UC Irvine UC Irvine Previously Published Works Title Arachidonate 5-lipoxygenase and its activating protein: prominent hippocampal expression and role in somatostatin signaling. Permalink https://escholarship.org/uc/item/5sg8x09h Journal Journal of neurochemistry, 66(1) ISSN 0022-3042 Authors Lammers, CH Schweitzer, P Facchinetti, P et al. Publication Date 1996 DOI 10.1046/j.1471-4159.1996.66010147.x License https://creativecommons.org/licenses/by/4.0/ 4.0 Peer reviewed eScholarship.org Powered by the California Digital Library University of California Journal of Neurochemistry Lippincott—Raven Publishers, Philadelphia © 1996 International Society for Neurochemistry Arachidonate 5-Lipoxygenase and Its Activating Protein: Prominent Hippocampal Expression and Role in Somatostatin Signaling *C...H. Lammers, tP. Schweitzer, ~ Facchinetti, *J..M. Arrang, 1S. G. Madamba, tO. R. Siggins, and *tD. Piomelli * Unite de Neurobiologie et Pharmacologie de I ‘INSERM, Paris, France; and tScripps Research Institute, La Jolla; and ~Neurosciences Institute, San Diego, California, U.S.A. Abstract: 5-Lipoxygenase—activating protein (FLAP) is 2~ an 18-kDa integral membrane protein required, in periph- andpie cofactorsATP (Ford-Hutchinsonto become fullyet ai., active,1994).includingRises inCain- eral cells, for the activation of 5-lipoxygenase (5-LO) and tracellular Ca2~cause the transiocation of 5-LO from for the resulting synthesis of leukotrienes from arachi- cytosoi to membrane, where the enzyme interacts with donic acid. In the brain, the leukotrienes have been impli- an 18-kDa integral membrane protein, 5-lipoxygen- cated in several pathophysiological events and in the ase—activating protein (FLAP), required for leuko- electrophysiological effect of somatostatin, yet the cellu- triene biosynthesis in peripheral cells (Dixon et al., lar origin and role of these messenger molecules are still 1990; Miller et al., 1990; Abramovitz et al., 1993). poorly understood. In the present study, we used reverse Arachidonic acid metabolism via the 5-LO pathway transcriptase—polymerase chain reaction, in situ hybrid- ization, and immunohistochemistry to demonstrate that may play important roles in the CNS (for review, see 5-LO and FLAP are expressed in various regions of the Piomelli, 1994). In vitro, preparations of brain tissue rat brain, including hippocampus, cerebellum, primary 01- convert exogenous and endogenous arachidonic acid factory cortex, superficial neocortex, thalamus, hypothal- to leukotrienes and to other 5-LO products (Lindgren amus, and brainstem. Highest levels of expression were et al., 1984; Adesuyi et al., 1985; Shimizu et al., 1987). observed in cerebellum and hippocampus. In the latter In vivo, leukotriene biosynthesis is stimulated by the we demonstrate the colocalization of 5-La and FLAP in intracerebroventricular administration of platelet-acti- CAl pyramidal neurons. Moreover, electrophysiological vating factor and is associated with the induction of experiments show that selective inhibition of FLAP with seizure activity, ischemia, and intracranial tumors the compound MK-886 (0.25—1 1sM) prevents the so- (Simmet and Peskar, 1990; Hynes et al., 1991). More- matostatin-induced augmentation of the hippocampal K~ over, LTC M-current. Our results provide necessary evidence for the 4 exerts multiple actions on neural cells, such presence and signaling role of 5-LO and FLAP in central as prolonged excitation of cerebellar Purkinje neurons neurons and strongly support their proposed participa- (Palmer et al., 1981), stimulation of luteinizing hor- tion in somatostatin-receptor transmembrane signaling. mone release from anterior pituitary cells (Kiesel et Key Words: Arachidonic acid— Leukotrienes—Signal al., 1991), and enhancement of the M-current (IM) in transduction— Hippocampus. the hippocampal CAl field (Schweitzer et al., 1990, J. Neurochem. 66, 147—152 (1996). 1993). The ‘M is a noninactivating time- and voltage-depen- dent K~current (Brown and Adams, 1980) that, in The leukotrienes, a family of biologically active me- tabolites of arachidonic acid, are synthesized through Received April 20, 1995; final revised manuscript received July a sequence of enzymatic reactions initiated by 5-lipox- 10, 1995; accepted July 13, 1995. ygenase (5-LO), a cytosolic dioxygenase that cata- The present address of Dr. C-H. Lammers is Max Planck Institute for Psychiatry, Clinical Institute, Munich, Germany. lyzes both the initial oxidation of arachidonic acid to Address correspondence and reprint requests to Dr. D. Piomelli 5-hydroperoxy-6,8,1 1,14-eicosatetraenoic acid and its at Neurosciences Institute, 10640 John J. Hopkins Drive, San Diego, subsequent dehydration to leukotriene A4 (LTA4). CA 92121, U.S.A. LTA4 is short lived and may be rapidly transformed Abbreviations used: FLAP, 5-lipoxygenase—activating protein; either into LTC4, by LTC4-synthase, or into LTB4, 154, M-current, a noninactivating time- and voltage-dependent K~ current; 5-LO, 5-lipoxygenase; LT, leukotriene; PCR, polymerase by LTA4-hydrolase (Samuelsson et al., 1987). Unlike chain reaction; PLA2, phospholipase A2 RT, reverse transcriptase; other mammalian lipoxygenases, 5-LO requires multi- SDS, sodium dodecyl sulfate; SSC, standard saline citrate. /47 148 C.