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Leukotriene C4 metabolism by hepatoma cells and liver
LEUKOTRIENE HEPATOMA
C4 M E T A B O L I S M CELLS AND LIVER
BY
DIETRICH KEPPLER, MICHAEL HUBER, GISBERT WECKBECKER, WOLFGANG HAGMANN, CLAUDIO DENZLINGER and ALBRECHT GUHLMANN Biochemisches Institut, University of Freiburg im Breisgau, D-7800 Freiburg, West Germany INTRODUCTION
The cysteinyl leukotrienes LTC4*, LTD4, and LTE4 are potent mediators of smooth muscle contraction and play an important role in inflammation, anaphylaxis (1-4), tissue injury (5), and shock (6-8). The relative potencies of LTC4, LTD4, and LTE4 differ in various biological systems. The order of molar potencies in the guinea pig ileum (4) and in the human bronchus (9) is LTD4 > LTC4 > LTE4. The N-acetylation product of LTE4, N-acetyl-LTE4, has been identified in rat feces (10, 11) and bile (12, 13), and represents the mercapturic acid derivative of cysteinyl leukotriene catabolism (13). NAcetyI-LTE4 is approximately 25-fold less active than LTD4 in the pulmonary pare nchymal strip (14) and 100-fold less active than LTC4 in the pig in vivo (15) indicating that conversion from LTD4 to N-acetyl-LTE4 in the mercapturic acid pathway is a stepwise deactivation of these highly active mediators. LTC4 catabolism comprises extracellular ectoenzyme-catalyzed reactions as well as intracellular conversions (10, 16, 17). The former include -/-glutamyltransferase (EC2.3.2.2) and dipeptidase (EC3.4.13.11) (Fig. 1); the intracellular reactions include the N-acetylation of LTE4 (17) and the formation of polar metabolites derived from the cysteinyl leukotrienes (13, 18, 19). Discrimination between extracellular and intracellular metabolism of LTC4 is possible by comparison of hepatocytes, which take up cysteinyl leukotrienes most actively (20, 21), with cells defective in the uptake system for these leukotrienes, such as the hepatoma cell line AS-30D (21). These hepatoma cells are useful in studies on ectoenzyme-catalyzed LTC4 catabolism and on inhibition of ~/-glutamyltransferase (21) and LTD4 dipeptidase in intact membranes. In vivo, the cysteinyl leukotrienes are rapidly eliminated from the circulating blood by hepatic (5, 6, 10, 13, 18-20) and renal (19, 22) uptake, followed by elimination of metabolites into bile (5, 6, 18) and urine (13, 18, 19, *The following abbreviations are used: LTA4, LTC(, LTD(, LTE(and LTE(NAc, leukotrienes A4, C . D . E~, and N-acetyI-E~,respectively; i.v., intravenous(ly); (RP-)HPLC, (reversed-phase) high-performance liquid chromatography; RIA, radioimmunoassay; HTMP, 4-hydroxy2,2,6,6-tetramethylpiperidine-l-oxyl. AZR-~
211
212
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CO-CH~ FIG. 1. Metabolism of LTC( in the mercapturic acid pathway. The glutathionyl derivative LTC4 is converted to the cysteinylglycine derivative LTD4 by ?-glutamyltransferase (3) and by dipeptidase to the cysteinyl derivative LTE4 (10). N-AcetyI-LTE4, the mercapturate, is a major LTC4 metabolite in bile (13) and feces (11) of the rat. In addition, metabolites more polar than LTC4 and a metabolite less polar than LTE4 are detected in bile and urine (19).
23, 24). Elucidation of the in vivo metabolism of LTC4 is an essential prerequisite for the quantitative analysis of cysteinyl leukotrienes under pathophysiological conditions (5, 6, 12, 13, 19) as well as for the development of effective leukotriene receptor antagonists (25, 26). MATERIALS
AND
METHODS
Hepatoma cells andhepatocytes. The AS-30D rat ascites hepatoma line was carried in Sprague Dawley rats (27). For studies on the uptake of cysteinyl leukotrienes, AS-30D cells were kept at 37°C in suspension culture on a gyratory shaker in a modified standard medium (27) containing in one liter
LEUKOTRIENEC4METABOLISM
213
9.0 g Swim's powdered medium, 1.0 g Pluronic F68, 0.4 mmol L-glutamine (added 30 rain prior to uptake determinations, except for the measurements of L-[14C]glutamineuptake), 0.06 mmol L-cystine, 25 mmol Na2HPO4, and 9 mmol NaHCO3. LTC4, LTD4, and LTE4 metabolism was studied in the absence of L-cystine. Cell suspensions were kept under CO2/air (1/20) at pH 7.4 at concentrations between 3 and 6 × 109 cells/l. Determination of apparent kinetic constants of LTD4 dipeptidase was carried out at 37°C in a medium containing in one liter 115 mmol NaC1, 5 mmol KC1, 21 mmol Na2HPO4, 5 mmol NaHEPO4, 6 mmol D-glucose, and 1 g Pluronic F68 at a pH of 7.4. Freshly isolated rat hepatocytes, kindly provided by Drs. G. Kurz and G. Fricker from the Chemisches Laboratorium of Freiburg University, were incubated at a concentration of 1 × 109 cells/1 under the same conditions as the hepatoma ceils (21). Details of the uptake measurements for cysteinyl leukotrienes were described previously (21). Analysis of leukotriene metabolites in bile and urine. Bile and urine were sampled continuously from anesthetized rats (13, 24) or cynomolgus monkeys (19) into ice-cold 90% aqueous methanol, containing 1 mmol/l HTMP and 0.5 mmol/1 EDTA at pH 7.4, and under argon to prevent oxidative degradation of leukotrienes. Details of bile collection, deproteinization, and separation of leukotriene metabolites by RP-HPLC were described (13, 19, 24). RP-HPLC was performed on a C 18-Hypersil column (4.6 x 250 mm, 5#m particles, Shandon, Runcorn, U.K.). The mobile phase consisted of methanol, water, and acetic acid (65/35/0.1 by vol) at pH 5.6. Radioactive leukotriene metabolites were analyzed by continuous detection of tritium in the HPLC eluent using the Berthold (Wildbad, FRG) liquid scintillation device LB 505. Radioimmunological assays for cysteinyl leukotrienes were performed in fractions of the HPLC eluate (5, 13, 19). Antibodies for the RIA were either obtained from New England Nuclear/DuPont, Boston, MA, or kindly provided by Dr. B. A. Peskar, Ruhr-Universit~it, Bochum, F.R.G. The commercial RIA for LTC4 from New England Nuclear had sufficient crossreactivities to allow for the analysis of LTC4 metabolites, particularly of N-acetyl-LTE4 which crossreacted with 34% relative to LTC4 (13). Leukotrienes and inhibitors. LTC4, LTD4, and LTE4 were obtained from Miles Scientific, Slough, U.K. [14,15-3H]LTC4, [14,15-3H]LTD4, and [14,153H]LTE4 (40 Ci/mmol each) were from New England Nuclear/DuPont, Boston, MA. N-AcetyI-LTE4 and N-acetyI-[3H]LTE4 were synthesized from LTE4 and [3H]LTE4, respectively, as described (13). L-PeniciUamine, Dpenicillamine, L-cysteine, and bestatin were from Sigma, St. Louis, MO. Cilastatin (MK 0791) was kindly donated by Merck Sharp and Dohme, Rahway, NJ. Dr. G. Neil (Upjohn Co., Kalamazoo, MI) kindly supplied us
