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Tissue distribution, elimination and metabolism of [3H] - leukotriene C4 in the American bullfrog, Rana catesbeiana
Prostaglandins 45:203-219, 1993
TISSUB DISTRIBUTION, ELIMINATION AND METABOLISM OF r3H] LEUKOTRIIZNE C4 IN THE AMERICAN BULLFROG, RANA CATBSBBIANA
C.A. Pfeifer, R.A. Furllla, K. Gronert, D.D. Goss X.B. Romig and C.A. Herman Department
of Biology, New Mexico State University Las Cruces, NM 89003
ABSTRACT Tissue distribution, elimination, and metabolism of r3H]leukotriene C4 were studied at 2.5 hours after injection in the conscious and anesthetized American bullfrog, Rana catesbeiana. Conscious frogs were injected via the dorsal lymph sac or the sciatic vein. Anesthetized frogs were injected via the abdominal vein. The organs containing the greatest percent of injected radioactivity at 2.5 hours after injection were liver, small intestine and kidney. Route of injection and anesthe ia appears to alter distribution and elimination of leukotrienes. [JHI-leukotrienes were eliminated into bladder water and bile. In addition, 7.8 f 2.2 and 5.2 % 2.5 percent of the injected radioactivity was found in the pan vater bathing the ventral surface of the venously and dorsally injected conscious frogs, respectively, suggesting transfer of radioactivity across the skin. At 2.5 hours, polar metabolites represented 50% of the radioactivity found in liver, bile, and bladder water. These polar metabolites were determined to be la-carboxy-19,20-dinor-leukotriene E4, 20-carboxy-leukotriene E4, and 20-hydroxy-leukotriene E4. Of the non-oxidized leukotrienes, bile contained mainly LTD4 while bladder water contained primarily LTE4. N-acetyl LTE4 was not detected in any samples. The tissue distribution, elimination and metabolism of leukotrienes in the bullfrog was similar to mammalian studies and suggests evolutionary conservation of leukotriene processing. INTRODUCTION The peptido-leukotrienes, LTC4, and its metabolites LTD4 and LTE4 are hormones derived from arachidonic acid and can cause potent hemodynamic and inflammatory effects (l-2). For many animal species, leukotrienes appear to play major roles in health and disease. A role for leukotrienes (LTs) in amphibia has been indicated by the endogenous production of LTs in inflammatory cells of several anuran amphibian species, including Rana catesbeiana in response to infection (3). Once formed, distribution, elimination, and metabolism of leukotrienes differs among animal species. Most studies have been performed in mammals (4-17, 25-33). Two studies have demonstrated that tritiated LTC4 and its metabolites were largely distributed to the liver and kidney in both the guinea pig (4) and mouse (5). respectively. Circulating peptidoleukotrienes are metabolized into LTD4 by gamma-glutamyl transpeptidase, and further to LTE4 by dipeptidase in mammalian blood. They are rapidly removed from the circulation by the hepatic and renal systems into bile and urine
Copyright
0 1993 Butterworth-Heinemann
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respectively (6). Monkey (7-g). dog (lo), domestic pig (ll), guinea pig (4.12) rat (13,14) and mouse (5) eliminate leukotrienes primarily via biliary excretion, while humans (9,13,15) eliminate LTs into bile and urine. LTE4 is the primary non-oxidized LT excreted in urine in most species but rats and mice further metabolize LTE4 to N-acetyl LTE4 (6,16,17). Little is known about how anuran amphibians distribute, in vitro and in vivo eliminate and metabolize leukotrienes. P evious -5 studi s in this laboratory have shown [ HI-LTC4 is metabolized only to [sHI-LTD4 by whole blood b vitro and when injected into cannulated bullfrogs (18,19). However mince3d bullfrog tissues inclu$ing heart (20) and lung (21) metab lized [ HI-LTC4 to [3H]-LTD4 and [ HI-LTE4. Despite the fact that [sHI-LTC4 was rapidly removed from the bullfrog circulation (19), the excretion and identity of LTC4 metabolites in bile and urine of amphibians in general is unknown. There are several distinct anatomical differences between amphibians and mammals which may alter the way these two classes process leukotrienes. Bullfrogs are poikilothermic, have a three chambered heart without coronary arteries and have renal and hepatic portal systems. Frog kidney tubules lack a loop of Henle and are unable to concentrate urine. The frog urinary bladder is important in water and electrolyte transport. The skin may also play a role in transport and elimination in these aquatic animals. All of these physiological differences may alter the way amphibians, such as bullfrogs, process leukotrienes. This study will characterize the tissue distribution and elimination of [3H]-LTC4 and its metabolites in the bullfrog. Because amphibians represent an older phylogenetic group than mammals, this study may provide insights into whether LT processing mechanisms have been evolutionarily conserved. METHODS Leukotriene (LT)C4, LTD4, LTE4, and N-acetyl LTE4 were generous gifts from The Merck Frosst Centre for Therapeutic Research (Pointe Claire-Dorval, Quebec, Canada) and were stored at -80° in ethanol (20 of LTE4, 16-carboxyUg/ml). The oxidized polar metabolites 17,18,19,20-tetranor-14,15-dihydro-LTE4 (16-carboxytetranordihydro(la-carboxydinor_LTE4), 20LTE4), 18-carboxy-19,20-dinor-LTE4 were purchased from Oxford carboxy-LTE4, and 20-hydroxy-LTE4 Biomedical Research, Inc. (Oxford, MI) and stored at -2O'C in ethanol at a concentration of 14.3 vg/ml. Tritiated leukotrienes [14* 15 (n)-3H]-LTC4, $53.4 Ci/mmol, [3H]-LTC4) and [14,-15(n)-3H]-LTD4 (39.3 Ci/mmol, [ HI-LTD4) were purchased from Amersham (Arlington Heights, IL) and New England Nuclear (Boston, MA), respectively. Ethyl-m-aminobenzoate (MS-222) and disodium salt-ethylenediamine tetraacetic acid (Na2EDTA) were obtained from Sigma Chemical Co. (St. Louis, MO) and tissue solubilizing solution, Solvable, was purchased from New England Nuclear (Boston, MA). Scintiverse E and high performance liquid
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Nuclear (Boston, MA). Scintiverse E and high performance liquid chromatography (HPLC) grade methanol and acetonitrile were purchased from Fisher Scientific (Dallas, TX). Sep-Pak (Cl8) extraction cartridges were purchased from Millipore Corp. (Milford, MA) and the free radical scavenger, 4-OH-TEMPO was purchased from Aldrich Chemical Co. (Milwaukee, WI). Animals Bullfrogs of both sexes, 284 * 18 grams (n = 18) were collected from ponds in Dona Ana County or purchased from Charles Sullivan Co., Nashville, TN. Frogs were force-fed beef liver three times weekly and housed at room temperature in a large plastic tank with constant running water. Two animal models, conscious and anesthetized, were used in this study to characterize [3H]-leukotriene distribution, elimination and organ metabolism. Conscious Ma. Bullfrogs (n=8) were anesthetized for surgery with a 0.3% solution of MS-222. A T-cannula (PE-90) was introduced into the sciatic vein and secured as previously described (19) allowing blood flow through the cannula. Following the surgery, muscle and skin Patency of the cannula was layers were closed with suture. maintained by flushing with heparinized (800 IU/ml) amphibian Ringer's solution (NaCl 113.5, KC1 3.5, NaHC03 2.4; CaC12 0.89 mM). Frogs were allowed to regain conciousness and stabilize for 12-24 hours before [3H]-LTC4 infusion. At the beginning of the experiment, the frog was placed into a plastic cage which was lowered into a pan with water to keep the ventral surface of the frog hydrated. A piece of foam was secured on top allowed the frog to move freely sideways but revented vertical movements. containing [p3H]-LTC4 (1.14 The injectate @i/kilogram-body weight (kg-bw)) and unlabelled LTC4 (3000 ng/ kgby evaporating the ethanol under N2 and bw) was prepared reconstituting in 50 pl amphibian Ringers. The injectate was slowly infused (20 seconds) into the sciatic vein, followed by a 400 pl bolus of amphibian Ringer's solution after which the end of the catheter was plugged with clay. To terminate the experiment, the frog was decapitated and pithed. Selected tissues, bladder water and bile from the gall bladder were collected. To protect against further [3H]-LT metabolism and degradation, bladder water and bile were placed into a cold solution of 45 mM serine borate - 10 mM cysteine in amphibian Ringer's solution, pH 7.4 (14) in a ratio of 1:2 and 1:5 respectively. To protect against further peroxidation, 30 pl and 20 ~1 of cold 1 mM 4-OH TEMPO and 0.18 M Na2EDTA aqueous solutions were added to 1 ml urine or 0.1 ml bile. Protected bile and bladder water samples were then stored at -8OOC. To determine if distribution and elimination were affected by a different route of injection, non-cannulated conscious frogs (n=3) were injected with the same dose of [3H]-LTC4/LTC4 into the dorsal
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Prostaglandins
lymph sac rather than intravenously into the sciatic vein. collection was as described above.
