Proc. Nati. Acad. Sci. USA Vol. 81, pp. 6212-6216, October 1984 Medical Sciences

Leukotrienes in the rat central nervous system (brain tissue/ metabolism/ pathway/ 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 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 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 and (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 and 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 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. The total amount of LTC4 remaining after re- ml anti-plasma diluted 1:400), with glutathione disulfide (8 chromatography was -90 pmol, as judged by radioimmu- nM to 800 ,uM in anti-plasma diluted 1:400), or with noassay. LTC4/bovine serum albumin conjugate (18). Approximately 150 ,ug of LTC4 was conjugated to 1.5 mg of bovine serum pmol of albumin. After dialysis, -30% of added LTC4 was re- LTD4 or LTE4 80 covered, resulting in a final concentration of -72 p.M LTC4 I in conjugated form in 1 ml of anti-plasma diluted 1:400. 4p 1 -so50 I 1 RESULTS LTD4 1 bdL 11 40 Isolation of Leukotrienes from Incubates of Whole Rat 0.5 pmol Brain. Sliced rat brains were incubated with arachidonic acid and ionophore A23187. The purified material was analyzed // 0.5 by RP-HPLC. Fig. 1 shows the UV absorbance profile (at rJX 0 0 280 nm) of eluted products. Peaks I, II, and III had elution 1 10 100l 1 I 2 LTE4 20 1.d 111 sens. x2 r16 1 pmol i f

0 E -12 x x

E o ._ 10 40 IlI x 8 us LTC4 LTD4 ,ul 11 LTE4 ,ul el 0 5 Pmol 5 pmol 3 pmol C. FIG. 2. Bioassay of material eluted in peaks II and III. Upper 4 -oE ta two tracings show typical contractions evoked by the samples and 0.10 standards of LTD4 or LTE4 on the guinea pig lung strip. Quanti- tation on the lung strip is shown in upper right-hand corner. Ab- O scissa, volumes of samples tested (both peaks were dissolved in a 0 10 20 30 40 50 total vol of 100 ul). Left ordinate, contractions (expressed as % of Elution time, min maximal responses in each preparation) evoked by samples of peak 11 (o) and III (o), respectively. Filled symbols show responses in FIG. 1. Upper curve shows a RP-HPLC chromatogram of an in- indomethacin-treated (10 ,uM) preparations. Right ordinate, con- jected mixture of LTC4, LTD4, and LTE4. Lower curve shows a traction responses evoked by standards of LTD4 or LTE4. Amount RP-HPLC chromatogram obtained after injection of the methanol of LTD4- or LTE4-like activity in peak II or III was extrapolated fraction of a silicic acid column chromatography of the chloroform from the linear volume response curves of each sample. Lower extract. Dotted line represents content of radioactivity in each tracings show contractions elicited by the samples and standards of HPLC fraction. (Inset) UV spectrum of material eluted in peak I. leukotrienes on the longitudinal muscle of the guinea pig ileum. Downloaded by guest on October 1, 2021 6214 Medical Sciences: Lindgren et al. Proc. Natl. Acad. Sci. USA 81 (1984)

A RIA of HPLC fractions B Bioassay 3- -40 +30 Q < FPL 55712 2~ GPLS -30~ E v2 -20 T A LTC4 1 pmol 10 A1 S 10 .1

GPIL

(-)

< 0 *4LTC, LTD, 50 TpII I 20 30 5 pmol 5 pmol Elution time, min FIG. 3. (A) RP-HPLC chromatogram (partially shown) obtained after rechromatography of peak I (Fig. 1). Dotted line represents the LTC4 content in each fraction as judged by radioimmunoassay. 