Partial purification and characterization of 12-lipoxygenase in bullfrog erythrocytes.

12-Lipoxygenase (12-LO) in bullfrog (Rana catesbeiana) erythrocytes was purified partially by ion exchange chromatography and affinity chromatography. Bullfrog 12-LO was a single chain protein with a pI of 7.1-7.8 and MW of 7.77 kDa. This enzyme did not show typical Michaelis Menten type kinetics. At low substrate concentrations, it had a lag phase and at higher substrate concentrations, the activity was inhibited. The product of linoleic acid (LA), 13-hydroperoxy-9, 11-octadecadienoic acid (13-HpODE), was an activator for the enzyme. When arachidonic acid (AA) was used as substrate, 13-HpODE also affected the Km of bullfrog 12-LO towards AA. The affinity of LA towards bullfrog 12-LO was higher than the affinity of AA. Suicide inactivation was much more rapid than that of any mammalian 12-LO reported. Hemoglobin (Hb) inhibited the activity of 12-LO partially and removing Hb eliminated this inhibition. Both Hb and Met-Hb inhibited the 12-LO activity but did not denatured completely the Hb, suggesting that the inhibition was a direct interaction between 12-LO and Hb protein chain and was not due to competition between 12-LO and Hb for oxygen. This study characterizes bullfrog 12-LO with respect to stability, optimal pH, suicide inactivation and interaction with Hb and provides important evolutionary information about this enzyme.

Light-regulated expression of the gsa gene encoding the chlorophyll biosynthetic enzyme glutamate 1-semialdehyde aminotransferase in carotenoid-deficient Chlamydomonas reinhardtii cells.

Expression of the Chlamydomonas reinhardtii gsa gene encoding the chlorophyll biosynthetic enzyme glutamate 1-semialdehyde aminotransferase was previously shown to be induced by blue light. Possible blue light photoreceptors include flavins and carotenoids. Light induction of gsa was investigated in carotenoid-deficient mutant C. reinhardtii cells. Strain CC-2682 cells are sensitive to light, produce only small amounts of chlorophyll, and do not exhibit phototaxis. Solvent extracts show the absence of carotenoids and carotenoid precursors beyond phytoene in dark-grown mutant cells. Although apparently devoid of carotenoids, the cells did show light induction of gsa. The gsa transcript level was very low in dark-grown cells but increased significantly after 2 h of exposure to dim (1.5 x 10(-5) mol m(-2) s(-1)) green (480-585 nm) light. This light regime was previously determined not to injure these photosensitive cells and to fully induce gsa in wild-type cells. Exposure to this light did not cause the mutant cells to produce measurable carotenoids or to become phototactic. Growth of the mutant cells in the presence of exogenous beta-carotene or all-trans retinol restored phototaxis but did not affect the degree of gsa induction by light. The induction of gsa by light in the absence of carotenoids, and the fact that incorporation of physiologically usable carotenoids (as indicated by the restoration of phototaxis) did not affect the degree of light induction, indicate that the photoreceptor for light induction of gsa in C. reinhardtii is not a carotenoid. The flavin antagonist diphenyleneiodonium blocked light induction of gsa in both wild-type and mutant cells under conditions where respiration was not inhibited. These results suggest that the photoreceptor or a signal transduction effector for light induction of the C. reinhardtii gsa gene is a flavoprotein.

Localization and changes in distribution of brain alpha 2- and beta-adrenoceptors in response to acclimation state in the American bullfrog (Rana catesbeiana).

Alpha (alpha)- and beta (beta)-adrenoceptors regulate physiological processes in vertebrates. This study determined the location of alpha 2- and beta-adrenoceptors in the brain of the American bullfrog, Rana catesbeiana, using autoradiography. As the density of receptors may be affected by environmental temperature, a comparative numerical analysis of adrenoceptors in the areas of localization with respect to warm and cold acclimation was also carried out. Areas of greatest concentration of alpha 2-adrenoceptors were the accessory olfactory bulb, medial pallium, and olfactory bulb. Adrenoceptor numbers were significantly decreased in the accessory olfactory bulb and medial pallium in cold-acclimated animals. beta-adrenoceptors were localized in the thalamus, cerebellum, medial pallium, and amygdala/ striatum. Cold acclimation decreased adrenoceptor density in medial pallium and torus semicircularis and increased adrenoceptor density in the thalamus and hypothalamic preoptic areas. Among the alpha 2- and beta-adrenoceptors, only four regions of overlap existed, the medial pallium, hypothalamic preoptic area, optic tract, and isthmic tegmentum. Otherwise, where there were alpha 2-adrenoceptors, there were few or no beta-adrenoceptors. No alpha 2- or beta-adrenoceptors were found in the pituitary and optic chiasm. The distribution of adrenoceptors in particular areas of the brain may have functional significance with respect to physiological changes which occur in response to hibernation.

