were amplified at 94°C (30 s), 60°C (1 min), and 72°C (1 min) for 35 cycles. PCR products were analyzed by electrophoresis in a 2% NuSieve agarose gel (FMC Bioproducts, Rockland, ME). Identity of PCR products was confirmed by restriction fragment analysis and sequencing. For sequence analysis, PCR products were extracted from the 2% agarose gel and submitted to cycle sequencing with Taq polymerase and dRhodamine-labeled dideoxy terminators, according to the manufacturer's instructions. The reaction products were analyzed with an automated 373A DNA sequencer (Applied Biosystems).Statistical analysis of the results was performed using the ANOVA and the paired and unpaired Student's t-test. Results are expressed as means ± SE, and differences were considered significant at a P value < 0.05.
The study was performed according to the criteria of the Investigation and Ethics Committee of the Hospital Clínic.
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RESULTS |
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To assess which COX isozymes are present in rat liver parenchymal
cells, total RNA from rat CC-1 cells was analyzed by RT-PCR for COX-1
and COX-2 mRNA expression. As shown in Fig.
1, CC-1 cells displayed constitutive mRNA
expression for COX-1. In contrast, these cells did not express COX-2
mRNA even after stimulation with LPS or interleukin 6 (Fig. 1).
Identical results were obtained in freshly isolated rat hepatocytes
(data not shown). These results were further confirmed by performing
the RT-PCR in parallel with positive sources of both COX-1 and COX-2
mRNA (RNA from RAW 264.7 macrophages and rat kidney; data not shown).
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To characterize which eicosanoids are produced by rat parenchymal liver
cells, biosynthesis of monohydroxy products (i.e., HETEs) and
TXB2 was monitored in freshly
isolated hepatocytes and CC-1 cells. Under resting conditions,
mono-HETE production by hepatocytes and CC-1 cells was not detected by
either RP-HPLC or ELISA. However, when these cells were exposed to
ionophore A-23187, significant amounts of 15-HETE and
TXB2 were detected by ELISA
(1-3 and 4-10 ng/106
cells, respectively). The chromatographic profile of materials obtained
from A-23187-stimulated CC-1 cells consistently revealed the presence
of a major product that coeluted with synthetic 15-HETE (Fig.
2). When the material eluting beneath this
peak was analyzed by UV spectroscopy, a smooth absorption curve with a
UV maximum at 234 nm was observed (data not shown). Under these
conditions, consistent formation of 12-HETE, but not 5-HETE, was also
observed (Fig. 2). Addition of ASA to either hepatocytes or to CC-1
cells resulted in a marked increase in the formation of 15-HETE (Fig. 2). As shown in Fig. 3, in CC-1 cells, ASA
induced a concentration-dependent increase in 15-HETE formation,
whereas it inhibited TXB2
biosynthesis (IC50 = 307 µM).
Incubation of CC-1 cells with either indomethacin, ibuprofen, valeryl
salicylate, or nimesulide did not significantly modify the amount of
15-HETE produced by CC-1 cells (Fig. 4). Taken together, these results indicate that rat hepatocytes and CC-1
cells generate 15-HETE and that ASA treatment augments its production.
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|
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The existence of the cytochrome P-450
system as an alternative enzymatic pathway to ASA-acetylated
COX-2-dependent ATL biosynthesis was recently suggested during the
interaction of airway epithelial cells and neutrophils (12). In the
current experiments, treatment of CC-1 cells with the cytochrome
P-450 inhibitor proadifen (SKF-525A) reduced ASA-induced release of 15-HETE by 58%, thus confirming that
cytochrome P-450 is likely to be
involved in 15-HETE biosynthesis (Fig. 5).
Moreover, pretreatment of CC-1 cells with phenobarbital, a
well-established inducer of cytochrome
P-450 activity, further increased
15-HETE biosynthesis in response to ASA, an effect that was also
inhibited by SKF-525 (Fig. 5). Phenobarbital alone induced a slight
increase in 15-HETE biosynthesis, but this effect did not reach
statistical significance (data not shown). Interestingly, resveratrol,
a phenolic compound with potent anti-oxidant and anti-proliferative
properties (11), prevented the release of 15-HETE elicited by ASA (Fig.
