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Leukotrienes in the Rat Central Nervous System

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Leukotrienes in the Rat Central Nervous System Proc. Nati. Acad. Sci. USA Vol. 81, pp. 6212-6216, October 1984 Medical Sciences Leukotrienes in the rat central nervous system (brain tissue/arachidonic acid metabolism/lipoxygenase pathway/leukotriene synthesis distribution/leukotriene C4 immunoreactivity) JAN AKE LINDGREN*, TOMAS HOKFELTt, SVEN-ERIK DAHLtNt, CARLO PATRONO§, AND BENGT SAMUELSSON* Departments of *Physiological Chemistry, tHistology, and tPhysiology, Karolinska Institutet, S-104 01 Stockholm, Sweden; and §Department of Pharmacology, Catholic University, 1-00168 Rome, Italy Contributed by Bengt Samuelsson, June 27, 1984 ABSTRACT Leukotrienes C4, D4, and E4 were isolated MATERIALS AND METHODS after incubation of rat brain tissue in vitro with the ionophore Materials. Synthetic LTC4, LTD4, and LTE4 were from A23187 and arachidonic acid. Identification of the compounds Upjohn. [14,15-3H2]LTC4 (20-60 Ci/mmol; 1 Ci = 37 GBq) was carried out using high-performance liquid chromatogra- was obtained from New England Nuclear, arachidonic acid phy, radioimmunoassay, and bioassay. Average production of was from Nu-Check Prep, and ionophore A23187 was from leukotrienes C4, D4, and E4 during 10 min of incubation was Calbiochem. Nordihydroguaiaretic acid and glutathione di- estimated to 25, 8, and 0.7 pmol per g of brain tissue (wet sulfide were obtained from Sigma. weight), respectively. Radioimmunoassay determinations in- Brain Tissue. Male rats (body weight 150-200 g, pathogen dicated in vitro biosynthesis of leukotriene C4 in most regions free strain; Anticimex, Stockholm) were anesthetized with of the sodium pentobarbital (40 mg/kg, i.p.) and perfused with 100 brain, with the highest levels obtained in the hypo- ml of oxygenated Tyrode's solution at room temperature. thalamus and the median eminence. In slices from the caudate The brains were rapidly removed and placed in ice-cold nucleus, ionophore A23187 caused a dose-dependent stimu- phosphate-buffered saline (Pi/NaCl). lation of leukotriene C4 formation with maximal effect at 5 Incubation Procedure. Frontal sections (thickness, -0.5 ,IM. Leukotriene C4 synthesis of rat brain tissue was inhibited mm) from the entire brain were sliced by hand with two razor by 30 jzM nordihydroguaiaretic acid. Finally, using the in- blades on a cooled stage. The slices were preincubated in direct immunofluorescence technique, nerve endings in the P1/NaCl (pH 7.4, 30 mg of brain tissue per ml) at 37°C for 10 median eminence and ceil bodies in the preoptic area reacting min prior to addition of ionophore A23187 and arachidonic with antibodies raised against leukotriene C4 were observed. acid (final concentrations, 5 and 75 ,M, respectively). After 10 min, the incubation mixture was filtered down into an equal volume of ethanol (containing [14,15-3H2]LTC4; Leukotrienes (LT) are bioactive compounds with proposed 250,000 dpm) at - 70°C. roles as important mediators of inflammation and allergy (1). In other experiments, thin slices (-0.3 mm) from various Formation of leukotrienes from arachidonic acid is initiated brain regions (see Results) were prepared by hand at a cooled by 5-lipoxygenation to 5(S)-hydroperoxy-eicosatetraenoic stage with the help of a razor blade and a frosted object slide. acid, which is further converted to an unstable epoxide Small round slices (diameter, 3 mm) were punched out from (LTA4) (2, 3). This intermediate can be enzymatically hy- the larger original slices and preincubated with or without drolyzed to LTB4 (4, 5). is nordihydroguaiaretic acid (30 gM) in 0.5 ml of P,/NaCl (pH Alternatively, LTA4 transformed, 7.4) at 37°C for 5 min. Thereafter, ionophore A23187 (0-20 by addition of glutathione at C-6, into LTC4 (6, 7). Stepwise ,uM) was added, and the samples were incubated for another enzymatic elimination of glutamic acid and glycine in the 10 min. In some experiments, arachidonic acid (75 ,uM) was peptide side chain leads to formation of LTD4 and LTE4, added together with the ionophore. The incubations were respectively (8, 9). Slow reacting substance of anaphylaxis, stopped by rapid freezing of the samples at - 70°C. After the proposed mediator of allergic reactions (10), has been thawing, samples were centrifuged at 1400 x g (10 min; 4°C) identified as an entity composed of LTC4, LTD4, and LTE4 to remove the tissue prior to radioimmunoassay. (1). Extraction, Purification, and Analytical Methods. The So far, leukotriene formation has almost exclusively been ethanol/H20 mixture was acidified with formic acid (to pH 4) described in leukocytes and lung tissue (1). On the other prior to extraction with chloroform, and the organic phase hand, production of arachidonic acid metabolites is widely was purified on a silicic acid column (Silicar CC-7) eluted distributed in various organ systems, including the central with chloroform, methanol/chloroform (20:80, vol/vol), and nervous system (11). LTC4 has been reported to produce a methanol. The