D. KEPPLER, et al.
214
with acivicin (L-(aS,5S)-a-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid, NSC-163501). RESULTS AND DISCUSSION
Cysteinyl Leukotriene Metabolism and Uptake by Hepatoma Cells and Hepatocytes Hepatocytes actively accumulated cysteinyl leukotrienes (Fig. 2, right). Previous studies demonstrated a common temperature- and energydependent transport of LTC4, LTD4, and LTE4 into hepatocytes (20). Within 20 min, hepatocyte suspensions incubated with [3H]LTC4 metabolized about 60% of this leukotriene to LTD4 (21%), metabolites more polar than LTC4 (33%), and small amounts of N-acetyl-LTE4 (4%) (21). AS-30D hepatoma cells, on the contrary, were completely deficient in the uptake of cysteinyl leukotrienes (Fig. 2, left). These hepatocyte-derived malignant cells converted labeled LTC4 to LTD4 and LTE4 (Fig. 3) but did not produce polar metabolites or N-acetyl-LTE4. Disruption of the hepatoma cells by sonication did not enhance the rate of peptide cleavage from LTC4 to LTE4 (Fig. 3). In control experiments, the hepatoma cells as well as the hepatoeytes took up L-[14C]glutamine and [14C]adenosine. These results indicate that
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FIG. 2. Comparativeuptake of [3H]-labeledLTC4and LTE4byAS-30Dhepatomacells(left)and hepatocytes(fight)in suspension.The concentrations of LTC4and LTE4were3 and 1.5 nmol/l, respectively.For comparisonand control, the uptakerates of L-[~4C]glutamine(0.4retool/I)and [t4C]adenosine (0.1 retool/l) weredetermined in parallel (21). Mean values_+S.E.M. (n=6) are given.
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FIG. 3. LTC4 metabolites in the medium of hepatoma cell suspensions. Cells were incubated in the presence of [3H]LTC4 (7.5 nmol/l) for 90 min. Supernatants were analyzed by RP-HPLC with continuous detection of radioactivity. Upper panel: untreated AS-30D cell control. Middle panel: inhibition of 3,-glutamyltransferase by acivicin (2 mmol/l) added 65 min prior to [3H]LTC4. Lower panel: cells disrupted by sonication 10 min prior to addition of [~H]LTC4 (21). Arrows indicate retention times of standard [3I-I]leukotrienes.
AS-30D cells have selectively lost the transport system for the uptake of cysteinyl leukotrienes. Moreover, ~/-glutamyltransferase and LTD4 dipeptidase, degrading LTC4 to LTD4 and LTE4, respectively, are located on the surface of these hepatoma cells and act as ectoenzymes (21). Formation of the intracellular metabolite N-acetyl-LTE4 (13, 17) could be observed, however, when hepatoma cells were disrupted by sonication and incubated with [3H]LTE4 in the presence of acetyl-CoA (1 mmol/l).
Inhibition of ~/-Glutamyltransferase and LTD4 Dipeptidase on Hepatoma Cells AS-30D cells, which are deficient in the uptake of cysteinyl leukotrienes, provide a useful system for studies on the in situ regulation and inhibition of ectoenzymes involved in LTC4 breakdown. 3~-Glutamyltransferase is irreversibly inhibited by the L-glutamine analog acivicin (28). This inhibitor of L-glutamine-dependent enzymes (29) effectively prevented the formation of LTD4 or other metabolites from LTC4 (Fig. 3).
216
D. KEPPLER, et al.
Cysteinylglycine-degradingdipeptidase (EC 3.4.13.11) (30) has been shown to hydrolyze LTD4 yielding LTE4 and glycine (31, 32). This enzyme, which differs from microsomal aminopeptidase (EC 3.4.11.2) (30, 31), is inhibited by various thiol compounds including D- and L-penicillamine (31). The LTD4 dipeptidase on the surface ofAS-30D hepatoma cells exhibited a high affinity for LTD4 with an apparent Km value of 6.6 #mol/1. D- and L-penicillamine were effective inhibitors with apparent Ki values of 0.46 and 0.21 mmol/1, respectively (Fig. 4). Cilastatin, which has been described as an inhibitor of renal LTD4 dipeptidase activity (33), is a comparatively weak inhibitor of the hepatoma cell L T D 4 dipeptidase (Fig. 4). Bestatin, which inhibits microsomal aminopeptidase but has no effect on dipeptidase (30), did not affect the L T D 4 dipeptidase on AS-30D cells even at bestatin concentrations as high as 5 mmol/l (Fig. 4). These concentrations did, however, inhibit the aminopeptidase activity on AS-30D hepatoma cells (data not shown). These studies indicate that microsomal aminopeptidase on AS-30D cells does not contribute to the formation of L T E 4 f r o m L T D 4. The inhibition of LTD4 dipeptidase by D- and L-penicillamine offers the possibility to interfere with the deactivation of LTD4, the biologically most potent of the cysteinyl leukotrienes.
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LEUKOTRIENEC4METABOLISM
217
Hepatobiliary Elimination of LTC4 Metabolites in the Rat Uptake of cysteinyl leukotrienes from the blood into hepatocytes a n d subsequent elimination into bile predominate in the rat over renal excretion (I0, 13, 24). One hour after i.v. injection of a tracer dose of[3H]LTC4, 77% of the radioactivity was recovered from rat bile and only 1.5% from urine (24). The pattern of LTC4 metabolites in bile changed with time (12, 13): LTD4 was the major product in the early phase, whereas later on metabolites formed within the hepatocytes, N-acetyl-LTE4 and polar metabolites, predominated. LTE4 comprised a very small fraction in rat bile (13) indicating that it was rapidly converted to N-acetyl-LTE4 and polar metabolites. A portion of the cysteinyl leukotrienes eliminated with rat bile undergoes enterohepatic circulation (23). The pattern of LTC4 metabolites in vivo was shifted towards a predominance of LTD4 when LTD4 dipeptidase was inhibited by Dpenicillamine (Fig. 5). Under this condition, LTD4 became the major cysteinyl leukotriene in the bile of rats. Moreover, this inhibition resulted in a retarded formation of N-acetyl-LTE4 and of polar leukotriene metabolites (Fig. 5). It is of interest that D-penicillamine, which is used in the therapy of rheumatoid emA iONTROL ooA D-PENICILLAMINE
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FIG. 5. Distribution of LTC4 metabolites in rat bile in vivo and the changes induced by D-penicillamine. [3H]LTC4 (3/~Ci or 75 pmol per kg body weight) was injected i.v. and bile collected continuously as described (13, 24). Labeled leukotriene metabolites were separated by RP-HPLC and defined by their retention times and by coelution with standards. Closed symbols represent the mean from 6 control rats; open symbols designate mean values from 6 animals treated i.v. with D-penicillamine (2 mmol/kg) 5 rain prior to [3H]LTC4. The metabofites accumulated in bile are expressed as percent of the bifiary radioactivity excreted within I hr. Polar LTs are leukotriene metabolites more polar than LTC4 on RP-HPLC.