-H. LAMMERS ET AL. hippocampal pyramidal neurons, is reduced by musca- hybridizations were performed in the presence of 50%35S-la-for- rinic receptor agonists (Halliwell and Adams, 1982) beledmamideantisenseand 10 oligonucleotidesmM dithiothreitol,(baseusing89—109syntheticfor 5-LO and and augmented by the neuropeptide somatostatin —6 to 14 for FLAP). We used as control a missense oligonu- (Moore et al., 1988). Pharmacological evidence indi- cleotide (5 ‘-ACA TAT CTG ACT TCG AAT GCC-3’). In cates that a 5-LO metabolite of arachidonic acid medi- some experiments, distribution of 5-LO mRNA was con- ates the effect of somatostatin; ‘M enhancement by so- firmed by using a riboprobe prepared with a Promega ribo- matostatin is prevented by drugs that inhibit phospholi- probe kit from a Smal—BamHI DNA fragment correspond- pase A ing to nucleotides 1,417—1,735 of human 5-LO sequence 2 (PLA2) or 5-LO activities and mimicked by (Dixon et al., 1988), obtained by excision of a recombinant applications of exogenous arachidonic acid or LTC4 5-LO pUC 13 plasmid vector (generously provided by Dr. but not by application of LTB4 (Schweitzer et al., 1990, A. Ford-Hutchinson, Merck-Frosst, Canada) and subeloned 1993). in pGEM-4Z. We confirmed distribution of FLAP mRNA Although these studies suggest a signaling role for by using two 32P-labeled oligonucleotide probes, correspond- leukotrienes in the CNS, it is not known whether the ing to nucleotides 86—132 and 285—331 of the rat FLAP enzymatic machinery necessary for leukotriene biosyn- sequence (Dixon et al., 1990). thesis is expressed in neurons and under what circum- Immunohistochemistry stances it may become activated. Here, we report that After a 5-mm fixation with 4% paraformaldehyde, sec- 5-LO and FLAP are coexpressed in rat brain neurons tions were rinsed in phosphate-buffered saline solution, incu- and enriched in discrete regions of the CNS including bated for 12 hat 4°Cwith FLAP antiserum (a gift of Dr. the hippocampus. To test whether FLAP is physiologi- A. Ford-Hutchinson) at a 1:500 dilution, rinsed, incubated cally functional, and involved in somatostatin modula- with biotin-labeled anti-rabbit protein antiserum, treated with avidin/biotin/horseradish peroxidase complex and devel- tion of ‘M~ we used the compound MK-886, a potent and selective FLAP inhibitor (Dixon et al., 1990). oped with diaminobenzidine for 5 mm. We find that the effect of somatostatin on ‘M in the Electrophysiology hippocampal slice preparation is abolished by MK- We performed intracellular recordings in rat hippocampal 886. Thus, our results provide strong support to a re- slices, as described previously (Schweitzer et al., 1993). In quirement of 5-LO and FLAP in transmembrane sig- brief, we cut transverse hippocampal slices (350—400 jim naling of somatostatin receptors in hippocampal neu- thick) and superfused them (2.0—4.0 mI/mm) with warm rons. (30—31°C),gassed (95% 02,5% C0 2) artificial CSF of the following composition (mM): NaCI 130, KC1 3.5, NaH2PO4 EXPERIMENTAL PROCEDURES 1.25, MgSO4 1.5, CaCl-, 2.0, NaHCO2 24, glucose 10. We Reverse transcriptase (RT) —polymerase chain dissolved MK-886 (a gift of Dr. A. Ford-Hutchinson) and reaction (PCR) analysis LTC4 (Biomol Research Laboratories, Plymouth Meeting, PA, U.S.A.) in 0.05% dimethyl sulfoxide, which alone had Poly (A) + mRNAs prepared from various regions of the no significant effect on ‘M~ We used sharp glass micropi- Wistar rat brain served as templates for eDNA synthesis pettes filled with 3 M KCI (tip resistances, 55—88 mIl) to using avian myeloblastosis virus RT (25 units, Boehringer). penetrate CA I pyramidal neurons for voltage clamp studies, We used the cDNAs obtained as templates to amplify, by using an Axoclamp preamplifier (Axon Instruments) in dis- PCR, DNA fragments corresponding to nucleotides —9 to continuous single-electrode voltage-clamp mode. We added 459 of rat FLAP sequence (Dixon et al., 1990) and nucleo- tetrodotoxin (0.5—1 pM) to block synaptic transmission and tides 442—1,028 of rat 5-LO sequence (Balcarek et al., Nat-dependent action potentials. We acquired current and 1988). PCR conditions were as follows: 92°C, 56°C,and voltage records by A/D sampling and acquisition software 72°C for I mm each; amplification of FLAP eDNA