5).
|
To test the hypothesis that parenchymal liver cell-derived 15-HETE is
subsequently transformed by transcellular routes to ATL, freshly
isolated rat hepatocytes were exposed to ASA and coincubated with NPC
in the presence of ionophore. Figure 6
depicts an RP-HPLC chromatographic profile of materials obtained from these coincubations, showing the presence of products with strong UV
absorbance at 300 nm. On-line spectral analysis of these products demonstrated that they each displayed a triplet of absorbing bands characteristic of conjugated tetraene-containing chromophores indicative of the LX basic structure (maximum at 301 nm and shoulders at 288 and 315 nm; Fig. 6,
insets). Both
LXA4 and novel ATL (e.g., 15-epi-LXA4) were identified in
the chromatographic profile on the basis of coelution with synthetic
materials and the presence of characteristic chromophores. In these
coincubations, ATL accounts for 65% of the total amount of
tetraene-containing compounds eluting within the LX
region. The presence of a chromophore absorbing at 271 nm (Fig. 6,
insets) suggests the existence of
conjugated triene-containing metabolites of
LXA4 and ATL (i.e.,
13,14-dihydro-LXA4; see Ref.
33). Taken together, these results indicate that, within the liver, ATL biosynthesis is likely to occur via transcellular routes.
|
To confirm that ATL biosynthesis also occurs in vivo, tissue
homogenates were obtained from rats treated with ASA, and their i15-epi-LXA4 levels were compared
with those from animals receiving placebo. As shown in Fig.
7,
i15-epi-LXA4 levels were
significantly higher in liver homogenates from rats receiving ASA.
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DISCUSSION |
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At therapeutic doses, orally ingested ASA is rapidly absorbed from the stomach and the upper small intestine, and in <30 min this NSAID reaches concentrations in plasma of 150-350 µg/ml (2, 6, 16, 29). In this range of clinically encountered concentrations, most ASA is deacetylated to form salicylate and acetic acid by esterases abundant in the liver cells and specifically in hepatocytes (1, 16, 18, 28). These cells also convert most of the salicylate to water-soluble conjugates (i.e., ester and ether glucuronides, salicyluric acid, and gentisic acid), which are rapidly cleared by the kidney (1, 18, 28). Although ASA has a serum half-life of <15 min, its actions (i.e., inhibition of platelet COX and TXB2 biosynthesis) have already been accomplished by the end of its passage through the portal circulation, rendering its pass into the systemic circulation unnecessary (28). Therefore, liver tissue is primarily exposed to ASA, and liver cells are likely to receive a higher impact of this acetylating NSAID.
The acetylation capability of ASA is a key step in the recently uncovered ATL pathway (13, 32). The novel biosynthetic pathway triggered by ASA is initiated by acetylation of the inducible COX isoform (COX-2), which switches the enzyme's catalytic activity from a PG synthase to a 15-LO. Thus PG biosynthesis by ASA-acetylated COX-2 is inhibited, and arachidonic acid is transformed to 15(R)-HETE (21). During the interaction of ASA-treated human endothelial or epithelial cells with neutrophils, 15-HETE is subsequently converted by transcellular routes to a new series of ATL (i.e., 15-epi-LXA4; see Refs. 12 and 13). In the current investigation, we demonstrate that ASA also stimulates 15-HETE biosynthesis in isolated rat hepatocytes and triggers ATL biosynthesis during the interaction of hepatocytes with NPC, indicating that biosynthesis of ATL in the liver is also likely to occur by transcellular routes.
The identity of the liver cell type or types responsible for 15-HETE and ATL formation and the precise mechanism of their biosynthesis are at present unknown. Given that hepatocytes are the predominant hepatic cell type and constitute >90% of liver cell mass (5), we first analyzed 15-HETE biosynthesis in these cells. Although we did not detect COX-2 expression in isolated rat hepatocytes and CC-1 cells (Fig. 1), ASA treatment resulted in 15-HETE formation (Figs. 2-4). Because previous findings showed that, in addition to the COX-2 pathway, the cytochrome P-450 system also contributes to 15-HETE and ATL generation during ASA treatment of human airway epithelial cell and polymorphonuclear neutrophil coincubations (12), we hypothesized that this alternative route of arachidonic acid metabolism may be operative in rat liver hepatocytes. This suggestion is based on the observation that 1) liver has the highest levels of cytochrome P-450 enzymatic activity (7, 38), 2) cytochrome P-450 is the most significant pathway of arachidonic acid metabolism in hepatocytes (30), 3) ASA is a well-known stimulus of cytochrome P-450 activity in isolated rat hepatocytes (14, 20), 4) oxygenation of arachidonic acid by cytochrome P-450 results in the stereoselective formation of 15-HETE (40% R and 60% S; see Ref. 8), and 5) adult human liver microsomes metabolize arachidonic acid by the cytochrome P-450 system and form 15-HETE, predominantly on the R configuration (26). Indeed, in this report, we show that proadifen (SKF-525A), a well-established and specific inhibitor of cytochrome P-450-dependent arachidonate metabolism (25), prevents ASA stimulation of 15-HETE biosynthesis by rat hepatocytes. Interestingly, resveratrol, an active compound found in wine, grapes, and other dietary sources with a particular affinity for liver tissue and with an inhibitory effect on cytochrome P-450 activity (11), also prevented ASA-induced 15-HETE release. Whether the ASA-induced increase in hepatic 15-HETE and ATL formation is a consequence of a direct effect of this NSAID on cytochrome P-450 activity or whether it works by shunting arachidonic acid from COX to the P-450 system is at present unknown.