218
D. KEPPLER, et al.
arthritis, markedly extended the biological half-life of LTD4 in vivo. This action of the drug may contribute to the adverse side effects after administration of D-penicillamine. Endogenous Cysteinyl Leukotrienes in the Rat Various stimuli including bacterial toxins (6, 12, 13, 19) and tissue injuries (5) elicit an enhanced systemic production of LTC4 and its metabolites. The in vivo production of these mediators can be measured in bile into which they are rapidly eliminated from the blood circulation (5, 6, 12, 13). LTE4 was the major metabolite in blood plasma (5, 19); it reached a concentration of about 1.7 nmol/1 plasma immediately after the tissue trauma required for sample collection from the abdominal aorta (5). In bile, N-acetyl-LTE4 was the major endogenous cysteinyl leukotriene detected by the sequential use of HPLC separation and RIA (Fig. 6) indicating that passage through the liver had
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FIG. 6. Endogenous cysteinyi leukotrienes in plasma and bile of the rat. Deproteinized samples were separated by RP-HPLC followed by determination of leukotriene concentrations in each fraction by RIA (5). Leukotriene production was elicited by the surgical trauma (abdominal incision, cannulation of the bile duct for bile collection, puncture of the aorta for blood collection). Arrows indicate retention times on HPLC of leukotriene standards. Broken lines at retention times shorter than 6 min indicate interference in the RIA because of high leukotriene binding in these fractions. Note the different ordinate scales with m u c h higher leukotriene concentrations in bile (bottom) as compared to plasma (top).
219
LEUKOTRIENEC4 METABOLISM
resulted in a biologically inactivated biliary metabolite. The concentration of endogenous cysteinyl leukotrienes in bile was up to 100 times as great as that in plasma (5). In addition to the formation of N-acetyl-LTE4, we have observed that this mercapturic acid derivative can be converted further to more polar leukotriene metabolites (13) which are not readily detected by the RIA.
LTC4 Metabolism in Primates After i.v. injection of [3H]LTC4 (10~tCi/kg body weight) in the monkey Macaca fascicularis, the radioactivity was rapidly eliminated from the circulating blood with an initial half-life of about 40 sec. Leukotriene elimination from the circulation by the liver exceeded renal elimination; within 5 hr, about 40% of the radioactivity was recovered from bile and about 20% from urine (19). This predominant hepatobiliary excretion in the monkey is in apparent disagreement with studies in man where urinary elimination of i.v. injected [3H]LTC, largely exceeded the excretion with feces (34). We therefore investigated whether intestinal reabsorption of leukotrienes excreted with bile contributes to the urinary elimination and whether enterohepatic circulation of cysteinyl leukotrienes occurs. As shown in Figure 7, intraduodenal administration of [3H]LTC4, which is metabolized in the intestine to [3H]LTD4 and [3H]LTE4, resulted within 8 hr in a recovery of about 8% of the tritium radioactivity in urine and about 5% in bile (19).
r 8./. after 8hr] I 5*/. after 8hr ] FIG. 7. Elimination of cysteinyl leukotrienes administered i.v. or into the duodenum of the monkey. Cysteinyl leukotrienes circulating in blood undergo hepatobiliary and urinary excretion, as well as enterohepatic circulation (19, 23). Open arrows indicate sites of tracer administration. Numbers represent the percentages of administered tritium recovered in bile or urine after i.v. (shaded boxes) or intraduodenal (open boxes) injection of [3H]LTC, (19).
The biological significance of the enterohepatic circulation of cysteinyl leukotrienes depends not only on the quantity of absorbed leukotrienes from the intestine but also on the biological activity of the metabolites. Analysis of the metabolite pattern in monkey bile provided no evidence for the formation of N-acetyl-LTE4 and, in contrast to the rat, LTE4 was a predominant metabolite (19). In addition to LTE4, LTD4 and metabolites more polar than AER-H*
220
D. KEPPLER,et al.
LTC4 were found in bile and urine (Fig. 8). The proportion of the most polar metabolites (LT 0.25 with a retention time on HPLC relative to LTC4 of 0.25) increased with time. LTE+, which has considerable biological activity (2-4, 35), has also been identified as a metabolite ofi.v, injected [3H]LTC4 in human urine (34). It is not yet known whether genetic polymorphism occurs in LTC4 catabolism in man, with some populations forming N-acetyl-LTE4 and others predominantly LTE4. Ethnic polymorphism has been widely recognized in the N-acetylation of drugs and other foreign compounds (36). ~
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FIG. 8. Relative percentages of biliary and urinary [3H]leukotriene metabolites after i.v. administration of [3H]LTC+ in the monkey. Samples were separated by RP-HPLC (13, 19). LT 0.25 and LT 0.45 designate leukotriene metabolites eluting from HPLC with mean retention times of 0.25 and 0.45 relative to [3H]LTC4, respectively (19). The percentage of metabolites is expressed relative to the total [3H]leukotriene metabolites in the respective sample. Mean values from 3 monkeys are given.
Endogenous Cysteinyl Leukotrienes in Primates Based on the tracer studies (Fig. 7), bile appears as the optimal compartment or body fluid for measurement of the systemic production of cysteinyl leukotrienes not only in rodents (5, 6) but also in primates. [3H]LTE4 appears as a predominant metabolite among the cysteinyl leukotrienes of defined structure in the tracer study (Fig. 8). LTE+ was the major endogenous cysteinyl leukotriene in the bile of monkeys treated with staphylococcal enterotoxin B (19), and in patients suffering from acute pancreatitis (Fig. 9). Both staphylococcal enterotoxin B intoxication and acute pancreatitis can be associated with shock symptoms in which cysteinyl leukotrienes may play a decisive role as mediators (5, 7, 8). The short half-life of cysteinyl leukotrienes in the blood circulation (5, 10, 19) precludes meaningful leukotriene measurements in blood plasma (19).
221
LEUKOTRIENE C4 METABOLISM 50
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FIG. 9. Endogenous cysteinyl leukotriene production in human acute pancreatitis: Measurement of LTE4 in bile. Bile was obtained during enteral retrograde cholangiography from a 67-year old patient suffering from acute pancreatitis. Control bile was obtained from the T-tube of a patient on the third day after gallbladder surgery. Leukotriene analysis in deproteinized bile samples was performed as described by the sequential use of HPLC and RIA (5, 19).