Sample
Anesthetized Model Due to the invasive nature of continuously collecting bile during the 2.5 hour period, an anesthetized model was used. Bullfrogs (n = 7) were anesthetized with a 0.3% solution of MS-222. Frogs were then placed on their back upon MS-222-soaked paper towels. MS-222 soaked towels were also placed on all exposed ventral areas, except for the surgical area, to maintain anesthesia. A midline incision was made from the xiphisternum to the lower abdomen to expose the gall bladder and abdominal vein. Saline-filled PE-20 tubing was then inserted into the abdo inal vein and secured in the direction of venous flow. Before [5HI-LTC4/LTC4 infusion, the bile duct was ligated, the gall bladder drained and a 26g syringe needle left pierced into the gall bladder sac. The mid-line incision was partially closed and covered with saline-soaked paper towels to maintain hydration. While anesthetized, a 50 111 bolus of [3H]-LTC4/LTC4 (same dose as the conscious model) followed by a 20 ~1 bolus of saline was infused into the abdominal vein. Every 30 minutes, bile was withdrawn from the gall bladder, the volume measured, and bile samples prepared for scintillation counting. After 2.5 hours, the frog was killed and selected tissues were collected. Tissue Collection &
Counting
Representative portions of tissue/organ ranging from 50 to 200 mg were collected and weighed. Ventricle, brain, urinary bladder, gall bladder, small intestine proximal (at site of bile duct junction), liver, kidneys, lung, spleen, and skeletal muscle (adductor magnus) were collected from both anesthetized and conscious frogs. AdditLonal tissues in the conscious model, small intestine distal (near junction of the large intestine), three lobes of the liver (separated), injection site (muscle tissue around T-cannulae), pectoral and pelvic skin and the eye were also collected. All tissues were minced and then solubilized by adding 1-2 ml Solvable and incubating in a 50°C water bath for 3 hours. Vials were cooled to room temperature and decolorized by adding 30% H202 (200 111). After 1 hour, 10 ml Scintiverse E was added, and the vials kept in the dark for 12-24 hours. All vials were then neutralized with 400 I.I~ 1 N HCl and gently shaken to enhance clarity. One hour Later vials were counted in a scintillation counter. Chemiluminescence and beta quenching were corrected for all samples. m
Homogenization
To characterize the t3H]-LT metabolites found in Liver, the tissue was removed, weighed, minced and placed in a homogenizing tube with 45 mM serine borate-10mM cysteine solution (15 ml), 0.18M The tissue was then Na2EDTA (1 ml) and 1 mM 4-OH-TEMPO (1 ml). The liver homogenized and centrifuged at 6000 x g at LO'C.
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Prostaglandins
supernatent radioactivity
was collected and stored in the supernatent was 40.0
Characterization
of Tritiated
Metabolites
at -8OOC. f 1.8%. Usinn
Recovery
of
RP-HPLC
Prior to RP-HPLC analysis, bile, bladder water and liver samples were extracted through Sep-Pak Cl8 cartridges. The cartridges were primed with 5 ml MeOH followed by 5 ml water, the sample was applied to the cartridge which was then rinsed with 5 ml water and metabolites eluted from the cartridge with 5 ml MeOH (22). The collected MeOH fraction was dried under N2 and reconstituted with a small volume of MeOH. Recovery of radioactivity following this procedure was 75.6 t 4.0%. Separation and characterization of LT metabolites was done on a Nova-Pat Cl8 column (Waters) and a Hewlett Packard 1040M Series II Diode Array Detector. The HPLC eluate was collected in l-minute fractions, 5 ml Scintiverse E was added and the samples were counted. Chemiluminescence and beta-quenching effects were corrected for all samples. Radioactive recovery in HPLC-collected fractions was compared to radioactivity of the injectate and found to be 78.5 + 5.8%. Samples sufficiently high in tritium radioactivity (> than 2000 cpm/sample) were separated by HPLC and detected by radiometric Quantitation of LT detection by Beta-RAM (IN/US Systems, Inc. NJ). radioactivity in tissue, bile and bladder was done by counting of Identification and prepared samples in a scintillation counter. in biological samples separated quantitation of r3H]-LT metabolites by the combined use of through the RP-HPLC system, was accomplished beta-counting of the HPLC l-min fractions, spectral UV analysis, and by radiometric detection. Three different mobile phase systems were used to identify LT metabolites. All three mobile phases were adjusted to pH 5.6 with Mobile phase I (H20:Acetonitrile:MeOH:acetic acid NaOH pellets. (53:26:20:1) at a flow rate of 0.5 ml/min) provided isocratic separation and resolution of the four primary LT metabolites. Typical retention times for LTC4, LTD4, LTE4, N-acetyl LTE4 were 13.8, 23.3, 28.9 and 26.0 minutes respectively. Polar metabolites were not separated and eluted in the first 2 to 6 minutes. Mobile phase II, MeOH:H20:acetic acid (62:37:1) derived from Mathews et al. (23), shifted the retention time of N-acetyl LTE4 with respect to the other primary LTs. Typical peak retention times for LTC4, LTD4, LTE4 and N-acetyl LTE4 were 15.3, 23.4, 28.4 and 21.8 minutes respectively. Polar metabolites eluted in the first 4-8 minutes and were not separated. Mobile phase III was a step-gradient mobile phase developed to separate the tritiated polar metabolite. For the first 19 minutes, the mobile phase was MeOH:H20:acetic acid (48:51:1) at a flow rate of 0.7 ml/min. Peak retention times for 16-carboxytetranordihydro-LTE4, 18-carboxydinor-LTE4, 20-carboxy-LTE4 and 20-hydroxy-LTE4 were 1.6, 2.8, 7.6 and 18.4 minutes respectively. At 19 minutes, the mobile phase was switched to mobile phase II at a flow rate of 0.7 ml/min.
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Prostaglandins
LTC4, LTD4, LTE4 and N-acetyl-LTE4 eluted with retention times of 29.8, 35.5, 38.7 and 34.2 minutes respectively. Validation Studies Validation studies were performed to determine the recovery and reproducibility of the isolation and identification procedures. [3H]LTC4 (lg.500 cpm) and LTD4 (22,000 cpm) were added separately into three different frog bile and bladder water samples containing serine borate-cysteine, 4-OH-TEMPO and EDTA. Samples then underwent freezing, Sep-pak processing, RP-HPLC separation, and counting of fractions as previously described. Radioactive profiles obta ned from these samples were identical to the original [3H]-LTC4 and [4HI-LTD4, indicating high reproducibility of the procedure and no metabolism of the leukotrienes. LTE4 (400 ng/ml) was added to 3 bladder water samples. Samples then also underwent freezing and Sep-Pak processing. Recovery of LTE4 was then determined by HPLC spectral analysis and peak area quantitation with known quantities of standard LTE4. Recovery of LTE4 was 69.0 t 3.0%. No other peaks were detected, indicating that 31 % of the sample was lost in handling and sample processing. These procedures and HPLC analysis also caused no significant metabolism or degradation of LTE4. Statistics Data were analyzed for significance using one way analysis of variance. Groups were compared using Tukeys HSD multiple range tes.4 for significance at P < .05. All data are expressed as mean standard error of the mean (S.E.M). RESULTS Distribution of Tritiated Metabolites The distribution pattern of [3H]-LTC4 and its [3HI-metabolites to specific tissues was examined in anesthetized and conscious frogs. In Figures 1 and 2, the top panel represents a grouping of tissues taken from the hepato-renal systems, and the bottom panel represents other tissues for the venously and dorsally injected conscious frogs, respectively. In Figure 1 (2.5 hr conscious frog injected by vei_), the percent of injected ra+dioactivitywas highest i,"liver (7.5 - 1.5%), The highest small intestine (3.5 - 0.8%) and kidney (1.6 - 0.7%) radioactive density (CPM/mg tissue) was found in gall bladder (16.1 f 4.4). Other tissues with relatively high radioactive density were urinary bladder, kidney, liver, small intestine, and spleen. Data from further orjan analysis in three animals femonstrated that the proximal (4.5 - 1.0) and distal ends (5.9 - 1.31 of the small intestine and the r+ight (3.1 + 1.1). middle (2.6 - 0.7) and left liver lobes (5.1 - 1.6, CPM/mg tissue) were not significantly different indicating organ homogeneity of radioactivity density. Percent of the radioactivity injected and CPM/mg tissue were low (below 0.2% and 0.8 CPM/mg tissue, respectively) for ventricle,
209
Prostaglandins
brain and muscle. Additional tissue samples (not shown in Figures lsite, pectoral and pelvic skin and the 2), taken from the injection eye also yielded low CPM/mg tissue values of 0.6 T 0.2, 2.3 t 1.0, 1.6 t 0.4 and 0.73 f 0.55 respectively. The total sum of in Figure 1, was radioactivity, shown for organs and tissues The total percent approximately 14.5% of the injected radioactivity. of injected radioactivity recovered from skeletal muscle and estimated at 0.05% of the injected radioactivity. (Stripped skeletal muscle tissue constitutes approximately 18% of the frog body weight).
i
I
I 20
Figure 1. Percent of the injected radioactivity measured in counts per minute (CPM) (white bar) and CPM/mg (black bar) for tissues and organs of conscious bullfrog injected with [3H]-LTC4 via the sciatic vein. Results represent mean f S.E.M. for 8 animals. The tissue distribution pattern for the conscious but dorsally injected frogs (Figure 2), was not significantly different from the conscious venously-injected frogs (Figure 1) in pattern and magnitude. Proximal and distal small intestinal segments and the liver lobes were also not significantly different in CPM/mg tissue from each other. The CF'M/mg tissue values we7 also low for injection s:te tissue (1.7 -0.1) and pectoral (0.7 - 0.3) and pelvic skin (0.9 - 0.3) (data not shown). The total sum of radioactivity collected for organs shown in Figure 2 was approximately 13.9% of the injected radioactivity.