Transcellular metabolism involves cell-cell interaction and processing of a metabolic intermediate generated by one cell (donor cell) by a vicinal cell (acceptor cell) for the production of an active eicosanoid that neither cell alone can generate (24). In view of ATL biosynthetic routes, once 15-HETE is synthesized in the hepatocyte, it must be released to the space of Disse and converted by transcellular metabolism to ATL by a nearby 5-LO containing NPC. Because of its strategic location in the space of Disse in close relationship with the hepatocytes, two potential acceptor liver cells may be considered: Kupffer cells and hepatic stellate cells (lipocytes, Ito cells; see Ref. 5). Classically, hepatic 5-LO activity has been ascribed to Kupffer cells and, theoretically, these liver resident macrophages possess the enzymatic machinery to transform hepatocyte-derived 15-HETE to ATL. However, in our experiments and to obtain pure hepatocyte cultures without contamination of resident macrophages, animals were first treated with GdCl3, a Kupffer cell-specific toxicant (19). Consequently, in our coincubations, the NPC pool is depleted of Kupffer cells. On the other hand, there is evidence that hepatic stellate cells contain the 5-LO protein, suggesting that this cell type may also be involved in the transcellular biosynthesis of ATL from hepatocyte-derived 15-HETE (3). Although additional studies are required to ascertain the precise contribution of each one of the NPC to ATL biosynthesis, it seems reasonable to assume that greater amounts of these novel compounds would be expected without Kupffer cell depletion. Furthermore, it is important to note that NPC (i.e., Kupffer and hepatic stellate cells) have COX-2 in place (data not shown), and they can therefore generate ATL from both endogenous and exogenous sources of 15-HETE.
The biosynthesis of ATL by liver cells may have significant physiological implications. ATL have numerous biological activities and exert modulatory actions in diverse cellular processes. Among these, those shown to be of relevance to leukocyte function include inhibition of both neutrophil adhesion to endothelial cells (13) and agonist-induced interleukin 8 secretion by enterocytes (17). In addition, 15-epi-LXB4, a member of these novel ATL, inhibits epithelial cell proliferation in vitro (12). Most of the in vitro effects of ATL occur in the nanomolar range concentrations (12, 13, 17). In vivo, stable analogs of ATL have been shown to inhibit leukocyte rolling and adherence in the rat mesenteric microvasculature (31). The in vivo physiological significance of ATL actions is further highlighted by the fact that topical application of ATL to mouse ears dramatically inhibits leukocyte infiltration (34, 35). The documented biological activities associated with ATL, together with the presence of ATL in vivo in murine peritonitis exudates (9), suggest an important functional role for these ASA-triggered eicosanoids. The demonstration of ATL as endogenous constituents of rat liver tissue (Fig. 7) provides further evidence to support the in vivo significance of these arachidonic acid metabolites. Although ATL released by hepatic cells may function as lipid autacoids that exert rapid and potent actions on nearby liver cells, these compounds may also be secreted in the circulation and display biological effects in extrahepatic tissues. Thus, because several arachidonic acid metabolites appear to be sufficiently stable to survive in the circulation, an enticing possibility is that effects of ATL generated in liver may extend to more distant organs. Further work is necessary to determine the functional significance of liver ATL biosynthesis.
In summary, in this study, we have shown that, in the presence of ASA, rat hepatocytes are able to initiate 15-HETE biosynthesis by COX-2-independent pathways. We also document the ASA-induced production of ATL during the interaction of hepatocytes with NPC and in vivo in rat liver tissue. Based on these data, we speculate that, before ASA is hydrolyzed extensively to salicylate in the liver and enters the systemic circulation, this NSAID triggers the release of potent biologically active eicosanoids (i.e., ATL) that may contribute to its broad range of beneficial actions.
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ACKNOWLEDGEMENTS |
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We are indebted to Jacinta Baena for contribution to this work and to Montse Bernat for technical assistance.
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FOOTNOTES |
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These studies were supported in part by Comisión Interministerial de Ciencia y Tecnología (SAF 97/0120 to J. Clària) and National Institutes of Health Grant GM-38765 (to C. N. Serhan), and were included in a Spanish-Italian Joint Research Program (HI97-68).
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. Clària, DNA Unit, Hospital Clínic, Villarroel 170, Barcelona 08036, Spain (E-mail: claria{at}medicina.ub.es).
Received 24 May 1999; accepted in final form 13 July 1999.
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