Urinary LTC4 metabolites may be analyzed (19), but structural identification of some of the metabolites (Fig. 8) should precede the measurement of endogenous cysteinyl leukotrienes in the urine of primates. SUMMARY
The metabolism of the glutathionyl leukotriene LTC4 in the mercapturic acid pathway was studied in suspensions of AS-30D hepatoma cells and hepatocytes, as well as in vivo in the bile duct-cannulated rat and in primates. 1. Isolated hepatocytes actively took up cysteinyl leukotrienes and metabolized LTC4 not only to LTD4 and LTE4 but also to N-acetyl-LTE4 and to metabolites more polar than LTC4. 2. AS-30D hepatoma cells are deficient in the transport system for the uptake of cysteinyl leukotrienes. Peptide cleavage of LTC4 to LTD4 and LTE4 was catalyzed by ectoenzymes of these cells. Inactivation of ~/-glutamyltransferase by acivicin and inhibition of LTD4 dipeptidase by penicillamine largely prevented further catabolism of LTC4 and LTD4, respectively. 3. [3H]LTC4 injected i.v. into rats was rapidly eliminated from the circulating blood, taken up by the liver, and excreted into bile where 77% of
222
D. KEPPLER, et al.
the administered radioactivity was recovered within 1 hr. The biliary LTC4 metabolites included LTD4, N-acetyl-LTE4, and metabolites more polar than LTC4. 4. Inhibition of [3H]LTC4 metabolism in vivo by i.v. penicillamine shifted the pattern of biliary cysteinyl leukotrienes; an extended half-life of [3H]LTD4 was associated with a retarded formation of N-acetyl-LTE4 and of polar metabolites. 5. Endogenous cysteinyl leukotrienes elicited by trauma were measured after HPLC separation by radioimmunologic analysis in plasma and bile of rats. The biliary concentration of these leukotrienes was up to 100 times as great as in plasma. N-AcetyI-LTE4 was the predominant endogenous metabolite in rat bile. 6. In the monkey Macaca fascicularis, cysteinyl leukotrienes were predominantly eliminated from blood via the liver into bile; renal excretion amounted to about 50% of the hepatobiliary elimination. Absorption of cysteinyl leukotrienes from the intestine resulted in enterohepatic circulation of these mediators. 7. Metabolites of [3H]LTC4 injected i.v. in the monkey were analyzed in bile and urine. In addition to polar metabolites and a small percentage of [3H]LTD4, [3H]LTE4 was a predominant metabolite particularly in bile. LTE4 was also the major endogenous cysteinyl leukotriene detected by radioimmunologic analysis in monkey bile. 8. LTE4 was the predominant endogenous cysteinyl leukotriene measured in human bile in patients suffering from acute pancreatitis. The detected amounts of LTE4 may be sufficient to induce known phenomena associated with acute pancreatitis including the shock-like reaction.
ACKNOWLEDGMENTS
The authors are indebted to Dr. H.-J. Brambs, Medizinische Universit~itsklinik Freiburg i. Br., for providing human bile samples. We are grateful to Ms. Stefanie K~stner and Ms. Andrea Hauser for their excellent assistance in the experimental work. The research outlined in this contribution was supported by grants from the Deutsche Forschungsgemeinschaft through SFB 154, Freiburg i. Br.
REFERENCES 1. B. SAMUELSSON, Leukotrienes: mediators of immediate hypersensitivity reactions and inflammation, Science 220, 568-575 (1983). 2. R.A. LEWIS and K. F. AUSTEN, The biologically active leukotrienes, J. Clin. Invest. 73, 889-897 (1984). 3. S. HAMMARSTR~)M, Leukotrienes, Annu. Rev. Biochem. 52, 355-377 (1983). 4. P.J. PIPER, Formation and actions of leukotrienes, Physiol. Rev. 64, 744-776 (1984).
LEUKOTRIENEC4METABOLISM
223
5. C. DENZLINGER, S. RAPF, W. HAGMANN and D. KEPFLER, Leukotrienes as mediators in tissue trauma, Science 230, 330-332 (1985). 6. W. HAGMANN, C. DENZLINGER and D. KEPPLER, Role ofpeptide leukotrienes and their hepatobiliary elimination in endotoxin action, Circ. Shock 14, 223-235 (1984). 7. G. FEUERSTEIN, Leukotrienes and the cardiovascular system, Prostaglandins 27, 781-802 (1984). 8. A.M. LEFER, Leukotrienes as mediators of ischemia and shock, Biochem. Pharmacol. 35, 123-127 (1986). 9. L. FENIUK, I. KENNEDY, C. J. WHELAN and G. WRIGHT, Evidence for the existence of multiple leukotriene (LT) receptors from studies on a range of isolated tissues, Prostaglandins 28, 675 (1984.). 10. S. HAMMARSTROM, L. ORNING and K. BERNSTROM, Metabolism of leukotrienes, Mol. Cell. Biochem. 69, 7-16 (1985). 11. L. ORNING, E: NORIN, B. GUSTAFSSON and S. HAMMARSTROM, In vivo metabolism of leukotriene C4 in germ-free and conventional rats, J. Biol. Chem. 261, 766-771 (1986). 12. W. HAGMANN, C. DENZLINGER and D. KEPPLER, Production of peptide leukotrienes in endotoxin shock, FEBS Lett. 180, 309-313 (1985). 13. W. HAGMANN, C. DENZLINGER, S. RAPP, G. WECKBECKER and D. KEPPLER, Identification of the major endogenous leukotriene metabolite in the bile of rats as N-acetyileukotriene E4. Prostaglandins 31, 239-251 (1986). 14. R.A. LEWIS, J. M. DRAZEN, K. F. AUSTEN, M. TODA, F. BRION, A. MARFAT and E. J. COREY, Contractile activities of structural analogs of leukotrienes C and D: Role of the polar substituents, Proc. Natl. Acad. Sci. U.S.A. 78, 4579-4583 (1981). 15. A. FOSTER, B. FITZSIMMONS and L. G. LETTS, The synthesis of N-acetyl-leukotriene E4 and its effects on cardiovascular and respiratory function of the anesthetized pig, Prostaglandins 31, 1077-1086 (1986). 16. D. KEPPLER, W. HAGMANN, S. RAPP, C. DENZLINGER and H. K. KOCH, The relation of leukotrienes to liver injury, Hepatology 5, 883-891 (1985). 17. K. BERNSTR~)M and S. HAMMARSTR~M, Metabolism ofleukotriene E4 by rat tissues: formation of N-acetyl leukotriene E4, Arch. Biochem. Biophys. 244, 486-491 (1986). 18. L.-E. APPELGREN and S. HAMMARSTROM, Distribution and metabolism of 3Hlabeled leukotriene C3 in the mouse, J. Biol. Chem. 257, 531-535 (1982). 19. C. DENZLINGER, A. GUHLMANN, P. H. SCHEUBER, D. WILKER, D. K. HAMMER and D. KEPPLER, Metabolism and analysis of cysteinyl leukotrienes in the monkey, J. Biol. Chem. 261, 15601-15606 (1986). 20. N. UEHARA, K. ORMSTAD, L. ORNING and S. HAMMARSTROM, Characteristics of the uptake of cysteine-containing leukotrienes by isolated hepatocytes, Biochim. Biophys. Acta 732, 69-74 (1983). 21. G. WECKBECKER and D. KEPPLER, Leukotriene C4 metabolism by hepatoma cells deficient in the uptake of cysteinyl leukotrienes, Fur. J. Biochem. 154, 559-562 (1986)... 22. K. ORMSTAD, N. UEHARA, S. ORRENIUS, L. ~)RNING and S. HAMMARSTROM, Uptake and metabolism of leukotriene C3 by isolated rat organs and cells, Biochem. Biophys. Res. Commun. 104, 1434-1440 (1982). 23. C. DENZLINGER, A. GUHLMANN, W. HAGMANN, P. H. SCHEUBER, F. SCHEYERL, D. WILKER, D. K. HAMMER and D. KEPPLER, Cysteinyl leukotrienes undergo enterohepatic circulation, Prostaglandins Leukotrienes Med. 21, 321-322 (1986). 24. M. HUBER, A. GUHLMANN, P. L. M. JANSEN and D. KEPPLER, Hereditary defect of hepatobiliary cysteinyl leukotriene elimination in mutant rats with defective hepatic anion excretion, Hepatology 7 (1987), in press. 25. J.H. MUSSER, A. F. KREFT and A. J. LEWIS, New developments concerning leukotriene antagonists: a review, Agents Actions 18, 332-341 (1986). 26. 3. H. FLEISCH, L. E. RINKEMA, K. D. HAISCH, D. McCULLOUGH, F. P. CARR and R. D. DILLARD, Evaluation of LY163443, l-[2-hydroxy-3-propyl-4-([4-(IH-tetrazol-5ylmethyl)phenoxy]methyl)phenyl]ethanone, as a pharmacologic antagonist of leukotrienes D4 and E4, Naunyn-Schmiedeberg's Arch. Pharmacol. 333, 70-77 (1986).