210
Prostaglandins
:idney
0
ventricle
amin
Liver
G
Lung
Figure 2. Percent of the injected radioactivity measured in counts per minute (CPM) (white bar) and CPM/mg (black bar) in tissues and organs of conscious bullfrogs injected with [3H]-LTC4 via the dorsal lymph sac. Results represent mean -1 S.E.M. for 3 animals. Anesthetized frogs in general showed similar distribution patterns when compared with the conscious frogs (data not shown). Radioactivity was similarly high in liver, kidney, and proximal small intestine, however liver and kidney contained significantly higher percent of the injectate values (25.5 *4.7 and 9.7 * 2.5 percent of injectate, respectively) than those tissues of conscious frogs. The cumulative radioactivity from all tissues was approximately 41.4% of the injected radioactive dose, and was significantly greater than the sum of all tissues from the conscious frogs. Elimination
of Tritiated
Leukotriene
Metabolftes
Figure 3 illustrates the total amount of leukotriene radioactivity eliminated into urinary bladder, bile and water bathing the ventral surface of both intravenously and dorsally-injected conscious frogs at 2.5 hr after injection. Elimination into bile was significantly lower for dorsally-injected frogs (1.1 * 0.7%) compared with intravenously-injected frogs (7.7 t 1.6% injectate), despite
211
Prostaglandins
identical tissue distribution patterns. The volume of bile collected from the gall bladder was not significantly different between the dorsally (277 * 75 ~1) and venously-injected (540 t 217 ~1) groups. injected Neither the bladder water volume nor the percent radioactivity in bladder water and bath water were different between to be contaminated by groups. Bathing water did not appear frog bladders were full at radioactive fecal pellets. In addition, suggesting that bathing water was the termination of the experiment, not contaminated by urinary contents in these frogs.
14
12
0
Vein
n
Dorsal
10
8
6
I
-1
u. tdaa. water
Batn water
Percent of the injected radioactivity (measured in counts per minute) in urinary bladder water, bile, and pan water of venously injected (white bar, n = 8) and dorsally injected (black bar, n = 3) bullfrogs. Results represent mean t S.E.M. Cumulative elimination of radioactivity into bile in the anesthetized frog was detectable but small (Figure 4). After 2.5 hr, 2.7 t 1.1% of the injected radioactivity was eliminated into the bile. This value was not significantly different from the dorsallyinjected frogs but was significantly less than the venously-injected frogs. Bile production rates were estimated at 28 t 6 pl/hr based on collections every 0.5 hr. Characterization
of
Tritiated
Metabolites
Bile, bladder water, and liver samples from conscious frogs were analyzed using mobile phase I, II and/or III to separate the tritiated LTC4 metabolites. Neither sufficient bile nor urine were obtained from the anesthetized frogs to characterize these metabolites.
212
Prostaglandins
4 3 Time (hrs) Figure 4. Cumulative elimination of radioactivity (percent of injected radioactivity in CPM) into bile of anesthetized frogs for Results represent mean * 2.5 hr following injection of [3H]-LTC4. S.E.M. for 7 animals. Figure 5 illustrates a large radioactive peak of polar metabolites as well as a peak with a retention time coinciding with the LTE4 radiometric detection. standard in bladder water using (Mobile phase II did not resolve the individual polar metabolites). Figure 6 shows the distribution of radioactivity collected in l-min fractions of a representative bile sample (Mobile phase III). Identification of leukotriene metabolites coeluting with leukotriene standard retention times are indicated above the histogram. Figure 7 summarizes the identity and relative quantities of [3H]-LTC4 metabolites in bile, liver, and bladder water samples for venously-injected conscious frogs analyzed usin Mobile phases I-III. 93 Less than 5% of the radioactivity remained as [ HI-LTC, after 2.5 hr in all samples. The absence of radioactivity coeluting with N-acetyl LTE4 and 16-carboxytetranordihydro-LTE4 suggested that these metabolites were not present. Liver had the ability to convert [3H]-LTD4 to [3H]-LTE4 and to further oxidize [3H]-LTE4 into the polar metabolites 18-carboxydinorand 20-hydroxy-LTE4, Bile had the same 20-carboxy-LTE4 LTE4, composition and quantity of i3H]-LT metabolites as liver. For bile, liver and bladder water, the sum of the radioactivity composed of the coyprised approximately 50% of the total three polar metabolites sample radioactivity (46.8 - 2.1, 47.8 * 12.7, and 51.4 t 6.16 %,
Prostaglandins
213
respectively). The quantity of 20-carboxy-LTEq was significantly higher than 20-hydroxy-LTEq and 18-carboxydinor-LTEq in bile, but was not significantly different from 20-hydroxy-LTE4 and 18-carboxydinorLTE4 quantities in liver and bladder water. In contrast to liver and bile, bladder water contained more [3H]-LTE4 than [3H]-LTD,.
0
10
5
15
20
25
30
35
40
Time (min)
Figure 5. Tracing from Beta-Ram detector of bladder water sample using Mobile phase II described in Methods. Polar metabolites (PM) were unresolved in this mobile phase and eluted early with the first radioactive peak. The second peak of radioactivity co-eluted with the LTE,+standard retention time.
350-1
I
II
Time (min)
Figure 6. Radioactive fractions collected from bile sample after RPHPLC separation and quantitated by scintillation counting. Mobile phase III as described in Methods was used for the separation. Radioactivity recovered in fractions comigrated with 18-carboxydinorLTE,+ (I), 20-carboxy-LTEq (II), 20-hydroxy-LTEq (III), LTD&, and LTEl, standard retention times.
Prostaglandins
214
n Bile
II
LTC 4
0
Liver
N
Bladder Water
LTD,
Figure 7. Percent of the injected radioactivity (black bar), liver (white bar) and bladder water 2.5 hours post-injection for venously-injected Extraction and analysis by RP-HPLC using Mobile described in Methods. Polar metabolites I, II, carboxydinor-LTE4. 20-carboxy-LTE4, and respectively.
LTE 4 recovered in bile (stripped bar) at conscious frogs. phase III was as and III were 18PO-hydroxy-LTE4,
DISCUSSION with mammals in The bullfrog shares some similarities leukotriene distribution, elimination, and metabolism. Distribution studies in mice using autoradiography found that radioactivity was highly distributed to bile, liver and kidney (5). It is difficult to compare tissue radioactivities directly, but the relative magnitudes between mouse and bullfrog were similar. The conscious and the anesthetized frogs showed similar tissue distribution patterns although the magnitude of radioactivity found in tissues was altered by anesthesia. Whether this phenomenon also occurs in mammals is unknown, but may be an important aspect because most studies of leukotriene metabolism use anesthetized animals. The route of administration affected elimination as evidenced by different biliary eliminations in venously versus dorsally injected frogs. In mice, intravenous versus intramuscular injection of leukotriene C,, did not make a difference in tissue distribution (5), but few other studies have compared routes of injection. The importance of renal elimination of leukotrienes in the bullfrog is still unclear. From Figures 1 and 2 it is clear that
Prostaglandins
215
distributed to the kidney in conscious radioactivity was highly frogs. Repeated attempts at collecting urine by cannulating the ureter ducts yielded very small (0 - 100 nl/hr) volumes of urine in recovered in the both anesthetized and conscious frogs. Radioactivity collected ureteral urine was negligible even up to 6 hr after [3H]urine collected in LTC4 injection in one animal. In Bufo marinus, this manner accounted for l/3 of the toad's body weight in 24 hr and contained nearly 55% of the injected radioactivity (24). The lack of urine output in frogs may help explain recovery of significant amounts of radioactivity in bladder water (figure 3). For both dorsally and venously-injected conscious frogs, the LTs eliminated into the bladder water was not significantly different. In frogs, the urinary bladder serves as a transportive membrane for electrolyte and water balance, in addition to its urine storage role. Ureters from the kidney empty into the cloaca and urine is subsequently transferred to the bladder for storage. The bladder can attain additional fluids by diffusion from extracellular fluid and from the cloaca1 contents. Thus, radioactive metabolites in the bladder water may not be exclusively of renal system origin. Whatever the origin of radioactivity, bladder water appears to be a major reservoir of leukotriene elimination. Elimination of [3H]-LTs into bile of conscious frogs was dependent upon the route of injection (Figure 3). Despite identical distribution of tritiated leukotrienes to the liver (Figures 1 and the dorsally-injected frog livers eliminated significantly less 21' [ HI-LTs into bile than the intravenously injected frogs. While final liver radioactivity at 2.5 hr was the same for both frog groups, radioactivity aquisition by the liver and subsequently into bile may have been slower during the 2.5 hr for the dorsally-injected frogs, probably because of more gradual infiltration of radioactivity into the circulation compared with direct intraveous injection. These results are similar to studies in Bufo marinus in which minimal amounts of radioactivity were found in bile (24). The anesthetized frog study was performed primarily to obtain data on continuous biliary elimination of [3H]-LTs (Figure 4). These data showed that biliary elimination of [3H]-LT~ by the anesthetized frog was slow, constant, and initiated within the first 30 minutes after [3H]-LTC4 injection. The total amount of LT radioactivity recovered after 2.5 hr was the same as for the dorsally-injected conscious Irogs, but significantly less than that obtained for the intravenously-injected conscious frogs. The route of injection for the anesthetized frogs was via the abdominal vein which first leads directly into the hepatic portal system and then to the heart. One might expect excretion into bile, therefore, to be greater, but this was not the case. However, biliary elimination may also have been slowed by the anesthetic, MS-222. Despite the differences between models, the biliary elimination of LTs by conscious and anesthetized frogs was far less after 2.5 hrs than most anesthetized mammals, including dog (10). domestic pig (11). monkey (9) and rat (17) which all eliminate primarily into bile. In rat, 28% and 50% of the radioactivity was eliminated in bile at 30 and 90 min following the intravenous injection of [3~]-~T~4 (17).