0 rV The amounts of [3H]LTC4 added in aliquots of the diluted HPLC o 8 7 6 5 4 fractions never exceeded 5% of [3H]LTC4 used in the assay pro- cedure. (B) Contractions evoked by samples of peak I on the guinea -log[A23187], M pig lung strip (GPLS) and ileum (GPIL), respectively. S, solvent control [EtOH/H20 (1:1 vol/vol)]. Concentration of FPL 55712, 1 FIG. 5. Effect of ionophore A23187 on LTC4 production in nu- JIM. cleus caudatus. Samples containing four slices each from the cau- date nucleus were preincubated in 0.5 ml of Pi/NaCl (pH 7.4) for 5 The fractions containing LTC4 were pooled, and the HPLC min prior to incubation with various concentrations of ionophore solvent was evaporated. Thereafter, the sample was dis- A23187 for 10 min. Each point represents the mean value of two solved in 1 ml of ethanol/water (1:1, vol/vol) and subjected duplicate determinations on each of two samples. to bioassay (Fig. 3B). This material had a pattern of contrac- tile activity that was indistinguishable from that of LTC4. chloroform extraction, silicic acid chromatography, and RP- Thus, the contractions elicited on the lung strip were slow in HPLC, 60%-70% of added radioactivity was still present in onset and resistant to indomethacin and receptor antagonists the peak corresponding to LTC4. In experiments in which for biogenic amines, but they were antagonized by FPL rechromatography of LTC4 was carried out, 40%-50% of 55712. Furthermore, on the ileum, the contractions induced added radioactivity was recovered. In addition, control ex- by samples of peak I were similar to those evoked by LTC4- periments suggested that 20%-30% of LTC4 was lost during i.e., with a slower rate of increase than the response to LTD4 the initial filtration. Assuming equal recovery of each leuko- (Fig. 3B) (cf. ref. 15). The total amount of LTC4-like activity triene, the average production of LTC4, LTD4, and LTE4 in peak I, as estimated by final quantitation on the lung strip, during 10 min of incubation was estimated to be 25, 8, and 0.7 was very close (=100 pmol) to the amount determined by pmol per g of brain tissue (wet weight), respectively. radioimmunoassay. Regional Distribution of LTC4 Production. Stimulation with The recovery of LTC4 during the purification procedure ionophore A23187 (5 AM) and arachidonic acid (75 AtM) was estimated by the use of radiolabeled compounds. After induced synthesis of LTC4 in slices of all regions tested (Fig. 4). Highest levels of LTC4 were produced by the hypo- thalamus (2.9 + 1.32 pmol per slice), the median eminence (2.1 + 0.92 pmol per slice), and nucleus accumbens/ tuberculum olfactorium (1.6 + 0.44 pmol per slice), whereas 0 2- the production in slices from the cerebellum was small (0.37 + 0.06 pmol per slice) (mean + SD). Background levels E T ol1T1 obtained with tissue incubated in the absence of ionophore u and exogenous arachidonic acid never exceeded 0.15 + 0.03 pmol per slice. LTC4 Production in the Caudate Nucleus. Ionophore A23187 (0-20 AM) in the absence of exogenous arachidonic C, acid caused a dose-dependent stimulation of LTC4 formation +dCOt+ c9 o tIq -q '2e9,;i in slices from the caudate nucleus (Fig. 5). At 0.1 ,M, the +~~~ ionophore caused a 2-fold increase in LTC4 levels. Maximal production was obtained at 5 ,uM ionophore (4-fold stimu- FIG. 4. Regional distribution of LTC4 biosynthesis in various lation). At higher concentrations, a marked decrease in stim- parts of the rat brain. One (Em. Med; median eminence) or two was slices from each of the indicated regions were preincubated in 0.5 ulatory effect observed. ml of Pi/NaCl (pH 7.4) for 5 min prior to a 10-min incubation with In other experiments, slices from the caudate nucleus were ionophore A23187 (5 uM) and arachidonic acid (75 jiM). Each mixed with slices from the cortex and the median eminence. LTC4 value is the mean + SD of duplicate determinations on sam- After preincubation with or without nordihydroguaiaretic ples from three different animals (n = 3). Statistical analyses were acid (30 AM) for 5 min, the samples were incubated with performed using Student's t test: n.s., not significant; *, P < 0.05; ionophore A23187 (5 AM) and arachidonic acid (75 pM) for **, P < 0.01; ***, P < 0.001 (differences in LTC4 values com- 10 min. In stimulated samples, an average level of 8.2 pmol pared to the value obtained in samples from the cerebellum). Cor- of LTC4 per ml was determined, as compared to 