224
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27. D. KEPPLER and D. F. SMITH, Nucleotide contents of ascites hepatoma cells and their changes induced by D-galactosamine, Cancer Res. 34, 705-711 (1974). 28. M . A . CAPRARO and R. P. HUGHEY, Use of acivicin in the determination of rate constants for turnover of rat renal ~/-glutamyltranspeptidase, J. Biol. Chem. 260, 3408-3412 (1985). 29. G. WEBER, Y. NATSUMEDA, M. S. LUI, M. A. FADERAN, J. J. LIEPNIEKS and W. L. ELLIOTT, Control of enzymic programs and nucleotide pattern in cancer cells by acivicin and tiazofurin, Adv. Enzyme Regul. 22, 69-93 (1984). 30. T. McINTYRE and N. P. CURTHOYS, Renal catabolism ofglutathione. Characterization of a particulate rat renal dipeptidase that catalyzes the hydrolysis of cysteinylglycine, J. Biol. Chem. 257, 11915-11921 (1982). 31. E.M. KOZAK and S. S. TATE, Glutathione-degrading enzymes of microviUus membranes, J. Biol. Chem. 257, 6322-6327 (1982). 32. M.E. ANDERSON, R. D. ALLISON and A. MEISTER, Interconversion of leukotrienes catalyzed by purified ~,-glutamyltranspeptidase: Concomitant formation of leukotriene D4 and ),-glutamyl amino acids, Proc. Natl. Acad. Sci. U.S.A. 79, 1088-1091 (1982). 33. M. KOLLER, J. BROM, M. RAULF and W. KONIG, Cilastatin (MK 0791) is a potent and specific inhibitor of the renal leukotriene D4-dipeptidase, Biochem. Biophys. Res. Commun. 131,974-979 (1985). 34. L. ORNING, L. KAIJSER and S. HAMMARSTROM, In vivo metabolism of leukotriene C4 in man: urinary excretion of leukotriene E4, Biochem. Biophys. Res. Commun. 130, 214-220 (1985). 35. X.-Y. HUA, S.-E. DAHLI~N, J. M. LUNDBERG, S. HAMMARSTROM and P. HEDQVIST, Leukotrienes C4, D~ and E4 cause widespread and extensive plasma extravasation in the guinea pig, Naunyn-Schmiedeberg's Arch. Pharmacol. 330, 136-141 (1985). 36. W.W. WEBER and D. W. HEIN, N-Acetylation pharmacogenetics, Pharmacol. Rev. 37, 25-79 (1985).
C4 M E T A B O L I S M CELLS AND LIVER
BY
DIETRICH KEPPLER, MICHAEL HUBER, GISBERT WECKBECKER, WOLFGANG HAGMANN, CLAUDIO DENZLINGER and ALBRECHT GUHLMANN Biochemisches Institut, University of Freiburg im Breisgau, D-7800 Freiburg, West Germany INTRODUCTION
The cysteinyl leukotrienes LTC4*, LTD4, and LTE4 are potent mediators of smooth muscle contraction and play an important role in inflammation, anaphylaxis (1-4), tissue injury (5), and shock (6-8). The relative potencies of LTC4, LTD4, and LTE4 differ in various biological systems. The order of molar potencies in the guinea pig ileum (4) and in the human bronchus (9) is LTD4 > LTC4 > LTE4. The N-acetylation product of LTE4, N-acetyl-LTE4, has been identified in rat feces (10, 11) and bile (12, 13), and represents the mercapturic acid derivative of cysteinyl leukotriene catabolism (13). NAcetyI-LTE4 is approximately 25-fold less active than LTD4 in the pulmonary pare nchymal strip (14) and 100-fold less active than LTC4 in the pig in vivo (15) indicating that conversion from LTD4 to N-acetyl-LTE4 in the mercapturic acid pathway is a stepwise deactivation of these highly active mediators. LTC4 catabolism comprises extracellular ectoenzyme-catalyzed reactions as well as intracellular conversions (10, 16, 17). The former include -/-glutamyltransferase (EC2.3.2.2) and dipeptidase (EC3.4.13.11) (Fig. 1); the intracellular reactions include the N-acetylation of LTE4 (17) and the formation of polar metabolites derived from the cysteinyl leukotrienes (13, 18, 19). Discrimination between extracellular and intracellular metabolism of LTC4 is possible by comparison of hepatocytes, which take up cysteinyl leukotrienes most actively (20, 21), with cells defective in the uptake system for these leukotrienes, such as the hepatoma cell line AS-30D (21). These hepatoma cells are useful in studies on ectoenzyme-catalyzed LTC4 catabolism and on inhibition of ~/-glutamyltransferase (21) and LTD4 dipeptidase in intact membranes. In vivo, the cysteinyl leukotrienes are rapidly eliminated from the circulating blood by hepatic (5, 6, 10, 13, 18-20) and renal (19, 22) uptake, followed by elimination of metabolites into bile (5, 6, 18) and urine (13, 18, 19, *The following abbreviations are used: LTA4, LTC(, LTD(, LTE(and LTE(NAc, leukotrienes A4, C . D . E~, and N-acetyI-E~,respectively; i.v., intravenous(ly); (RP-)HPLC, (reversed-phase) high-performance liquid chromatography; RIA, radioimmunoassay; HTMP, 4-hydroxy2,2,6,6-tetramethylpiperidine-l-oxyl. AZR-~
211
212
D. KEPPLER, et al. 0H I
I
/
/
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CO-CH~ FIG. 1. Metabolism of LTC( in the mercapturic acid pathway. The glutathionyl derivative LTC4 is converted to the cysteinylglycine derivative LTD4 by ?-glutamyltransferase (3) and by dipeptidase to the cysteinyl derivative LTE4 (10). N-AcetyI-LTE4, the mercapturate, is a major LTC4 metabolite in bile (13) and feces (11) of the rat. In addition, metabolites more polar than LTC4 and a metabolite less polar than LTE4 are detected in bile and urine (19).