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Prostaglandins
The decreased biliary elimination of LTs found for the bullfrog may be correlated with its lower bile flow when compared with the anesthetized dog and rat. Bile flow in the anesthetized frog was estimated to be 99 ul/hr-kg, and was less than for the dog (336 Pl/hr-kg (10) and much less than for rat (4980 pl/hr-kg (17). Because it is an amphibian, the slower bile production in the bullfrog may be due to lower metabolic rate and/or decreased perfusion of the liver. Likewise, while extraction of [3H]-LTs out of the circulation by liver appears to be efficient because the liver contained the highest percent of injected radioactivity (figures 1 and 2), the elimination of [3H]-LTs into the bile may be slower. In mammals, export of peptide-LTs into the bile appears to be energy dependent and a ratedetermining step in biliary elimination (25). Radioactivity found in the bath water may be from the transfer and elimination of injected LT radioactivity across the skin and into the external environment. This unique transport also found in Bufo marinus (24) and may be another important elimination route in amphibians in which the skin functions as a transport medium. Like many mammals, but unlike rat and mouse (5,13,16,26), bullfrogs appear to be unable to N-acetylate LTE4 further to N-acetyl LTE4. N-acetyl LTE4 was not found in bladder water, bile and liver samples. Unlike its circulatory system (19). the bullfrog renal system appears to have dipeptidase causing the further conversion of LTD4 to LTE4. Significant dipeptidase activity has been reported in mammalian kidney and liver (4,26-28). The absence of dipeptidase within the vascular system is unique to the bullfrog and has not been reported in mammalian studies. Leukotriene C4 is essentially deactivated when converted to LTD4 in the bullfrog as LTD4 and LTE4 have little hemodynamic activity (18.19). In mammals, both LTC4 and and metabolic LTD4 have potent cardiovascular activity (29) deactivation does not occur until the less active LTE4 and N-acetyl LTE4 are formed. Dipeptidase activity in the bullfrog may be greater in the renal system than in the hepatic system, because of significantly greater found in bladder water compared with bile and liver. LTE4 amounts Sites other than renal cells, such as the cloaca1 surfaces and urinary bladder epithelium may have contributed to the formation of metabolizing [3H]-LT metabolites found in bladder water. Indeed, enzymes, particularly gamma-glutamyl transpeptidase and dipeptidase, may also be found in bullfrog urine, metabolism was however, prevented under the conditions of this study. These enzymes have been reported in human urine ex vivo (30). Identical metabolite compositions found in liver and bile (figure 7) suggest that the liver extracts [3H]-LTD4 from the blood which is then converted LTE4 and its omega and beta-oxidation products 20-hydroxy-LTE4, 20-carboxy-LTE4 and 18-carboxydinor-LTE4. Extensive liver These metabolites were then excreted into the bile. metabolism of leukotrienes and excretion of these metabolites have been reported in mammals (9,14,17,31-33). Omega and beta-oxidation products were also found in bullfrog bladder water (Figure 7). Oxidation products of LTE4 and N-acetyl LTE4 have been reported for
217
Prostaglandins
mammalian kidney (17) Beta oxidation in the bullfrog may be slower than in mammals. After 2.5 hrs, only 18-carboxydinor-LTE4, the first beta oxidation product, was detected, with no signs of 16carboxytetranordihydro-LTE4. In monkey, 16_carboxytetranordihydroLTE4 was already a major metabolite found in bile and urine by 2 hr after the injection of tritiated LTC4 (9). The bullfrog is an interesting model for the study of leukotriene distribution, elimination, and metabolism. Despite major differences in the amphibian physiological organ systems, the distribution of radioactivity was similar to mammals. Like mammals, the frog eliminates in both urine and bile, however, there appears to be an additional unique route of elimination th ough the skin. Overall, with slight variations, metabolism of [ 5HI-LTC4 in the pathways and produce bullfrog appears to follow the same enzymatic the same metabolites as its mammalian counterparts, suggesting that leukotriene processing has been conserved over evolutionary time. ACKNOWLEDGMENTS This research was supported by National Science Foundation Grant DCB-9018542 to C.A.H and D.D.G. was supported by the NSF Research Experiences for Undergraduates program. The authors would like to thank Bonnie Valentich and Susan Sandoval for excellent technical assistance and Dr. Peter Herman for critical reading of the manuscript. REFERENCES 1.
Samuelsson, Leukotrienes
B., S. Hammarstrom, R.C. Murphy, and and SRS-A. Allergy (Copenhagen) 35:375.
2.
Needleman, Arachidonic
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Green, F.A. -In viva generation of lipoxygenase products and toads. Biochem. Biophys. Res. Commun 148:1533. 1987.
4.
Hammarstrom, S. Metabolism of leukotriene J. Biol. Chem. 256:9573. 1981.
5.
Applzgren, L.E. and Hammarstrom, H-labeled leukotriene C3 of 257:531. 1982.
6.
Hammarstrom, leukotrienes:
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Denzlinger, Hammer, and leukotrienes
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Tagari, P.. A. Foster, D. Delorme, Y. Metabolism and excretion of exogenous 1989. Prostaglandins x:629.
P., J. Turk, B.A. Jakschik, and acid metabolism. Annu. Rev. Biochem.
S., L. Review.
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P. Borgeat. 1980.
A.R. =:69.
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Morrison. 1986. in frogs
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S. Distribution and metabolism in the mouse. J. Biol. Chem.
Orning, and K. Bernstrom. Metabolism Mol. Cell. Biochem. 69:7. 1985.
of
C., A. Guhlmann, P.H. Scheuber, D. Wilker, D. Metabolism and analysis of cysteinyl D. Keppler. in the monkey. J. Biol. Chem. 2:15601. 1986. G$,rard, and J. Rokach. [ HI-LTC4 in primates.
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9.
Huber, M., J. Muller, I. Leier, G. Jedlitschky, H.A. Ball, K.P. Moore, G.W. Taylor, R. Williams, and D. Keppler. Metabolism of cysteinyl leukotrienes in monkey and man. Eur. .I. Biochem. -:309. 1990.
10.
Pfeifer, C.A., G.D. Bottoms, M.A. Johnson, and J. Fessler, J. Leukotriene C4 disposition and metabolism in the anesthetized and endotoxemic dog. Circ. Shock. 33~68. 1991
11.
Ezra, D., A. Foster, M. Cirano, J. Rokach, and L.G. Letts. Biliary and urinary excretion of peptide leukotrienes in the domestic pig. Prostaglandins 33:717. 1987.
12. Foster, A. Dagenais, F. Letts, G. and Rokach, J. Metabolism of leukotriene C4 in the anesthetized guinea pig. Prostaglandins, Leukotrienes Essential Fatty Acids x:93. 1991. 13. Hammarstrom, S., L.E. Oming, K. Bernstrom. B. Gustafsson, E. Norin, and L. Kaijser. Metabolism of leukotriene C4 in rats and humans. In: Advances in Prostaglandin, Thromboxane and Leukotriene Research (0. Hayaishi and S. Yamamoto, eds). Raven Press, New York, 1985. p. 185. 14. Foster, A., B. Fitssimmons, J. Rokach, and G. Letts. Evidence of in vivo w- oxidation of peptide leukotrienes in the rat: Biliary excretion of 20-C02H N-acetyl LTE4. Biochem. Biophys. Res. Commun. =:1237. 1987. 15. Orning, L.E., L. Kaijser, and S. Hammarstrom. In vivo metabolism of leukotriene C4 in man: Urinary excretion of leukotriene E4. Biochem. Biophys. Res. Commun. 130:214. 1985. 16. Hammarstrom, S., L. Orning, and K. Bernstrom. Metabolism and excretion of cysteinyl-leukotrienes. Advances in Prostaglandin, Thromboxane, and Leukotriene Research ed. by U. Zor et al. Raven Press, N.Y. pp 383. 1986. 17. Hammarstrom, S., L. Orning, and A. Keppler. Metabolism of cysteinyl leukotrienes to novel polar metabolites in the rat and endogenous formation of leukotriene D4 during systemic anaphylaxis in the guinea pig. Ann. N.Y. Acad. Sci. s:43. 1988. 18. Herman, C.A., D.O. Robleto, S.T. Reitmeyer, L.A. Martinez, and R.P. Herman. Cardiovascular effects of leukotrienes in the bullfrog. Biomed. Biochim. Acta u:S178. 1988. 19
Herman, C.A., G.A. Charlton, and R.L. Cranfill. Metabolism and cardiovascular effects of leukotrienes in warm- and coldacclimated American bullfrogs (w catesbeiana). Am. J. Physiol. m:~834. 1991.