1.8 pmol of tex ant., anterior cortex; Cortex post., posterior cortex; Hippoc., LTC4 per ml in samples preincubated with nordihydroguai- hippocampus; Acc. + tub. olf., nucleus accumbens and tubercu- lum olfactorium; Caud., caudate nucleus; Thal. ant., anterior aretic acid (78% inhibition). In unstimulated controls, the thalamus; Thal. post., posterior thalamus; Hypothal., hypo- average level of LTC4 was 0.6 pmol/ml. thalamus; Em. med., median eminence; S. nigra, substantia nigra; Immunohistochemical Determination of LTC4 in Rat Brain. P.A.G., periaqueductal grey matter; Cereb., cerebellum; Med. Two patterns of immunoreactivity were observed after in- obl., medulla oblongata. cubation with LTC4 anti-plasma. In the lateral part of the Downloaded by guest on October 1, 2021 Medical Sciences: Lindgren et al. Proc. Natl. Acad. Sci. USA 81 (1984) 6215

I FIG. 6. Immunofluorescence micrographs of the median eminence (a and b), supramammillary region (c), medial preoptic area (d), re- ticular thalamic nucleus (e), and parietal cortex (f ) after incubation with LTC4 anti-plasma (a, c-f ) or LTC4 anti-plasma preabsorbed with LTC4/bovine serum albumin conjugate (b). a and b are consecutive sections. Asterisk denotes third ventricle. LTC4 immunoreactive fibers are seen mainly in the lateral external layer of the median eminence (arrowheads in a) with single nerves in the supramammillary region (c). Fluorescent cell bodies (arrowheads in d) are seen in the preoptic area. Note that the nuclei lack fluorescence (d). No fibers are seen after incubation with control serum (b). (e and f ) Strongly fluorescent cell bodies are seen in many brain areas. The specificity of this fluores- cence is uncertain, because it can be blocked only partially by absorption with LTC4/bovine serum albumin conjugate. (Bars = 25 Aum.) external layer of the median eminence a medium-dense The LTC4 formation induced by ionophore A23187 was dose plexus of LTC4 immunoreactive fibers was observed with dependent, and leukotriene production could be inhibited by anti-plasma dilutions up to 1:3200 (Fig. 6a). The fibers were the lipoxygenase inhibitor nordihydroguaiaretic acid (19). strongly varicose and terminated close to the portal vessels. Radioimmunological determinations indicated that the ca- Single fibers with the same appearance were also observed in pacity to produce LTC4 was widely distributed in the brain. other brain regions, including many areas in the hypo- However, the detected amounts of LTC4 varied among dif- thalamus (Fig. 6c), septum, and the subfornical organ. In ferent brain regions, with the highest levels found in the addition, weakly fluorescent cell bodies were seen in the hypothalamus and parts of the basal ganglia. The results medial preoptic area (Fig. 6d). Both fibers and cell bodies suggest distinct differences in biosynthetic capacity between were also observed after incubation with LTC4 anti-plasma the investigated brain regions; however, differences in ca- preabsorbed with LTC4, but not after preabsorption with tabolism of LTC4 may also contribute. LTC4/bovine serum albumin conjugate (Fig. 6b). This fluo- Evidence for the presence of LTC4 was also obtained in rescence was also completely abolished by preabsorption of the immunohistochemical analysis, demonstrating immuno- the LTC4 anti-plasma with 800 AM glutathione disulfide but reactive fibers in the median eminence and cell bodies in the not with 80 AtM. Using higher concentrations of antibodies preoptic area. So far, however, this immunofluorescence (up to 1:800 dilution), numerous fluorescent cell bodies were could only be abolished by absorption with LTC4/bovine seen in brain 6 e This serum albumin conjugate but not by LTC4 alone. Control many regions (Fig. andf). fluorescence experiments revealed that this lack of effect of LTC4 alone was not affected by absorption with LTC4 or glutathione was not