Liver microsomes from the yellow rat snake (Elaphe obsoleta) and American bullfrog (Rana catesbeiana) oxidize polyunsaturated fatty acids by NADPH-dependent hydroxylation and epoxidation.

Polyunsaturated fatty acids (PUFAs) can be oxygenated by mammalian hepatic P450s to a series of metabolites. The most prominent of these are formed by omega- and (omega-1)-hydroxylation, epoxidation of the double bonds or bisallylic hydroxylation. The object of the present investigation was to determine whether similar oxygenations are catalyzed by liver microsomes of the yellow rat snake (Elaphe obsoleta) and the American bullfrog (Rana catesbeiana). Liver microsomes were incubated with [1-14C]-labeled arachidonic (AA), eicosapentaenoic (EPA), and linoleic acids (LA) in the presence or absence of 1 mM NADPH, and the major metabolites were analyzed by reverse-phase and straight-phase high performance liquid chromatography and capillary gas chromatography-mass spectroscopy. No metabolites were produced in the absence of NADPH. Profiles of metabolites were different depending on the organism and the acclimation state. In all incubations, EPA was the most effective substrate utilized and LA the least effective. The major products from EPA were 19-HEPE, 13-HEPE, and 20-HEPE from cold-acclimated (5 degrees C), warm-acclimated (22 degrees C) frogs, and snakes (22 degrees C), respectively. In contrast, 20-HETE production from AA was greater than 19-HETE in all three. Cold-acclimated frog liver microsomes produced significantly more of all metabolites when compared with microsomes from warm-acclimated frogs. We conclude that amphibian and snake liver can catalyze epoxidation and hydroxylation of PUFAs and that products are species-specific and acclimation-state dependent.

Eicosanoid synthesis by purified thrombocytes and erythrocytes from warm- and cold-acclimated American bullfrogs (Rana catesbeiana).

Amphibian blood plays an important role in eicosanoid synthesis. Although clotting frog blood produces eicosanoids, the cellular source of prostaglandins and thromboxanes in bullfrog blood is unknown. Thromboxane (TX)B2 synthesis from purified thrombocytes was affected by 30-day cold-acclimation at 5 degrees, but not PGE2 or leukotriene (LT) synthesis. Although no cyclooxygenase activity has been found in human erythrocytes, frog erythrocytes were capable of forming cyclooxygenase products, but the amounts were lower than those produced by thrombocytes. Additionally, there was no effect of cold exposure on eicosanoid production by isolated erythrocytes. Similar to some mammalian nucleated white blood cells, nucleated bullfrog thrombocytes and erythrocytes produced leukotrienes. The production of eicosanoids by thrombocytes was stimulated by A23187 and thrombin. Erythrocytes were stimulated by A23187. Control synthesis by erythrocytes and thrombocytes was inhibited by 5 microM indomethacin (cyclooxygenase pathway) or nordihydroguaiaretic acid (5-lipoxygenase pathway) and cyclooxygenase products were increased in the presence of nordihydroguaiaretic acid. Thrombin stimulation of eicosanoid production by thrombocytes was inhibited when the inhibitors were present prior to the final centrifugation of the cell isolation. The results suggest that cold exposure can affect eicosanoid synthesis in thrombocytes, but not erythrocytes, and that thrombocytes are a major source of eicosanoids in bullfrogs. The production of cyclooxygenase and lipoxygenase products by nucleated erythrocytes and thrombocytes suggests a role for these compounds in hemostasis and inflammatory responses in these animals.

Cardiovascular effects of homologous bradykinin in rainbow trout.