20
Herman, R.P., R.S. Heller, C.M. Canavan, and C.A. Herman. Leukotriene C4 action and metabolism in the isolated, perfused bullfrog heart. Can. J. Physiol. Pharmacol. 6&:980. 1988.
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21.
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Heller, R.S., R.P. Herman, and metabolism of peptide leukotrienes Can. J. Physiol. Pharmacol. a:309.
S.A. and 22. Galton, bioconversion of %:617. 1987.
P.J. Piper. leukotrienes
C,A. Herman. The action and in the isolated bullfrog lung. 1989.
The role in the
23. Mathews, W. J. Rokach, and R.C. Murphy. by high-pressure liquid chromatography. 1981. 24.
of blood vessels in the pig. Br. J. Pharmacol.
Analysis of leukotrienes Anal. Biochem. -:96.
Pfeifer, C.A., R.A. Furilla, K. Gronert, D.D. Goss, and C.A. Herman. Tissue distribution, elimination, and metabolism of [3H]leukotriene C4 in the marine toad, Bufo marinus. Can. J. Physiol. Pharmacol. in nress 1992.
25. Keppler, D., A. Guhlmann, F. Oberdorfer, K. Krauss, J. Muller, H. Ostertag, and M. Huber. Generation and metabolism of cysteinyl leukotrienes u m. in the Understanding and In Advances Treatment of Asthma, Vol. 629 of the Annals of the New York Academy of Science 1991. p 100. 26. Fauler, Frolich. perfused
J. A. Wiemeyer, M. Yoshisawa, H.J. Schurek, and J.C. Metabolism of cysteinyl leukotrienes by the isolated rat kidney. Prostaglandins Q:239. 1991
and S. 27. Ormstad, K., N. Uehara. S. Orrenius, L. Orning, Hammarstrom. Uptake and metabolism of leukotriene C3 in isolated rat organs and cells. Biochem. Biophys. Res. Commun. 104:1434. 1982. 28. Bernstrom, K. and S. Hammarstrom. Metabolism of leukotriene porcine kidney. J. Biol. Chem. =:9579. 1981. 29. Feuerstein, G. Prostaglandins.
Leukotrienes =:7al. 1984.
and
the
cardiovascular
D4 by
system.
30. Beyer, G., C.O. Meese, and U. Klotz. Stability of leukotrienes C4 and D4 in human urine. Prostaglandins, Leukotrienes, and Medicine. 2:229. 1987. 31. Keppler, D., M. Huber, G. Weckbecker, W. Hagmann, C. Denzlinger, and A. Guhlmann. Leukotriene C4 metabolism by hepatoma cells and liver. Adv. Enzyme Regulation z:211. 1987. 32. Delorme, D., A. Foster, Y. Girard, and J. Rokach. Synthesis of Boxidation products as potential leukotriene metabolites and their detection in the bile of anesthetized rat. Prostaglandins x:291. 1988. 33. Orning, L. w - Hydroxylation of N-acetylleukotriene E4 liver microsomes. Biochem. Biophys. Res. Commun. 143:337. Editor:
R.
Krell
Received:
8-19-92
Accepted:
by rat 1987. 12-2-92
TISSUB DISTRIBUTION, ELIMINATION AND METABOLISM OF r3H] LEUKOTRIIZNE C4 IN THE AMERICAN BULLFROG, RANA CATBSBBIANA
C.A. Pfeifer, R.A. Furllla, K. Gronert, D.D. Goss X.B. Romig and C.A. Herman Department
of Biology, New Mexico State University Las Cruces, NM 89003
ABSTRACT Tissue distribution, elimination, and metabolism of r3H]leukotriene C4 were studied at 2.5 hours after injection in the conscious and anesthetized American bullfrog, Rana catesbeiana. Conscious frogs were injected via the dorsal lymph sac or the sciatic vein. Anesthetized frogs were injected via the abdominal vein. The organs containing the greatest percent of injected radioactivity at 2.5 hours after injection were liver, small intestine and kidney. Route of injection and anesthe ia appears to alter distribution and elimination of leukotrienes. [JHI-leukotrienes were eliminated into bladder water and bile. In addition, 7.8 f 2.2 and 5.2 % 2.5 percent of the injected radioactivity was found in the pan vater bathing the ventral surface of the venously and dorsally injected conscious frogs, respectively, suggesting transfer of radioactivity across the skin. At 2.5 hours, polar metabolites represented 50% of the radioactivity found in liver, bile, and bladder water. These polar metabolites were determined to be la-carboxy-19,20-dinor-leukotriene E4, 20-carboxy-leukotriene E4, and 20-hydroxy-leukotriene E4. Of the non-oxidized leukotrienes, bile contained mainly LTD4 while bladder water contained primarily LTE4. N-acetyl LTE4 was not detected in any samples. The tissue distribution, elimination and metabolism of leukotrienes in the bullfrog was similar to mammalian studies and suggests evolutionary conservation of leukotriene processing. INTRODUCTION The peptido-leukotrienes, LTC4, and its metabolites LTD4 and LTE4 are hormones derived from arachidonic acid and can cause potent hemodynamic and inflammatory effects (l-2). For many animal species, leukotrienes appear to play major roles in health and disease. A role for leukotrienes (LTs) in amphibia has been indicated by the endogenous production of LTs in inflammatory cells of several anuran amphibian species, including Rana catesbeiana in response to infection (3). Once formed, distribution, elimination, and metabolism of leukotrienes differs among animal species. Most studies have been performed in mammals (4-17, 25-33). Two studies have demonstrated that tritiated LTC4 and its metabolites were largely distributed to the liver and kidney in both the guinea pig (4) and mouse (5). respectively. Circulating peptidoleukotrienes are metabolized into LTD4 by gamma-glutamyl transpeptidase, and further to LTE4 by dipeptidase in mammalian blood. They are rapidly removed from the circulation by the hepatic and renal systems into bile and urine
Copyright
0 1993 Butterworth-Heinemann
204
Prostaglandins
respectively (6). Monkey (7-g). dog (lo), domestic pig (ll), guinea pig (4.12) rat (13,14) and mouse (5) eliminate leukotrienes primarily via biliary excretion, while humans (9,13,15) eliminate LTs into bile and urine. LTE4 is the primary non-oxidized LT excreted in urine in most species but rats and mice further metabolize LTE4 to N-acetyl LTE4 (6,16,17). Little is known about how anuran amphibians distribute, in vitro and in vivo eliminate and metabolize leukotrienes. P evious -5 studi s in this laboratory have shown [ HI-LTC4 is metabolized only to [sHI-LTD4 by whole blood b vitro and when injected into cannulated bullfrogs (18,19). However mince3d bullfrog tissues inclu$ing heart (20) and lung (21) metab lized [ HI-LTC4 to [3H]-LTD4 and [ HI-LTE4. Despite the fact that [sHI-LTC4 was rapidly removed from the bullfrog circulation (19), the excretion and identity of LTC4 metabolites in bile and urine of amphibians in general is unknown. There are several distinct anatomical differences between amphibians and mammals which may alter the way these two classes process leukotrienes. Bullfrogs are poikilothermic, have a three chambered heart without coronary arteries and have renal and hepatic portal systems. Frog kidney tubules lack a loop of Henle and are unable to concentrate urine. The frog urinary bladder is important in water and electrolyte transport. The skin may also play a role in transport and elimination in these aquatic animals. All of these physiological differences may alter the way amphibians, such as bullfrogs, process leukotrienes. This study will characterize the tissue distribution and elimination of [3H]-LTC4 and its metabolites in the bullfrog. Because amphibians represent an older phylogenetic group than mammals, this study may provide insights into whether LT processing mechanisms have been evolutionarily conserved. METHODS Leukotriene (LT)C4, LTD4, LTE4, and N-acetyl LTE4 were generous gifts from The Merck Frosst Centre for Therapeutic Research (Pointe Claire-Dorval, Quebec, Canada) and were stored at -80° in ethanol (20 of LTE4, 16-carboxyUg/ml). The oxidized polar metabolites 17,18,19,20-tetranor-14,15-dihydro-LTE4 (16-carboxytetranordihydro(la-carboxydinor_LTE4), 20LTE4), 18-carboxy-19,20-dinor-LTE4 were purchased from Oxford carboxy-LTE4, and 20-hydroxy-LTE4 Biomedical Research, Inc. (Oxford, MI) and stored at -2O'C in ethanol at a concentration of 14.3 vg/ml. Tritiated leukotrienes [14* 15 (n)-3H]-LTC4, $53.4 Ci/mmol, [3H]-LTC4) and [14,-15(n)-3H]-LTD4 (39.3 Ci/mmol, [ HI-LTD4) were purchased from Amersham (Arlington Heights, IL) and New England Nuclear (Boston, MA), respectively. Ethyl-m-aminobenzoate (MS-222) and disodium salt-ethylenediamine tetraacetic acid (Na2EDTA) were obtained from Sigma Chemical Co. (St. Louis, MO) and tissue solubilizing solution, Solvable, was purchased from New England Nuclear (Boston, MA). Scintiverse E and high performance liquid
Prostaglandins
205