due to break-down of the compound during the disulfide (up to 800 AuM), but a detectable decrease was incubation (unpublished data). A somewhat similar situation observed when using anti-plasma preabsorbed with has previously been encountered in immunohistochemical LTC4/bovine serum albumin conjugate. Absorption with studies on serotonin-containing neurons. Thus, much higher bovine serum albumin had no effect on the LTC4-like im- concentrations of serotonin alone had to be used to abolish munoreactivity. the immunoreaction as compared to serotonin conjugated to bovine serum albumin (20). The fluorescence of widely dis- DISCUSSION tributed cell bodies could not be abolished by absorption by LTC4 and only to a certain degree by the LTC4/bovine The present results demonstrate the formation of LTC4, serum albumin conjugate. The finding that the immunoreac- LTD4, and LTE4 in the rat brain. Identification of the prod- tion in the median eminence was sensitive to high concen- ucts was based on data from HPLC, UV spectroscopy, trations of glutathione disulfide (10 times higher than bioassay, and a specific radioimmunoassay against LTC4. LTC4/bovine serum albumin conjugate), taken together with Downloaded by guest on October 1, 2021 6216 Medical Sciences: Lindgren et al. Proc. Natl. Acad. Sci. USA 81 (1984) the lack of effect of LTC4, makes the identity of the im- 00217, 04X-02887, and 03P-06949), Karolinska Institutet Research munoreactivity uncertain. Further investigations are re- Funds, and Alice and Knut Wallenberg Foundation. quired to establish the nature of the antigen reacting with our LTC4 anti-plasma (18). 1. Samuelsson, B. (1983) Science 220, 568-575. Arachidonic acid metabolism by brain tissue was first 2. Borgeat, P., Hamberg, M. & Samuelsson, B. (1976) J. Biol. described 20 yr ago (21). Numerous studies have Chem. 251, 7816-7820, and correction (1977) 252, 8872. demon- 3. Borgeat, P. & Samuelsson, B. (1979) Proc. Natl. Acad. Sci. strated the formation of cyclooxygenase products such as USA 76, 3213-3217. D2, E2, and F2, and in nervous 4. Borgeat, P. & Samuelsson, B. (1979) Proc. Natl. Acad. Sci. tissue (11), whereas prostacyclin formation in brain from USA 76, 2148-2152. various species originates mainly from the vasculature (11). 5. Corey, E. J., Marfat, A., Goto, G. & Brion, F. (1981) J. Am. The physiological role of prostaglandins in the central nerv- Chem. Soc. 102, 7984-7985. ous system is still incompletely known. However, E-type 6. Murphy, R. C., Hammarstrom, S. & Samuelsson, B. (1979) prostaglandins may be involved in generation of hyperther- Proc. Natl. Acad. Sci. USA 76, 4275-4279. mia (22) and have a general action to modulate secretion of 7. Morris, H. R., Taylor, G. W., Piper, P. J. & Tippins, J. R. neurotransmitters (23). In addition, these prostaglandins have (1980) Nature (London) 285, 104-106. 8. Orning, L. & Hammarstrom, S. (1980) J. Biol. Chem. 255, an anticonvulsant effect (11), which may relate to an excit- 8023-8026. atory effect on certain brain stem neurons (24) and cerebellar 9. Bernstrom, K. & Hammarstrom, S. (1981) J. Biol. Chem. 256, Purkinje cells (25). A major formed in the 9579-9582. central nervous system is prostaglandin D2, which seems to 10. Austen, F. (1978) J. Immunol. 121, 793-805. have regulatory effects on pyrogen-altered body temperature 11. Wolfe, L. S. (1982) J. Neurochem. 38, 1-14. and sleep pattern (26). 12. Palmer, M. R., Matheus, R., Murphy, R. C. & Hoffer, B. J. More recently, epoxy acids formed by a NADPH-depen- (1980) Neurosci. Lett. 18, 173-180. dent cytochrome P-450 linked monooxidase pathway have 13. Lindgren, J. A., Hammarstrom, S. & Goetzl, E. J. (1983) been described in the rat hypothalamic microsomal fraction FEBS Lett. 152, 83-88. 14. Dahldn, S.-E. (1983) Acta Physiol. Scand. 118, Suppl. 512, (27). The major metabolite formed by this pathway, 5,6- 1-51. epoxy-eicosatrienoic acid, stimulated release of somato- 15. Bjorck, T., Dahldn, S.