Bradykinins have only recently been identified in fish, and a detailed analysis of their cardiovascular actions is lacking. The present study examines the cardiovascular effects of trout bradykinin ([Arg0,Trp5,Leu8]bradykinin; tBK) in conscious trout, Oncorhynchus mykiss. tBK (1-10 nmol/kg body wt bolus) produced triphasic pressor-depressor-pressor responses. In phase 1, cardiac output (CO), ventral aortic (P(VA)), dorsal aortic (P(DA)), and central venous pressure increased, whereas systemic (R(S)) and gill resistance (R(G)) were unchanged. In phase 2, R(G) increased, whereas R(S), CO, and heart rate decreased, reducing P(VA) and P(DA). Plasma prostaglandin E2 and the prostacyclin metabolite, 6-ketoprostaglandin F1alpha, were significantly elevated during phase 2, whereas leukotrienes C4 and B4 and thromboxane B2 were unaffected. Phase 3 was produced by an increased CO and R(S) and the return of R(G) to control. Phase 1 pressor response was not blocked by inhibitors of cyclooxygenase, angiotensin-converting enzyme (ACE) or alpha-adrenoceptors (alpha-AD), whereas phase 2 depressor and plasma prostaglandin responses were prevented by cyclooxygenase inhibition. Phase 3 was partially blocked by ACE and alpha-AD inhibitors and is a response to the preceding hypotension. In vitro, tBK only decreased vascular resistance in the perfused splanchnic or skeletal muscle-kidney preparations. These results show that although tBK has multiple effects on the trout cardiovascular system, none of the effects are due to direct tBK stimulation of vascular smooth muscle. Phase 2 vasodilation has features consistent with release of vasodilator prostaglandins while the mechanism of phase 1 constriction is unknown.

Biosynthesis of thromboxane by snake (Elaphe obsoleta) erythrocytes and the requirement of eicosanoid production for blood clotting.

Lower vertebrates provide important insights into the evolution of eicosanoid synthesis and function. Whole snake blood, purified nucleated erythrocytes, and isolated leukocytes activated by clotting or A23187 produced thromboxane, PGE2, and 5-lipoxygenase products. Indomethacin's complete inhibition of clotting suggests eicosanoids produced by these cells are important in snake blood hemostasis.

Characterization of adrenoceptor types and subtypes in American bullfrogs acclimated to warm or cold temperature.

While indirect evidence suggested that the responsiveness of frog adrenoceptors changes in response to temperature, direct measurement of adrenoceptor binding following acclimation to warm and cold temperatures had not been done. In the present study, the radioligands [3H]prazosin, [3H]RX821002, and [125I]cyanopindolol were used to label and quantify alpha 1-, alpha 2-, and beta-adrenoceptors in bullfrogs acclimated to warm or cold environments. The number of alpha 1-, alpha 2-, and beta-adrenoceptors in atrium, ventricle, and kidney membranes was not significantly different between warm- and cold-acclimated frogs. Characterization of receptor subtypes using pharmacological antagonists demonstrated that alpha 2-adrenoceptors in frog spinal cord and kidney were of the same pharmacological subtype, which is similar to the mammalian alpha 2A-subtype. The beta-adrenoceptor in frog ventricle, atrium, and kidney was the beta 2-subtype. These results suggest that while the alpha 1-, alpha 2-, and beta-adrenoceptor types have evolved in the frog, multiple subtypes of adrenoceptors are not necessary for physiological regulation in this species.

Thrombocytes are the predominant source of endogenous sulfidopeptide leukotrienes in the American bullfrog (Rana catesbeiana).

Nucleated bullfrog erythrocytes have 5-lipoxygenase (LO) and are the first non-mammalian cell to exhibit endogenous sulfidopeptide leukotriene (LT) synthesis. Non-nucleated mammalian platelets lack 5-LO, but contribute significantly to LTC4 production by transcellular synthesis. However, nucleated bullfrog thrombocytes have not been examined for 5-LO activity. Endogenous leukotriene synthesis by bullfrog thrombocytes and mixed leukocytes was analyzed by reverse-phase high-performance liquid chromatography (RP-HPLC). Calcium ionophore activated (A23187) leukocytes demonstrated 5-LO, 12-LO, and 15-LO activity. Spectral analysis demonstrated synthesis of LTB4, LTB4 isomers, 15(S)-monohydroxyicosatetraenoic acid (HETE), 5(S),12(S)-diHETE, 5(S),15(S)-di-HETE, lipoxin A4 (LXA4) and LXB4. Thrombocytes synthesized large quantities of sulfidopeptide leukotrienes but no lipoxins. Sulfidopeptide leukotriene and LTB4 radioimmunoassay analysis and the radiological RP-HPLC profile of [3H]AA metabolism further confirmed synthesis. Incubations with [3H]LTC4 demonstrated slow and incomplete conversion to [3H]LTD4. Thrombocyte leukotriene profile changed over time revealing a significant shift from the LTC4 synthase to LTA4 hydrolase pathway, corresponding with release of large amounts of LTA4. Thrombocytes potentially play a pivotal role in inflammatory and cardiovascular responses. 5-LO activity in amphibian homologs to mammalian platelets and erythrocytes compared with the lack of activity in the mammalian counterparts may correspond to the loss of the nucleus in the evolution of these cells.