material eluted in the methanol fraction was prolonged excitation of cerebellar Purkinje neurons (12), subjected to reversed-phase high-performance liquid chro- suggesting a role for leukotrieties in brain function. In the matography (RP-HPLC). A Nucleosil 5 C18 column (250 x present study, the possible occurrence of leukotrienes in the 4.6 mm, Macherey-Nagel) was eluted at 1 ml/min with a central nervous system was investigated. This report de- mobile phase of methanol/water/acetic acid (62:38:0.02, scribes the isolation of LTC4, LTD4, and LTE4 from rat brain vol/vol) containing 0.03% disodium EDTA. Eluted com- incubations and the regional distribution of LTC4 biosyn- pounds were continuously monitored with a UV detector at thesis 280 nm. Before use, the column was rinsed overnight with in the brain. In addition, immunohistochemical evi- 0.5% disodium EDTA (pH 8.5) in methanol/water (10:90, dence for a possible occurrence of LTC4-like immunoreac- vol/vol) and thereafter for at least 60 min with water to tivity in nerve endings of the median eminence is presented. remove excess disodium EDTA. The recovery of LTC4 was The publication costs of this article were defrayed in part by page charge Abbreviations: LTC4, LTD4, and LTE4, leukotrienes C4, D4, and E4, payment. This article must therefore be hereby marked "advertisement" respectively; RP-HPLC, reversed-phase high-performance liquid in accordance with 18 U.S.C. §1734 solely to indicate this fact. chromatography. 6212 Downloaded by guest on October 1, 2021 Medical Sciences: Lindgren et al. Proc. Natl. Acad. Sci. USA 81 (1984) 6213 determined by measurement of radioactivity in the HPLC times corresponding to standards of LTC4, LTD4, and LTE4, fractions. respectively. Peak I also coeluted with [3H]LTC4 added to Levels of LTC4 were determined by radioimmunoassay the sample after incubation. UV spectroscopy of compound (13). I showed maximal absorbance at 280 nm with shoulders at Bioassay. Strips oflung parenchyma and longitudinal ileum 270 and 290 nm (Fig. 1). The material that cochromato- muscle were prepared from guinea pigs and used under graphed with LTD4 (peak II) or LTE4 (peak III) was dis- nonflow conditions (14, 15). Briefly, the lung strip affords a solved in 100 ,ul of ethanol/water (1:1, vol/vol) after evapo- high sensitivity (detection limit, 0.1 pmol) but cannot dis- ration of the HPLC solvent. Samples of both compounds tinguish between LTC4, LTD4, or LTE4 (cf. ref. 14), whereas elicited dose-related contractions of the lung strip (Fig. 2). the ileum is 1/10th as sensitive but reacts differently to each Furthermore, the contractions were unaffected by indometh- leukotriene. Thus, LTC4 and LTD4 cause contractions with acin or by receptor antagonists for acetylcholine and bio- different time-courses on the ileum, and LTE4 is recognized genic amines, but they were susceptible to antagonism by in this preparation because of its lower potency (cf. ref. 15). FPL 55712. Quantitation on the lung strip indicated that Therefore, quantitation of HPLC-purified material was per- peaks II and III contained -60 and 5 pmol of LTC4-like formed on the lung strip and the results were expressed as activity, respectively (Fig. 2). The material in peak II caused pmol of LTC4-like activity, whereas the ileum was used to contractions on the ileum that had a time-course and relative further characterize the material. potency consistent with the presence of LTD4. The ileum Immunohistochemistry. Rats of either sex were sacrificed assay also suggested that peak II contained 60-70 pmol of and perfused for 10 min with 0.9% saline at room tempera- LTD4 (see above). Finally, on the ileum, the material in peak ture, followed by ice-cold formalin containing picric acid III was considerably less active than equal amounts of ma- (16). The brains were dissected out and immersed in the III same fixative for 90 min, rinsed, cut in 14-gm sections on a terial eluted in peak II, giving further evidence that peak cryostat, and processed for the indirect immunofluorescence contained LTE4, because this leukotriene is less potent than technique of Coons and collaborators (17). Briefly, the sec- LTD4 in the ileum but not in the lung strip (Fig. 2) (14, 15). tions were incubated for 18-24 hr at 40C with LTC4 antiplas- Therefore, considered together, it is reasonable to conclude ma (18), diluted 1:200 to 1:3200, rinsed, incubated with that peaks II and III contained 60 and 5 pmol of LTD4 and fluorescein isothiocyanate-conjugated swine anti-rabbit anti- LTE4, respectively. bodies (1:10) (Dakopatts, Copenhagen, Denmark), rinsed, After rechromatography of peak I on RP-HPLC, the frac- mounted, and examined in a fluorescence microscope. Con- tions were analyzed for LTC4 by radioimmunoassay. As trols were prepared by incubating sections with LTC4 anti- shown in Fig. 3A, the UV-absorbing (280 nm) fractions also plasma preabsorbed with an excess of LTC4 (65 uM in anti- contained immunologically active material binding to the plasma diluted 1:400), with bovine serum albumin (5 mg per antibody.
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