23, 24). Elucidation of the in vivo metabolism of LTC4 is an essential prerequisite for the quantitative analysis of cysteinyl leukotrienes under pathophysiological conditions (5, 6, 12, 13, 19) as well as for the development of effective leukotriene receptor antagonists (25, 26). MATERIALS
AND
METHODS
Hepatoma cells andhepatocytes. The AS-30D rat ascites hepatoma line was carried in Sprague Dawley rats (27). For studies on the uptake of cysteinyl leukotrienes, AS-30D cells were kept at 37°C in suspension culture on a gyratory shaker in a modified standard medium (27) containing in one liter
LEUKOTRIENEC4METABOLISM
213
9.0 g Swim's powdered medium, 1.0 g Pluronic F68, 0.4 mmol L-glutamine (added 30 rain prior to uptake determinations, except for the measurements of L-[14C]glutamineuptake), 0.06 mmol L-cystine, 25 mmol Na2HPO4, and 9 mmol NaHCO3. LTC4, LTD4, and LTE4 metabolism was studied in the absence of L-cystine. Cell suspensions were kept under CO2/air (1/20) at pH 7.4 at concentrations between 3 and 6 × 109 cells/l. Determination of apparent kinetic constants of LTD4 dipeptidase was carried out at 37°C in a medium containing in one liter 115 mmol NaC1, 5 mmol KC1, 21 mmol Na2HPO4, 5 mmol NaHEPO4, 6 mmol D-glucose, and 1 g Pluronic F68 at a pH of 7.4. Freshly isolated rat hepatocytes, kindly provided by Drs. G. Kurz and G. Fricker from the Chemisches Laboratorium of Freiburg University, were incubated at a concentration of 1 × 109 cells/1 under the same conditions as the hepatoma ceils (21). Details of the uptake measurements for cysteinyl leukotrienes were described previously (21). Analysis of leukotriene metabolites in bile and urine. Bile and urine were sampled continuously from anesthetized rats (13, 24) or cynomolgus monkeys (19) into ice-cold 90% aqueous methanol, containing 1 mmol/l HTMP and 0.5 mmol/1 EDTA at pH 7.4, and under argon to prevent oxidative degradation of leukotrienes. Details of bile collection, deproteinization, and separation of leukotriene metabolites by RP-HPLC were described (13, 19, 24). RP-HPLC was performed on a C 18-Hypersil column (4.6 x 250 mm, 5#m particles, Shandon, Runcorn, U.K.). The mobile phase consisted of methanol, water, and acetic acid (65/35/0.1 by vol) at pH 5.6. Radioactive leukotriene metabolites were analyzed by continuous detection of tritium in the HPLC eluent using the Berthold (Wildbad, FRG) liquid scintillation device LB 505. Radioimmunological assays for cysteinyl leukotrienes were performed in fractions of the HPLC eluate (5, 13, 19). Antibodies for the RIA were either obtained from New England Nuclear/DuPont, Boston, MA, or kindly provided by Dr. B. A. Peskar, Ruhr-Universit~it, Bochum, F.R.G. The commercial RIA for LTC4 from New England Nuclear had sufficient crossreactivities to allow for the analysis of LTC4 metabolites, particularly of N-acetyl-LTE4 which crossreacted with 34% relative to LTC4 (13). Leukotrienes and inhibitors. LTC4, LTD4, and LTE4 were obtained from Miles Scientific, Slough, U.K. [14,15-3H]LTC4, [14,15-3H]LTD4, and [14,153H]LTE4 (40 Ci/mmol each) were from New England Nuclear/DuPont, Boston, MA. N-AcetyI-LTE4 and N-acetyI-[3H]LTE4 were synthesized from LTE4 and [3H]LTE4, respectively, as described (13). L-PeniciUamine, Dpenicillamine, L-cysteine, and bestatin were from Sigma, St. Louis, MO. Cilastatin (MK 0791) was kindly donated by Merck Sharp and Dohme, Rahway, NJ. Dr. G. Neil (Upjohn Co., Kalamazoo, MI) kindly supplied us
D. KEPPLER, et al.
214
with acivicin (L-(aS,5S)-a-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid, NSC-163501). RESULTS AND DISCUSSION
Cysteinyl Leukotriene Metabolism and Uptake by Hepatoma Cells and Hepatocytes Hepatocytes actively accumulated cysteinyl leukotrienes (Fig. 2, right). Previous studies demonstrated a common temperature- and energydependent transport of LTC4, LTD4, and LTE4 into hepatocytes (20). Within 20 min, hepatocyte suspensions incubated with [3H]LTC4 metabolized about 60% of this leukotriene to LTD4 (21%), metabolites more polar than LTC4 (33%), and small amounts of N-acetyl-LTE4 (4%) (21). AS-30D hepatoma cells, on the contrary, were completely deficient in the uptake of cysteinyl leukotrienes (Fig. 2, left). These hepatocyte-derived malignant cells converted labeled LTC4 to LTD4 and LTE4 (Fig. 3) but did not produce polar metabolites or N-acetyl-LTE4. Disruption of the hepatoma cells by sonication did not enhance the rate of peptide cleavage from LTC4 to LTE4 (Fig. 3). In control experiments, the hepatoma cells as well as the hepatoeytes took up L-[14C]glutamine and [14C]adenosine. These results indicate that
HEPATOMA CELLS
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FIG. 2. Comparativeuptake of [3H]-labeledLTC4and LTE4byAS-30Dhepatomacells(left)and hepatocytes(fight)in suspension.The concentrations of LTC4and LTE4were3 and 1.5 nmol/l, respectively.For comparisonand control, the uptakerates of L-[~4C]glutamine(0.4retool/I)and [t4C]adenosine (0.1 retool/l) weredetermined in parallel (21). Mean values_+S.E.M. (n=6) are given.
LEUKOTRIENE C, METABOLISM .
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FIG. 3. LTC4 metabolites in the medium of hepatoma cell suspensions. Cells were incubated in the presence of [3H]LTC4 (7.5 nmol/l) for 90 min. Supernatants were analyzed by RP-HPLC with continuous detection of radioactivity. Upper panel: untreated AS-30D cell control. Middle panel: inhibition of 3,-glutamyltransferase by acivicin (2 mmol/l) added 65 min prior to [3H]LTC4. Lower panel: cells disrupted by sonication 10 min prior to addition of [~H]LTC4 (21). Arrows indicate retention times of standard [3I-I]leukotrienes.
AS-30D cells have selectively lost the transport system for the uptake of cysteinyl leukotrienes. Moreover, ~/-glutamyltransferase and LTD4 dipeptidase, degrading LTC4 to LTD4 and LTE4, respectively, are located on the surface of these hepatoma cells and act as ectoenzymes (21). Formation of the intracellular metabolite N-acetyl-LTE4 (13, 17) could be observed, however, when hepatoma cells were disrupted by sonication and incubated with [3H]LTE4 in the presence of acetyl-CoA (1 mmol/l).