Endogenous sulfidopeptide leukotriene synthesis and 12-lipoxygenase activity in bullfrog (Rana catesbeiana) erythrocytes.

Endogenous leukotriene (LT) synthesis by mammalian inflammatory cells requires both 5-lipoxygenase (5-LO) and 5-lipoxygenase-activating protein. Other myeloid cells, like erythrocytes, have an incomplete 5-lipoxygenase pathway and synthesize leukotrienes transcellularly. Several studies indicate that sulfidopeptide leukotrienes have important physiological functions in bullfrogs and receptors have been characterized. Calcium ionophore activated bullfrog blood was analyzed by reverse phase-high-performance liquid chromatography (RP-HPLC). Endogenous metabolites consisted of 5-LO products including leukotriene D4. Other metabolites also suggested 12-lipoxygenase activity. Following purification, metabolites from activated erythrocytes were analyzed by RP-HPLC coupled with radioimmunoassay. Erythrocytes demonstrated endogenous synthesis of LTD4 which was inhibited by non-selective (NDGA) and specific (MK886) 5-lipoxygenase inhibitors. Experiments with partially purified erythrocyte cytosol further confirmed 5-LO activity and revealed 12-lipoxygenase activity. HPLC analysis of [1-14C]arachidonic acid labeled metabolites from activated erythrocytes indicates that most of the available substrate is converted to 12-hydroxy-eicosatetraenoic acid (12-HETE). These novel findings indicate that, in contrast to mammals, bullfrog erythrocytes endogenously synthesize LTD4 and large quantities of 12-HETE giving them the potential to contribute directly to inflammatory responses. The evolutionary loss of the nucleus in mammalian erythrocytes appears to be associated with the inability to synthesize leukotrienes endogenously.

Cardiovascular effects of N-methyl leukotriene C4, a nonmetabolizable leukotriene C4 analogue, and the antagonism of leukotriene-induced hypotension by Ro 23-3544, in the American bullfrog, Rana catesbeiana.

Although some leukotriene antagonists have been reported to block leukotriene (LT) C4 responses in vivo, it is difficult to determine whether those antagonists block the effect of LTC4 directly or act via blocking the action of LTD4, as LTC4 is metabolized to LTD4 rapidly in vivo. In this study, the dose-response curves of N-methyl LTC4 (NMLTC4), the nonmetabolizable LTC4 analogue, and the peptidoleukotrienes (LTC4, LTD4, and LTE4) were obtained in the absence and presence of the leukotriene antagonist Ro 23-3544 in cannulated frogs. The more potent effect of NMLTC4 suggests that receptors that preferentially bind LTC4 exist in frog vascular smooth muscle and the previously reported LTC4 effect is a combination of LTC4 and its less potent metabolite LTD4. The NMLTC4- and LTC4-induced hypotensive effects were antagonized by Ro 23-3544. Ro 23-3544 also antagonized the effects induced by high doses of LTD4 and LTE4. Ro 23-3544 had no effect on duration of response and did not affect heart rate responses to LTC4 at low dose of the antagonist. The data suggest that receptors that preferentially bind LTC4 in bullfrog vascular smooth muscle regulate the hypotensive effect and that they can be antagonized by Ro 23-3544.

The American bullfrog (Rana catesbeiana) exhibits differential cardiovascular responses to LTC4 after short- and long-term cold exposure.

Mean arterial pressure (MAP) and heart rate (HR) responses to leukotriene (LT)C4 have been studied in warm (W-A) and cold-acclimated (C-A) America bullfrogs, Rana catesbeiana, but nothing is known about how the length of cold treatment affects the response to LTC4 or how responses are affected by return to room temperature following cold exposure. In this study, two groups of frogs were placed at 5 degrees either for 30 days (C-A) or for 1 day (W-C) and LTC4 dose-response curves obtained at 5 degrees. After the frogs were removed from cold, the baseline MAP and HR were monitored for 120 min, and dose-response curves were repeated at 2, 24, and 48 hr. The LTC4 dose-response curves were also obtained during the three experimental days on frogs which had never been placed at cold temperature (W-A). C-A frogs showed greater MAP response than W-C frogs at 5 degrees, and also showed the greatest MAP response of the three groups 48 hr after the animals were removed from cold temperature. These data suggest that up regulation of LTC4 receptors may have occurred in C-A frogs. Cold exposure alone eliminated reflex HR response to LTC4 in both C-A and W-C groups. Longer duration of action was also observed in these groups. The results demonstrate that short-term cold exposure can affect the response to LTC4 in frogs, but that physiological adjustments occur in frogs undergoing long-term exposure.