Nuclear (Boston, MA). Scintiverse E and high performance liquid chromatography (HPLC) grade methanol and acetonitrile were purchased from Fisher Scientific (Dallas, TX). Sep-Pak (Cl8) extraction cartridges were purchased from Millipore Corp. (Milford, MA) and the free radical scavenger, 4-OH-TEMPO was purchased from Aldrich Chemical Co. (Milwaukee, WI). Animals Bullfrogs of both sexes, 284 * 18 grams (n = 18) were collected from ponds in Dona Ana County or purchased from Charles Sullivan Co., Nashville, TN. Frogs were force-fed beef liver three times weekly and housed at room temperature in a large plastic tank with constant running water. Two animal models, conscious and anesthetized, were used in this study to characterize [3H]-leukotriene distribution, elimination and organ metabolism. Conscious Ma. Bullfrogs (n=8) were anesthetized for surgery with a 0.3% solution of MS-222. A T-cannula (PE-90) was introduced into the sciatic vein and secured as previously described (19) allowing blood flow through the cannula. Following the surgery, muscle and skin Patency of the cannula was layers were closed with suture. maintained by flushing with heparinized (800 IU/ml) amphibian Ringer's solution (NaCl 113.5, KC1 3.5, NaHC03 2.4; CaC12 0.89 mM). Frogs were allowed to regain conciousness and stabilize for 12-24 hours before [3H]-LTC4 infusion. At the beginning of the experiment, the frog was placed into a plastic cage which was lowered into a pan with water to keep the ventral surface of the frog hydrated. A piece of foam was secured on top allowed the frog to move freely sideways but revented vertical movements. containing [p3H]-LTC4 (1.14 The injectate @i/kilogram-body weight (kg-bw)) and unlabelled LTC4 (3000 ng/ kgby evaporating the ethanol under N2 and bw) was prepared reconstituting in 50 pl amphibian Ringers. The injectate was slowly infused (20 seconds) into the sciatic vein, followed by a 400 pl bolus of amphibian Ringer's solution after which the end of the catheter was plugged with clay. To terminate the experiment, the frog was decapitated and pithed. Selected tissues, bladder water and bile from the gall bladder were collected. To protect against further [3H]-LT metabolism and degradation, bladder water and bile were placed into a cold solution of 45 mM serine borate - 10 mM cysteine in amphibian Ringer's solution, pH 7.4 (14) in a ratio of 1:2 and 1:5 respectively. To protect against further peroxidation, 30 pl and 20 ~1 of cold 1 mM 4-OH TEMPO and 0.18 M Na2EDTA aqueous solutions were added to 1 ml urine or 0.1 ml bile. Protected bile and bladder water samples were then stored at -8OOC. To determine if distribution and elimination were affected by a different route of injection, non-cannulated conscious frogs (n=3) were injected with the same dose of [3H]-LTC4/LTC4 into the dorsal
206
Prostaglandins
lymph sac rather than intravenously into the sciatic vein. collection was as described above.
Sample
Anesthetized Model Due to the invasive nature of continuously collecting bile during the 2.5 hour period, an anesthetized model was used. Bullfrogs (n = 7) were anesthetized with a 0.3% solution of MS-222. Frogs were then placed on their back upon MS-222-soaked paper towels. MS-222 soaked towels were also placed on all exposed ventral areas, except for the surgical area, to maintain anesthesia. A midline incision was made from the xiphisternum to the lower abdomen to expose the gall bladder and abdominal vein. Saline-filled PE-20 tubing was then inserted into the abdo inal vein and secured in the direction of venous flow. Before [5HI-LTC4/LTC4 infusion, the bile duct was ligated, the gall bladder drained and a 26g syringe needle left pierced into the gall bladder sac. The mid-line incision was partially closed and covered with saline-soaked paper towels to maintain hydration. While anesthetized, a 50 111 bolus of [3H]-LTC4/LTC4 (same dose as the conscious model) followed by a 20 ~1 bolus of saline was infused into the abdominal vein. Every 30 minutes, bile was withdrawn from the gall bladder, the volume measured, and bile samples prepared for scintillation counting. After 2.5 hours, the frog was killed and selected tissues were collected. Tissue Collection &
Counting
Representative portions of tissue/organ ranging from 50 to 200 mg were collected and weighed. Ventricle, brain, urinary bladder, gall bladder, small intestine proximal (at site of bile duct junction), liver, kidneys, lung, spleen, and skeletal muscle (adductor magnus) were collected from both anesthetized and conscious frogs. AdditLonal tissues in the conscious model, small intestine distal (near junction of the large intestine), three lobes of the liver (separated), injection site (muscle tissue around T-cannulae), pectoral and pelvic skin and the eye were also collected. All tissues were minced and then solubilized by adding 1-2 ml Solvable and incubating in a 50°C water bath for 3 hours. Vials were cooled to room temperature and decolorized by adding 30% H202 (200 111). After 1 hour, 10 ml Scintiverse E was added, and the vials kept in the dark for 12-24 hours. All vials were then neutralized with 400 I.I~ 1 N HCl and gently shaken to enhance clarity. One hour Later vials were counted in a scintillation counter. Chemiluminescence and beta quenching were corrected for all samples. m
Homogenization
To characterize the t3H]-LT metabolites found in Liver, the tissue was removed, weighed, minced and placed in a homogenizing tube with 45 mM serine borate-10mM cysteine solution (15 ml), 0.18M The tissue was then Na2EDTA (1 ml) and 1 mM 4-OH-TEMPO (1 ml). The liver homogenized and centrifuged at 6000 x g at LO'C.
207
Prostaglandins
supernatent radioactivity
was collected and stored in the supernatent was 40.0
Characterization
of Tritiated
Metabolites
at -8OOC. f 1.8%. Usinn
Recovery
of
RP-HPLC
Prior to RP-HPLC analysis, bile, bladder water and liver samples were extracted through Sep-Pak Cl8 cartridges. The cartridges were primed with 5 ml MeOH followed by 5 ml water, the sample was applied to the cartridge which was then rinsed with 5 ml water and metabolites eluted from the cartridge with 5 ml MeOH (22). The collected MeOH fraction was dried under N2 and reconstituted with a small volume of MeOH. Recovery of radioactivity following this procedure was 75.6 t 4.0%. Separation and characterization of LT metabolites was done on a Nova-Pat Cl8 column (Waters) and a Hewlett Packard 1040M Series II Diode Array Detector. The HPLC eluate was collected in l-minute fractions, 5 ml Scintiverse E was added and the samples were counted. Chemiluminescence and beta-quenching effects were corrected for all samples. Radioactive recovery in HPLC-collected fractions was compared to radioactivity of the injectate and found to be 78.5 + 5.8%. Samples sufficiently high in tritium radioactivity (> than 2000 cpm/sample) were separated by HPLC and detected by radiometric Quantitation of LT detection by Beta-RAM (IN/US Systems, Inc. NJ). radioactivity in tissue, bile and bladder was done by counting of Identification and prepared samples in a scintillation counter. in biological samples separated quantitation of r3H]-LT metabolites by the combined use of through the RP-HPLC system, was accomplished beta-counting of the HPLC l-min fractions, spectral UV analysis, and by radiometric detection. Three different mobile phase systems were used to identify LT metabolites. All three mobile phases were adjusted to pH 5.6 with Mobile phase I (H20:Acetonitrile:MeOH:acetic acid NaOH pellets. (53:26:20:1) at a flow rate of 0.5 ml/min) provided isocratic separation and resolution of the four primary LT metabolites. Typical retention times for LTC4, LTD4, LTE4, N-acetyl LTE4 were 13.8, 23.3, 28.9 and 26.0 minutes respectively. Polar metabolites were not separated and eluted in the first 2 to 6 minutes. Mobile phase II, MeOH:H20:acetic acid (62:37:1) derived from Mathews et al. (23), shifted the retention time of N-acetyl LTE4 with respect to the other primary LTs. Typical peak retention times for LTC4, LTD4, LTE4 and N-acetyl LTE4 were 15.3, 23.4, 28.4 and 21.8 minutes respectively. Polar metabolites eluted in the first 4-8 minutes and were not separated. Mobile phase III was a step-gradient mobile phase developed to separate the tritiated polar metabolite. For the first 19 minutes, the mobile phase was MeOH:H20:acetic acid (48:51:1) at a flow rate of 0.7 ml/min. Peak retention times for 16-carboxytetranordihydro-LTE4, 18-carboxydinor-LTE4, 20-carboxy-LTE4 and 20-hydroxy-LTE4 were 1.6, 2.8, 7.6 and 18.4 minutes respectively. At 19 minutes, the mobile phase was switched to mobile phase II at a flow rate of 0.7 ml/min.