-E., Gustavsson, L.-E. & Hedqvist, P. statin and luteinizing hormone-releasing hormone from the (1984) Acta Physiol. Scand., in press. hypothalamic median eminence in vitro (27) and luteinizing 16. Zamboni, L. & de Martino, C. (1967) J. Cell Biol. 35, 148A hormone release from dispersed anterior pituitary cells (28). (abstr.). Lipoxygenase activity in the brain has been indicated by 17. Coons, A. H. (1958) in General Cytochemical Methods, ed. the formation of 12-hydroxyicosatetraenoic acid in brain Danielli, J. F. (Academic, New York), pp. 399-422. cortex (29, 30). The present study documents the presence of 18. Aehringhaus, U., Wolbling, R. H., Konig, W., Patrono, C., a 5-lipoxygenase pathway and the of brain Peskar, B. M. & Peskar, B. A. (1982) FEBS Lett. 146, 111-114. ability tissue to 19. Tappel, A. L., Lundberg, W. 0. & Boyer, P. D. (1953) Arch. form cysteinyl-containing leukotrienes. Leukotriene forma- Biochem. Biophys. 42, 293-304. tion by leukocytes present in the brain vessels is not likely, 20. Steinbusch, H., Verhofstad, A. & Joosten, H. (1978) Neuro- because the animals were perfused with buffer. Generation science 3, 811-819. ofleukotrienes by vascular tissue has recently been indicated 21. Samuelsson, B. (1964) Biochim. Biophys. Acta 84, 218-219. (31, 32). However, the present immunohistochemical data 22. Feldberg, W. & Milton, A. S. (1978) in Handbook of Experi- suggest leukotriene production by nervous tissue. mental Pharmacology, eds. Vane, J. R. & Ferreira, S. H. The (Springer, Berlin), Vol. 50, pp. 615-656. observation that LTC4 was formed in the cerebellum 23. Hedqvist, P. (1977) Annu. Rev. Pharmacol. Toxicol. 17, may have functional implications, because LTC4 and LTD4 259-279. cause a conspicuously prolonged excitation of cerebellar 24. Quesney, L. F., Gloor, P., Wolfe, L. S. & Jozsef, S. (1975) Purkinje cells (12, 33). However, the highest amounts of Prostaglandins 10, 383-393. LTC4 were detected in the hypothalamus and median emi- 25. Siggins, G., Hoffer, B. & Bloom, F. (1971) Ann. N.Y. Acad. nence. It is of particular interest in this context that low Sci. 180, 302-323. 26. Hayaishi, 0. (1983) in Advances in Prostaglandin, Thrombox- concentrations of LTC4 (0.01 pM) specifically evoked re- ane and Leukotriene Research, eds. Samuelsson, B., Paoletti, lease of luteinizing hormone from dispersed anterior pitui- R. & Ramwell, P. (Raven, New York), Vol. 12, pp. 333-337. tary cells (unpublished observations). Furthermore, the dis- 27. Capdevila, J., Chacos, N., Falck, J. R., Manna, S., Negro- tribution of the LTC4 immunoreactive fibers in the external Vilar, A. & Ojeda, S. R. (1983) Endocrinology 113, 421-423. layer of the median eminence was identical to that of lut- 28. Snyder, G. D., Capdevila, J., Chacos, N., Manna, S. & Falck, einizing hormone-releasing hormone. Taken together, the J. R. (1983) Proc. Natl. Acad. Sci. USA 80, 3504-3507. 29. Sautebin, L., Spagnuolo, C., Galli, C. & Galli, G. (1978) present findings indicate that the leukotrienes may have a Prostaglandins 16, 985-988. role as messengers or modulators of central nervous activity 30. Spagnuolo, C., Sautebin, L., Galli, G., Racegui, G., Galli, C., and neuroendocrine events. Mazzani, S. & Ginesso, M. (1979) Prostaglandins 18, 53-61. 31. Piper, P. J., Letts, L. G. & Galton, S. A. (1983) Prostaglan- We wish to thank Ms. Margareta Maxe, Anne Peters, Waldtraut dins 25, 591-599. Hiort, Sandra Kleinau, and Lilian Franzen for skillful technical 32. Wolbling, R. H., Aehringhaus, U., Peskar, B. M. & Peskar, assistance. This work was supported by grants from the Swedish B. A. (1983) Prostaglandins 25, 823-828. Medical Research Council (projects 03X-06805, 03P-06347, 03X- 33. Palmer, M. R., Mathews, R., Hoffer, B. J. & Murphy, R. C. (1981) J. Pharmacol. Exp. Ther. 219, 91-96. Downloaded by guest on October 1, 2021