Leukotriene C4-stimulated contractions in bullfrog lung are affected by cold acclimation and calcium antagonists.

Leukotriene C4 (LTC4) contracts isolated bullfrog lung. This study examined effects of cold-acclimation and the involvement of extracellular and intracellular Ca++ on the contractile response to LTC4. The response to LTC4 was greater in lungs from warm-acclimated (22 degrees C) frogs compared with cold-acclimated (5 degrees C) frogs at incubation temperatures of both 22 degrees C and 5 degrees C. LTC4, LTC5, and N-methyl LTC4 were equally effective in stimulating lung contraction at concentrations from 1-100 nM. Nicardipine (3 microM) partially antagonized the response to LTC4, but verapamil, nifedipine, or nitrendipine at the same concentration was ineffective. Ethylene glycol tetraacetic acid (EGTA, 0.3 mM) prevented the response to 30 nM LTC4, but the response was restored when the lung was retested in EGTA-free medium containing Ca++, suggesting that extracellular Ca++ was involved in the response. Caffeine (10 mM) or thapsigargin (1 mM) inhibited the responses to LTC4, suggesting a role for intracellular Ca++ in the contraction.

Eicosanoid synthesis by warm- and cold-acclimated American bullfrog (Rana catesbeiana) brain.

Previous studies in bullfrogs have demonstrated the presence of leukotriene (LT)C4 binding sites in the brain. However, synthesis of eicosanoids by brain tissue has not been examined. Because prostaglandin (PG) synthesis differs in warm- and cold-acclimated bullfrog lung tissue, this study compared the synthesis of prostaglandins and leukotrienes in brains from warm-(22 degrees C) and cold-acclimated (5 degrees C) animals. Initial experiments determined that leukotriene and prostaglandin production rates were greatest during the initial 30 min time period. Therefore, tissues were incubated in Munsick's solution and gassed with 95% O2, 5% CO2 for 30 min. Media were analyzed by radioimmunoassay for LTC4, LTB4, PGE2, PGF2 alpha, TXB2, and 6-keto PGF1 alpha. In warm-acclimated bullfrog brains, production was as follows: LTC4 > PGE2 > 6-keto PGF1 alpha, thromboxane (TX)B2, LTB4, and PGF2 alpha. Brain tissues from cold-acclimated animals incubated at 22 degrees C produced significantly greater quantities of PGE2 and 6-keto PGF1 alpha than did brains from warm-acclimated animals. Stimulation of TXB2 levels was observed when the animal was stunned with a blow to the head prior to decapitation. Indomethacin, a cyclooxygenase inhibitor, decreased prostaglandin but not leukotriene synthesis. Epinephrine (4 x 10(-8) M), the amphibian sympathetic postganglionic neurotransmitter, stimulated leukotriene synthesis by brains from warm-acclimated bullfrogs, and the effect was blocked with the 5-lipoxygenase inhibitor MK-886 (5 x 10(-5) M). These results clearly indicate that the bullfrog brain synthesized both leukotrienes and prostaglandins. Further studies are necessary to determine their function in the amphibian central nervous system.

Metabolism and cardiovascular effects of leukotrienes in the marine toad Bufo marinus.