208
Prostaglandins
LTC4, LTD4, LTE4 and N-acetyl-LTE4 eluted with retention times of 29.8, 35.5, 38.7 and 34.2 minutes respectively. Validation Studies Validation studies were performed to determine the recovery and reproducibility of the isolation and identification procedures. [3H]LTC4 (lg.500 cpm) and LTD4 (22,000 cpm) were added separately into three different frog bile and bladder water samples containing serine borate-cysteine, 4-OH-TEMPO and EDTA. Samples then underwent freezing, Sep-pak processing, RP-HPLC separation, and counting of fractions as previously described. Radioactive profiles obta ned from these samples were identical to the original [3H]-LTC4 and [4HI-LTD4, indicating high reproducibility of the procedure and no metabolism of the leukotrienes. LTE4 (400 ng/ml) was added to 3 bladder water samples. Samples then also underwent freezing and Sep-Pak processing. Recovery of LTE4 was then determined by HPLC spectral analysis and peak area quantitation with known quantities of standard LTE4. Recovery of LTE4 was 69.0 t 3.0%. No other peaks were detected, indicating that 31 % of the sample was lost in handling and sample processing. These procedures and HPLC analysis also caused no significant metabolism or degradation of LTE4. Statistics Data were analyzed for significance using one way analysis of variance. Groups were compared using Tukeys HSD multiple range tes.4 for significance at P < .05. All data are expressed as mean standard error of the mean (S.E.M). RESULTS Distribution of Tritiated Metabolites The distribution pattern of [3H]-LTC4 and its [3HI-metabolites to specific tissues was examined in anesthetized and conscious frogs. In Figures 1 and 2, the top panel represents a grouping of tissues taken from the hepato-renal systems, and the bottom panel represents other tissues for the venously and dorsally injected conscious frogs, respectively. In Figure 1 (2.5 hr conscious frog injected by vei_), the percent of injected ra+dioactivitywas highest i,"liver (7.5 - 1.5%), The highest small intestine (3.5 - 0.8%) and kidney (1.6 - 0.7%) radioactive density (CPM/mg tissue) was found in gall bladder (16.1 f 4.4). Other tissues with relatively high radioactive density were urinary bladder, kidney, liver, small intestine, and spleen. Data from further orjan analysis in three animals femonstrated that the proximal (4.5 - 1.0) and distal ends (5.9 - 1.31 of the small intestine and the r+ight (3.1 + 1.1). middle (2.6 - 0.7) and left liver lobes (5.1 - 1.6, CPM/mg tissue) were not significantly different indicating organ homogeneity of radioactivity density. Percent of the radioactivity injected and CPM/mg tissue were low (below 0.2% and 0.8 CPM/mg tissue, respectively) for ventricle,
209
Prostaglandins
brain and muscle. Additional tissue samples (not shown in Figures lsite, pectoral and pelvic skin and the 2), taken from the injection eye also yielded low CPM/mg tissue values of 0.6 T 0.2, 2.3 t 1.0, 1.6 t 0.4 and 0.73 f 0.55 respectively. The total sum of in Figure 1, was radioactivity, shown for organs and tissues The total percent approximately 14.5% of the injected radioactivity. of injected radioactivity recovered from skeletal muscle and estimated at 0.05% of the injected radioactivity. (Stripped skeletal muscle tissue constitutes approximately 18% of the frog body weight).
i
I
I 20
Figure 1. Percent of the injected radioactivity measured in counts per minute (CPM) (white bar) and CPM/mg (black bar) for tissues and organs of conscious bullfrog injected with [3H]-LTC4 via the sciatic vein. Results represent mean f S.E.M. for 8 animals. The tissue distribution pattern for the conscious but dorsally injected frogs (Figure 2), was not significantly different from the conscious venously-injected frogs (Figure 1) in pattern and magnitude. Proximal and distal small intestinal segments and the liver lobes were also not significantly different in CPM/mg tissue from each other. The CF'M/mg tissue values we7 also low for injection s:te tissue (1.7 -0.1) and pectoral (0.7 - 0.3) and pelvic skin (0.9 - 0.3) (data not shown). The total sum of radioactivity collected for organs shown in Figure 2 was approximately 13.9% of the injected radioactivity.
210
Prostaglandins
:idney
0
ventricle
amin
Liver
G
Lung
Figure 2. Percent of the injected radioactivity measured in counts per minute (CPM) (white bar) and CPM/mg (black bar) in tissues and organs of conscious bullfrogs injected with [3H]-LTC4 via the dorsal lymph sac. Results represent mean -1 S.E.M. for 3 animals. Anesthetized frogs in general showed similar distribution patterns when compared with the conscious frogs (data not shown). Radioactivity was similarly high in liver, kidney, and proximal small intestine, however liver and kidney contained significantly higher percent of the injectate values (25.5 *4.7 and 9.7 * 2.5 percent of injectate, respectively) than those tissues of conscious frogs. The cumulative radioactivity from all tissues was approximately 41.4% of the injected radioactive dose, and was significantly greater than the sum of all tissues from the conscious frogs. Elimination
of Tritiated
Leukotriene
Metabolftes
Figure 3 illustrates the total amount of leukotriene radioactivity eliminated into urinary bladder, bile and water bathing the ventral surface of both intravenously and dorsally-injected conscious frogs at 2.5 hr after injection. Elimination into bile was significantly lower for dorsally-injected frogs (1.1 * 0.7%) compared with intravenously-injected frogs (7.7 t 1.6% injectate), despite
211
Prostaglandins
identical tissue distribution patterns. The volume of bile collected from the gall bladder was not significantly different between the dorsally (277 * 75 ~1) and venously-injected (540 t 217 ~1) groups. injected Neither the bladder water volume nor the percent radioactivity in bladder water and bath water were different between to be contaminated by groups. Bathing water did not appear frog bladders were full at radioactive fecal pellets. In addition, suggesting that bathing water was the termination of the experiment, not contaminated by urinary contents in these frogs.
14
12
0
Vein
n
Dorsal
10
8
6
I
-1
u. tdaa. water
Batn water
Percent of the injected radioactivity (measured in counts per minute) in urinary bladder water, bile, and pan water of venously injected (white bar, n = 8) and dorsally injected (black bar, n = 3) bullfrogs. Results represent mean t S.E.M. Cumulative elimination of radioactivity into bile in the anesthetized frog was detectable but small (Figure 4). After 2.5 hr, 2.7 t 1.1% of the injected radioactivity was eliminated into the bile. This value was not significantly different from the dorsallyinjected frogs but was significantly less than the venously-injected frogs. Bile production rates were estimated at 28 t 6 pl/hr based on collections every 0.5 hr. Characterization
of
Tritiated
Metabolites
Bile, bladder water, and liver samples from conscious frogs were analyzed using mobile phase I, II and/or III to separate the tritiated LTC4 metabolites. Neither sufficient bile nor urine were obtained from the anesthetized frogs to characterize these metabolites.
212
Prostaglandins
4 3 Time (hrs) Figure 4. Cumulative elimination of radioactivity (percent of injected radioactivity in CPM) into bile of anesthetized frogs for Results represent mean * 2.5 hr following injection of [3H]-LTC4. S.E.M. for 7 animals. Figure 5 illustrates a large radioactive peak of polar metabolites as well as a peak with a retention time coinciding with the LTE4 radiometric detection. standard in bladder water using (Mobile phase II did not resolve the individual polar metabolites). Figure 6 shows the distribution of radioactivity collected in l-min fractions of a representative bile sample (Mobile phase III). Identification of leukotriene metabolites coeluting with leukotriene standard retention times are indicated above the histogram. Figure 7 summarizes the identity and relative quantities of [3H]-LTC4 metabolites in bile, liver, and bladder water samples for venously-injected conscious frogs analyzed usin Mobile phases I-III. 93 Less than 5% of the radioactivity remained as [ HI-LTC, after 2.5 hr in all samples. The absence of radioactivity coeluting with N-acetyl LTE4 and 16-carboxytetranordihydro-LTE4 suggested that these metabolites were not present. Liver had the ability to convert [3H]-LTD4 to [3H]-LTE4 and to further oxidize [3H]-LTE4 into the polar metabolites 18-carboxydinorand 20-hydroxy-LTE4, Bile had the same 20-carboxy-LTE4 LTE4, composition and quantity of i3H]-LT metabolites as liver. For bile, liver and bladder water, the sum of the radioactivity composed of the coyprised approximately 50% of the total three polar metabolites sample radioactivity (46.8 - 2.1, 47.8 * 12.7, and 51.4 t 6.16 %,
Prostaglandins
213
respectively). The quantity of 20-carboxy-LTEq was significantly higher than 20-hydroxy-LTEq and 18-carboxydinor-LTEq in bile, but was not significantly different from 20-hydroxy-LTE4 and 18-carboxydinorLTE4 quantities in liver and bladder water. In contrast to liver and bile, bladder water contained more [3H]-LTE4 than [3H]-LTD,.
0
10
5
15
20
25
30
35
40
Time (min)
Figure 5. Tracing from Beta-Ram detector of bladder water sample using Mobile phase II described in Methods. Polar metabolites (PM) were unresolved in this mobile phase and eluted early with the first radioactive peak. The second peak of radioactivity co-eluted with the LTE,+standard retention time.