Inhibition of ~/-Glutamyltransferase and LTD4 Dipeptidase on Hepatoma Cells AS-30D cells, which are deficient in the uptake of cysteinyl leukotrienes, provide a useful system for studies on the in situ regulation and inhibition of ectoenzymes involved in LTC4 breakdown. 3~-Glutamyltransferase is irreversibly inhibited by the L-glutamine analog acivicin (28). This inhibitor of L-glutamine-dependent enzymes (29) effectively prevented the formation of LTD4 or other metabolites from LTC4 (Fig. 3).
216
D. KEPPLER, et al.
Cysteinylglycine-degradingdipeptidase (EC 3.4.13.11) (30) has been shown to hydrolyze LTD4 yielding LTE4 and glycine (31, 32). This enzyme, which differs from microsomal aminopeptidase (EC 3.4.11.2) (30, 31), is inhibited by various thiol compounds including D- and L-penicillamine (31). The LTD4 dipeptidase on the surface ofAS-30D hepatoma cells exhibited a high affinity for LTD4 with an apparent Km value of 6.6 #mol/1. D- and L-penicillamine were effective inhibitors with apparent Ki values of 0.46 and 0.21 mmol/1, respectively (Fig. 4). Cilastatin, which has been described as an inhibitor of renal LTD4 dipeptidase activity (33), is a comparatively weak inhibitor of the hepatoma cell L T D 4 dipeptidase (Fig. 4). Bestatin, which inhibits microsomal aminopeptidase but has no effect on dipeptidase (30), did not affect the L T D 4 dipeptidase on AS-30D cells even at bestatin concentrations as high as 5 mmol/l (Fig. 4). These concentrations did, however, inhibit the aminopeptidase activity on AS-30D hepatoma cells (data not shown). These studies indicate that microsomal aminopeptidase on AS-30D cells does not contribute to the formation of L T E 4 f r o m L T D 4. The inhibition of LTD4 dipeptidase by D- and L-penicillamine offers the possibility to interfere with the deactivation of LTD4, the biologically most potent of the cysteinyl leukotrienes.
100
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INHIBITORCONCENTRATION (mmotlt) FIG. 4. Inhibition of LTD4 dipeptidas¢ on the surface of hepatoma cells by thiol compounds. The inhihitors were added to the cell suspension 30 min prior to [~H]LTD4 (1.5 nmol/1). After 2 hr at 37°C, remaining [3H]LTD4 and formed [3HILTE 4were separated by RP-HPLC. The inhibition of LTD4 dipeptidase is expressed as percent of the activity in untreated controls. Mean values from 3 experiments are given.
LEUKOTRIENEC4METABOLISM
217
Hepatobiliary Elimination of LTC4 Metabolites in the Rat Uptake of cysteinyl leukotrienes from the blood into hepatocytes a n d subsequent elimination into bile predominate in the rat over renal excretion (I0, 13, 24). One hour after i.v. injection of a tracer dose of[3H]LTC4, 77% of the radioactivity was recovered from rat bile and only 1.5% from urine (24). The pattern of LTC4 metabolites in bile changed with time (12, 13): LTD4 was the major product in the early phase, whereas later on metabolites formed within the hepatocytes, N-acetyl-LTE4 and polar metabolites, predominated. LTE4 comprised a very small fraction in rat bile (13) indicating that it was rapidly converted to N-acetyl-LTE4 and polar metabolites. A portion of the cysteinyl leukotrienes eliminated with rat bile undergoes enterohepatic circulation (23). The pattern of LTC4 metabolites in vivo was shifted towards a predominance of LTD4 when LTD4 dipeptidase was inhibited by Dpenicillamine (Fig. 5). Under this condition, LTD4 became the major cysteinyl leukotriene in the bile of rats. Moreover, this inhibition resulted in a retarded formation of N-acetyl-LTE4 and of polar leukotriene metabolites (Fig. 5). It is of interest that D-penicillamine, which is used in the therapy of rheumatoid emA iONTROL ooA D-PENICILLAMINE
>
LT~
3[ LT~
~ 20 N ~ 10 0
3'o TIMEAFTER[3H]LTC4i.v.(rain)
FIG. 5. Distribution of LTC4 metabolites in rat bile in vivo and the changes induced by D-penicillamine. [3H]LTC4 (3/~Ci or 75 pmol per kg body weight) was injected i.v. and bile collected continuously as described (13, 24). Labeled leukotriene metabolites were separated by RP-HPLC and defined by their retention times and by coelution with standards. Closed symbols represent the mean from 6 control rats; open symbols designate mean values from 6 animals treated i.v. with D-penicillamine (2 mmol/kg) 5 rain prior to [3H]LTC4. The metabofites accumulated in bile are expressed as percent of the bifiary radioactivity excreted within I hr. Polar LTs are leukotriene metabolites more polar than LTC4 on RP-HPLC.
218
D. KEPPLER, et al.
arthritis, markedly extended the biological half-life of LTD4 in vivo. This action of the drug may contribute to the adverse side effects after administration of D-penicillamine. Endogenous Cysteinyl Leukotrienes in the Rat Various stimuli including bacterial toxins (6, 12, 13, 19) and tissue injuries (5) elicit an enhanced systemic production of LTC4 and its metabolites. The in vivo production of these mediators can be measured in bile into which they are rapidly eliminated from the blood circulation (5, 6, 12, 13). LTE4 was the major metabolite in blood plasma (5, 19); it reached a concentration of about 1.7 nmol/1 plasma immediately after the tissue trauma required for sample collection from the abdominal aorta (5). In bile, N-acetyl-LTE4 was the major endogenous cysteinyl leukotriene detected by the sequential use of HPLC separation and RIA (Fig. 6) indicating that passage through the liver had
LT ,LTE, AcITO,ITE,
0.5 " E
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40
FIG. 6. Endogenous cysteinyi leukotrienes in plasma and bile of the rat. Deproteinized samples were separated by RP-HPLC followed by determination of leukotriene concentrations in each fraction by RIA (5). Leukotriene production was elicited by the surgical trauma (abdominal incision, cannulation of the bile duct for bile collection, puncture of the aorta for blood collection). Arrows indicate retention times on HPLC of leukotriene standards. Broken lines at retention times shorter than 6 min indicate interference in the RIA because of high leukotriene binding in these fractions. Note the different ordinate scales with m u c h higher leukotriene concentrations in bile (bottom) as compared to plasma (top).
219
LEUKOTRIENEC4 METABOLISM
resulted in a biologically inactivated biliary metabolite. The concentration of endogenous cysteinyl leukotrienes in bile was up to 100 times as great as that in plasma (5). In addition to the formation of N-acetyl-LTE4, we have observed that this mercapturic acid derivative can be converted further to more polar leukotriene metabolites (13) which are not readily detected by the RIA.