Leukotriene (LT) metabolism and physiology have been studied extensively in mammals; however, little is known of their roles in nonmammalian vertebrates. This study examines the cardiovascular effects of leukotrienes on blood pressure and heart rate in the conscious and cannulated marine toad, Bufo marinus. The sulfidopeptide leukotrienes, LTC4, LTD4, and LTE4 elicited hypotension with equal potency. However, with respect to heart rate changes and duration of action, the responses to LTC4 and LTD4 were greater and lasted longer than those to LTE4. The nonpeptide leukotriene, LTB4, had significantly less potent effects on heart rate and blood pressure. The leukotriene-induced increases in heart rate with 1000 and 300 ng/kg body wt LTC4 and LTD4 were blocked with 5 mg/kg body wt propranolol, a beta-antagonist, suggesting sympathetic reflex regulation of heart rate. Metabolism of [3H]LTC4 to [3H]LTD4 and [3H]LTE4 occurred rapidly in blood, with complete conversion to [3H]LTE4 within 5 min. Conversion was slower in plasma, with 18.9 +/- 0.5% of the radioactivity associated with [3H]LTC4 still remaining after 120 min. The toad is more similar to mammals than the bullfrog with respect to the metabolism of leukotrienes. In contrast to mammals, leukotrienes have hypotensive effects in both toad and bullfrog, although the order of potency differs. The effectiveness of the sulfidopeptide leukotrienes in eliciting hypotension at low doses (1 ng/kg body wt) suggests that these compounds may be important cardiovascular regulators in the toad.

Tissue distribution, elimination and metabolism of [3H]-leukotriene C4 in the American bullfrog, Rana catesbeiana.

Tissue distribution, elimination, and metabolism of [3H]-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 anesthesia appears to alter distribution and elimination of leukotrienes. [3H]-leukotrienes were eliminated into bladder water and bile. In addition, 7.8 +/- 2.2 and 5.2 +/- 2.5 percent of the injected radioactivity was found in the pan water 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 18-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.

Leukotriene B4 and leukotriene B5 have binding sites on lung membranes and cause contraction of bullfrog lung.

Leukotriene (LT)B4 and LTB5 cause contraction of isolated bullfrog lung. LTB4 receptors were characterized in membranes prepared from bullfrog lung. Binding of [3H]LTB4 was maximal at 5 min and was reversible with the addition of 1000-fold excess LTB4. Scatchard analysis indicated a single class of binding sites with a Kd of 2.22 nM and a Bmax of 1228.86 fmol/mg protein. The Kd and the Bmax values in the presence of guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) were 2.76 nM and 1289.61 fmol/mg protein, respectively. The Ki values for LTB4, LTB5 and 20(OH)-LTB4 were 5.5, 30.5 and 144.0 nM, respectively, whereas 20(COOH)-LTB4 was ineffective in preventing binding of [3H] LTB4 from 10(-9) to 10(-5) M. The peptide leukotrienes LTC4, LTD4 and LTE4 failed to inhibit the specific binding of [3H]LTB4. GTP gamma S in concentrations from 10(-10) to 10(-4) M did not affect the binding of 5 nM [3H]LTB4. Neither the mammalian LTD4 antagonist LY171883 nor the mammalian LTB4 antagonist LY255283 was an effective competitor for the bullfrog lung LTB4 receptor. In addition, sulfhydryl-modifying reagents NEM and PCMP did not affect LTB4 binding as they do in mammalian membrane preparations. The LTB4 receptor shows some differences from the described mammalian receptor. The cell type containing the LTB4 receptor remains to be determined.

Tissue distribution, elimination, and metabolism of [3H]leukotriene C4 by the conscious marine toad, Bufo marinus.

Tissue distribution, elimination, and metabolism of 3H-labelled leukotriene (LT) C4 were studied in ureter-catheterized conscious marine toads, Bufo marinus. Six and 24 h after injection, organs containing the highest percent of injected radioactivity were small intestine, liver, and kidney. Radioactivity declined in these organs at 24 h by approximately threefold. Peak elimination time for radioactivity in the urine was between 2 and 4 h after the injection. During the 24-h collection period, 55.2 +/- 0.2% of the injected radioactivity was eliminated in the urine. Polar metabolites represented 40.3 +/- 1.1, 57.3 +/- 5.6, and 62.8 +/- 1.6% of the radioactivity at 2, 4, and 6 h, respectively. The primary urinary polar metabolite was 20-carboxy-LTE4, with 18-carboxydinor-LTE4 and 20-hydroxy-LTE4 also present. [3H]LTE4 decreased from 37.2 +/- 1.8% at 2 h to 15.8 +/- 3.3 and 15.0 +/- 2.1% of the radioactivity at 4 and 6 h, respectively. Bile radioactivity was low. N-Acetyl-LTE4 was not detected in urine or bile samples. Radioactivity in the pan water was 14.3 +/- 2.4 and 15.8 +/- 2.5% of the injected radioactivity, at 6 and 24 h, respectively, suggesting that the skin was a route for excretion of leukotrienes. The marine toad is an interesting model demonstrating both similarities and differences from mammals in distribution, elimination, and metabolism of peptide leukotrienes.