350-1
I
II
Time (min)
Figure 6. Radioactive fractions collected from bile sample after RPHPLC separation and quantitated by scintillation counting. Mobile phase III as described in Methods was used for the separation. Radioactivity recovered in fractions comigrated with 18-carboxydinorLTE,+ (I), 20-carboxy-LTEq (II), 20-hydroxy-LTEq (III), LTD&, and LTEl, standard retention times.
Prostaglandins
214
n Bile
II
LTC 4
0
Liver
N
Bladder Water
LTD,
Figure 7. Percent of the injected radioactivity (black bar), liver (white bar) and bladder water 2.5 hours post-injection for venously-injected Extraction and analysis by RP-HPLC using Mobile described in Methods. Polar metabolites I, II, carboxydinor-LTE4. 20-carboxy-LTE4, and respectively.
LTE 4 recovered in bile (stripped bar) at conscious frogs. phase III was as and III were 18PO-hydroxy-LTE4,
DISCUSSION with mammals in The bullfrog shares some similarities leukotriene distribution, elimination, and metabolism. Distribution studies in mice using autoradiography found that radioactivity was highly distributed to bile, liver and kidney (5). It is difficult to compare tissue radioactivities directly, but the relative magnitudes between mouse and bullfrog were similar. The conscious and the anesthetized frogs showed similar tissue distribution patterns although the magnitude of radioactivity found in tissues was altered by anesthesia. Whether this phenomenon also occurs in mammals is unknown, but may be an important aspect because most studies of leukotriene metabolism use anesthetized animals. The route of administration affected elimination as evidenced by different biliary eliminations in venously versus dorsally injected frogs. In mice, intravenous versus intramuscular injection of leukotriene C,, did not make a difference in tissue distribution (5), but few other studies have compared routes of injection. The importance of renal elimination of leukotrienes in the bullfrog is still unclear. From Figures 1 and 2 it is clear that
Prostaglandins
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distributed to the kidney in conscious radioactivity was highly frogs. Repeated attempts at collecting urine by cannulating the ureter ducts yielded very small (0 - 100 nl/hr) volumes of urine in recovered in the both anesthetized and conscious frogs. Radioactivity collected ureteral urine was negligible even up to 6 hr after [3H]urine collected in LTC4 injection in one animal. In Bufo marinus, this manner accounted for l/3 of the toad's body weight in 24 hr and contained nearly 55% of the injected radioactivity (24). The lack of urine output in frogs may help explain recovery of significant amounts of radioactivity in bladder water (figure 3). For both dorsally and venously-injected conscious frogs, the LTs eliminated into the bladder water was not significantly different. In frogs, the urinary bladder serves as a transportive membrane for electrolyte and water balance, in addition to its urine storage role. Ureters from the kidney empty into the cloaca and urine is subsequently transferred to the bladder for storage. The bladder can attain additional fluids by diffusion from extracellular fluid and from the cloaca1 contents. Thus, radioactive metabolites in the bladder water may not be exclusively of renal system origin. Whatever the origin of radioactivity, bladder water appears to be a major reservoir of leukotriene elimination. Elimination of [3H]-LTs into bile of conscious frogs was dependent upon the route of injection (Figure 3). Despite identical distribution of tritiated leukotrienes to the liver (Figures 1 and the dorsally-injected frog livers eliminated significantly less 21' [ HI-LTs into bile than the intravenously injected frogs. While final liver radioactivity at 2.5 hr was the same for both frog groups, radioactivity aquisition by the liver and subsequently into bile may have been slower during the 2.5 hr for the dorsally-injected frogs, probably because of more gradual infiltration of radioactivity into the circulation compared with direct intraveous injection. These results are similar to studies in Bufo marinus in which minimal amounts of radioactivity were found in bile (24). The anesthetized frog study was performed primarily to obtain data on continuous biliary elimination of [3H]-LTs (Figure 4). These data showed that biliary elimination of [3H]-LT~ by the anesthetized frog was slow, constant, and initiated within the first 30 minutes after [3H]-LTC4 injection. The total amount of LT radioactivity recovered after 2.5 hr was the same as for the dorsally-injected conscious Irogs, but significantly less than that obtained for the intravenously-injected conscious frogs. The route of injection for the anesthetized frogs was via the abdominal vein which first leads directly into the hepatic portal system and then to the heart. One might expect excretion into bile, therefore, to be greater, but this was not the case. However, biliary elimination may also have been slowed by the anesthetic, MS-222. Despite the differences between models, the biliary elimination of LTs by conscious and anesthetized frogs was far less after 2.5 hrs than most anesthetized mammals, including dog (10). domestic pig (11). monkey (9) and rat (17) which all eliminate primarily into bile. In rat, 28% and 50% of the radioactivity was eliminated in bile at 30 and 90 min following the intravenous injection of [3~]-~T~4 (17).
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Prostaglandins
The decreased biliary elimination of LTs found for the bullfrog may be correlated with its lower bile flow when compared with the anesthetized dog and rat. Bile flow in the anesthetized frog was estimated to be 99 ul/hr-kg, and was less than for the dog (336 Pl/hr-kg (10) and much less than for rat (4980 pl/hr-kg (17). Because it is an amphibian, the slower bile production in the bullfrog may be due to lower metabolic rate and/or decreased perfusion of the liver. Likewise, while extraction of [3H]-LTs out of the circulation by liver appears to be efficient because the liver contained the highest percent of injected radioactivity (figures 1 and 2), the elimination of [3H]-LTs into the bile may be slower. In mammals, export of peptide-LTs into the bile appears to be energy dependent and a ratedetermining step in biliary elimination (25). Radioactivity found in the bath water may be from the transfer and elimination of injected LT radioactivity across the skin and into the external environment. This unique transport also found in Bufo marinus (24) and may be another important elimination route in amphibians in which the skin functions as a transport medium. Like many mammals, but unlike rat and mouse (5,13,16,26), bullfrogs appear to be unable to N-acetylate LTE4 further to N-acetyl LTE4. N-acetyl LTE4 was not found in bladder water, bile and liver samples. Unlike its circulatory system (19). the bullfrog renal system appears to have dipeptidase causing the further conversion of LTD4 to LTE4. Significant dipeptidase activity has been reported in mammalian kidney and liver (4,26-28). The absence of dipeptidase within the vascular system is unique to the bullfrog and has not been reported in mammalian studies. Leukotriene C4 is essentially deactivated when converted to LTD4 in the bullfrog as LTD4 and LTE4 have little hemodynamic activity (18.19). In mammals, both LTC4 and and metabolic LTD4 have potent cardiovascular activity (29) deactivation does not occur until the less active LTE4 and N-acetyl LTE4 are formed. Dipeptidase activity in the bullfrog may be greater in the renal system than in the hepatic system, because of significantly greater found in bladder water compared with bile and liver. LTE4 amounts Sites other than renal cells, such as the cloaca1 surfaces and urinary bladder epithelium may have contributed to the formation of metabolizing [3H]-LT metabolites found in bladder water. Indeed, enzymes, particularly gamma-glutamyl transpeptidase and dipeptidase, may also be found in bullfrog urine, metabolism was however, prevented under the conditions of this study. These enzymes have been reported in human urine ex vivo (30). Identical metabolite compositions found in liver and bile (figure 7) suggest that the liver extracts [3H]-LTD4 from the blood which is then converted LTE4 and its omega and beta-oxidation products 20-hydroxy-LTE4, 20-carboxy-LTE4 and 18-carboxydinor-LTE4. Extensive liver These metabolites were then excreted into the bile. metabolism of leukotrienes and excretion of these metabolites have been reported in mammals (9,14,17,31-33). Omega and beta-oxidation products were also found in bullfrog bladder water (Figure 7). Oxidation products of LTE4 and N-acetyl LTE4 have been reported for
217
Prostaglandins
mammalian kidney (17) Beta oxidation in the bullfrog may be slower than in mammals. After 2.5 hrs, only 18-carboxydinor-LTE4, the first beta oxidation product, was detected, with no signs of 16carboxytetranordihydro-LTE4. In monkey, 16_carboxytetranordihydroLTE4 was already a major metabolite found in bile and urine by 2 hr after the injection of tritiated LTC4 (9). The bullfrog is an interesting model for the study of leukotriene distribution, elimination, and metabolism. Despite major differences in the amphibian physiological organ systems, the distribution of radioactivity was similar to mammals. Like mammals, the frog eliminates in both urine and bile, however, there appears to be an additional unique route of elimination th ough the skin. Overall, with slight variations, metabolism of [ 5HI-LTC4 in the pathways and produce bullfrog appears to follow the same enzymatic the same metabolites as its mammalian counterparts, suggesting that leukotriene processing has been conserved over evolutionary time. ACKNOWLEDGMENTS This research was supported by National Science Foundation Grant DCB-9018542 to C.A.H and D.D.G. was supported by the NSF Research Experiences for Undergraduates program. The authors would like to thank Bonnie Valentich and Susan Sandoval for excellent technical assistance and Dr. Peter Herman for critical reading of the manuscript. REFERENCES 1.
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Krell
Received:
8-19-92
Accepted:
by rat 1987. 12-2-92