LTC4 Metabolism in Primates After i.v. injection of [3H]LTC4 (10~tCi/kg body weight) in the monkey Macaca fascicularis, the radioactivity was rapidly eliminated from the circulating blood with an initial half-life of about 40 sec. Leukotriene elimination from the circulation by the liver exceeded renal elimination; within 5 hr, about 40% of the radioactivity was recovered from bile and about 20% from urine (19). This predominant hepatobiliary excretion in the monkey is in apparent disagreement with studies in man where urinary elimination of i.v. injected [3H]LTC, largely exceeded the excretion with feces (34). We therefore investigated whether intestinal reabsorption of leukotrienes excreted with bile contributes to the urinary elimination and whether enterohepatic circulation of cysteinyl leukotrienes occurs. As shown in Figure 7, intraduodenal administration of [3H]LTC4, which is metabolized in the intestine to [3H]LTD4 and [3H]LTE4, resulted within 8 hr in a recovery of about 8% of the tritium radioactivity in urine and about 5% in bile (19).
r 8./. after 8hr] I 5*/. after 8hr ] FIG. 7. Elimination of cysteinyl leukotrienes administered i.v. or into the duodenum of the monkey. Cysteinyl leukotrienes circulating in blood undergo hepatobiliary and urinary excretion, as well as enterohepatic circulation (19, 23). Open arrows indicate sites of tracer administration. Numbers represent the percentages of administered tritium recovered in bile or urine after i.v. (shaded boxes) or intraduodenal (open boxes) injection of [3H]LTC, (19).
The biological significance of the enterohepatic circulation of cysteinyl leukotrienes depends not only on the quantity of absorbed leukotrienes from the intestine but also on the biological activity of the metabolites. Analysis of the metabolite pattern in monkey bile provided no evidence for the formation of N-acetyl-LTE4 and, in contrast to the rat, LTE4 was a predominant metabolite (19). In addition to LTE4, LTD4 and metabolites more polar than AER-H*
220
D. KEPPLER,et al.
LTC4 were found in bile and urine (Fig. 8). The proportion of the most polar metabolites (LT 0.25 with a retention time on HPLC relative to LTC4 of 0.25) increased with time. LTE+, which has considerable biological activity (2-4, 35), has also been identified as a metabolite ofi.v, injected [3H]LTC4 in human urine (34). It is not yet known whether genetic polymorphism occurs in LTC4 catabolism in man, with some populations forming N-acetyl-LTE4 and others predominantly LTE4. Ethnic polymorphism has been widely recognized in the N-acetylation of drugs and other foreign compounds (36). ~
V
f
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TIMEAFTER [+H]LTCv.i.v (hr
FIG. 8. Relative percentages of biliary and urinary [3H]leukotriene metabolites after i.v. administration of [3H]LTC+ in the monkey. Samples were separated by RP-HPLC (13, 19). LT 0.25 and LT 0.45 designate leukotriene metabolites eluting from HPLC with mean retention times of 0.25 and 0.45 relative to [3H]LTC4, respectively (19). The percentage of metabolites is expressed relative to the total [3H]leukotriene metabolites in the respective sample. Mean values from 3 monkeys are given.
Endogenous Cysteinyl Leukotrienes in Primates Based on the tracer studies (Fig. 7), bile appears as the optimal compartment or body fluid for measurement of the systemic production of cysteinyl leukotrienes not only in rodents (5, 6) but also in primates. [3H]LTE4 appears as a predominant metabolite among the cysteinyl leukotrienes of defined structure in the tracer study (Fig. 8). LTE+ was the major endogenous cysteinyl leukotriene in the bile of monkeys treated with staphylococcal enterotoxin B (19), and in patients suffering from acute pancreatitis (Fig. 9). Both staphylococcal enterotoxin B intoxication and acute pancreatitis can be associated with shock symptoms in which cysteinyl leukotrienes may play a decisive role as mediators (5, 7, 8). The short half-life of cysteinyl leukotrienes in the blood circulation (5, 10, 19) precludes meaningful leukotriene measurements in blood plasma (19).
221
LEUKOTRIENE C4 METABOLISM 50
25 ACUTEHUMAN PANCREATTIS
40
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FIG. 9. Endogenous cysteinyl leukotriene production in human acute pancreatitis: Measurement of LTE4 in bile. Bile was obtained during enteral retrograde cholangiography from a 67-year old patient suffering from acute pancreatitis. Control bile was obtained from the T-tube of a patient on the third day after gallbladder surgery. Leukotriene analysis in deproteinized bile samples was performed as described by the sequential use of HPLC and RIA (5, 19).
Urinary LTC4 metabolites may be analyzed (19), but structural identification of some of the metabolites (Fig. 8) should precede the measurement of endogenous cysteinyl leukotrienes in the urine of primates. SUMMARY
The metabolism of the glutathionyl leukotriene LTC4 in the mercapturic acid pathway was studied in suspensions of AS-30D hepatoma cells and hepatocytes, as well as in vivo in the bile duct-cannulated rat and in primates. 1. Isolated hepatocytes actively took up cysteinyl leukotrienes and metabolized LTC4 not only to LTD4 and LTE4 but also to N-acetyl-LTE4 and to metabolites more polar than LTC4. 2. AS-30D hepatoma cells are deficient in the transport system for the uptake of cysteinyl leukotrienes. Peptide cleavage of LTC4 to LTD4 and LTE4 was catalyzed by ectoenzymes of these cells. Inactivation of ~/-glutamyltransferase by acivicin and inhibition of LTD4 dipeptidase by penicillamine largely prevented further catabolism of LTC4 and LTD4, respectively. 3. [3H]LTC4 injected i.v. into rats was rapidly eliminated from the circulating blood, taken up by the liver, and excreted into bile where 77% of
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the administered radioactivity was recovered within 1 hr. The biliary LTC4 metabolites included LTD4, N-acetyl-LTE4, and metabolites more polar than LTC4. 4. Inhibition of [3H]LTC4 metabolism in vivo by i.v. penicillamine shifted the pattern of biliary cysteinyl leukotrienes; an extended half-life of [3H]LTD4 was associated with a retarded formation of N-acetyl-LTE4 and of polar metabolites. 5. Endogenous cysteinyl leukotrienes elicited by trauma were measured after HPLC separation by radioimmunologic analysis in plasma and bile of rats. The biliary concentration of these leukotrienes was up to 100 times as great as in plasma. N-AcetyI-LTE4 was the predominant endogenous metabolite in rat bile. 6. In the monkey Macaca fascicularis, cysteinyl leukotrienes were predominantly eliminated from blood via the liver into bile; renal excretion amounted to about 50% of the hepatobiliary elimination. Absorption of cysteinyl leukotrienes from the intestine resulted in enterohepatic circulation of these mediators. 7. Metabolites of [3H]LTC4 injected i.v. in the monkey were analyzed in bile and urine. In addition to polar metabolites and a small percentage of [3H]LTD4, [3H]LTE4 was a predominant metabolite particularly in bile. LTE4 was also the major endogenous cysteinyl leukotriene detected by radioimmunologic analysis in monkey bile. 8. LTE4 was the predominant endogenous cysteinyl leukotriene measured in human bile in patients suffering from acute pancreatitis. The detected amounts of LTE4 may be sufficient to induce known phenomena associated with acute pancreatitis including the shock-like reaction.
ACKNOWLEDGMENTS
The authors are indebted to Dr. H.-J. Brambs, Medizinische Universit~itsklinik Freiburg i. Br., for providing human bile samples. We are grateful to Ms. Stefanie K~stner and Ms. Andrea Hauser for their excellent assistance in the experimental work. The research outlined in this contribution was supported by grants from the Deutsche Forschungsgemeinschaft through SFB 154, Freiburg i. Br.
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