Arachidonate 5-Lipoxygenase and Its Activating Protein: Prominent Hippocampal Expression and Role in Somatostatin Signaling

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

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 hybridization, 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-dependent K~current (Brown and Adams, 1980) that, in

The leukotrienes, a family of biologically active metabolites 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.

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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 was (Pelamp, Axon Instruments) and measured ‘Nt amplitude performed for 15 cycles and that of 5-LO eDNA for 20 with software (Clampfit, Axon Instruments) that fitted expo- cycles. We subjected the PCR products to agarose (1%) nential curves of the 154 relaxation peak of the initial (quasi- gel electrophoresis and transferred them onto nitrocellulose. instantaneous) current. Prehybridization was at 56°C for 2hinlOx Denhardt’s solution/20 mM Na2HPO4/NaH2PO4 buffer, pH 6.8/5x standard saline citrate (SSC)/l% sodium dodecyl sulfate RESULTS (SDS)/denaturated salmon sperm DNA (0.1 mg/ml). We performed hybridization for 3 h at 56°C,in prehybridization32P-labeled 46- We localized 5-La mRNA35S-labeledin rat CNSoligonucleotideby in situ solutionmer oligonucleotidecontaining 10%probesdextran(5 X I sulfate~6 dpmlml)and corresponding probe.hybridization,We foundusing the highesta labeling densities in hip- to nucleotides 86—132 (FLAP-I) and 285—331 (FLAP-2) pocampus and cerebellum and moderate labeling in of rat FLAP sequence (Dixon et al., 1990) and nucleotides primary olfactory cortex, superficial neocortex, thala- 818—864 of rat 5-LO sequence (Balcarek et al., 1988). We mus, hypothalamus, and brainstem (Fig. lA). In situ washed blots twice with 2X SSC/0.l% SDS/l0>< Den- hybridization with a 35S-labeled FLAP probe revealed hardt’s solution/lU mM Na a distribution of transcripts that appeared to overlap 2HPO4/NaH,P04 buffer, pH 6.8, at 56°Cfor 30 mm. with that of 5-L0, with highest hybridization signals in hippocampus and cerebellum. In addition, clear signals In situ hybridization were visible in primary olfactory cortex, neocortex, We prepared and incubated brain sections essentially as brainstem, and thalamus (Fig. 1B). By contrast, only described previously (Bouthenet et al., 1991). For FLAP,

I. Neurochem., Vol. 66, No. I, /996 5-LIPOXYGENASE AND FLAP IN BRAIN NEURONS 149

To localize FLAP protein, we used a rabbit poly- clonal antiserum selective for FLAP (Dixon et al., 1990). We assessed the selectivity of theimmunostain- ing procedure by (I) showing that the antiserum stained a single band of 18 kDa on western blots of rat brain particulate fractions, (2) replacing the primary antiserum with preimmune serum (not shown), and (3) demonstrating that FLAP antiserum and ~S- labeled FLAP probe labeled the same neuronal elements. Both FLAP mRNA and FLAP immunostaining were dense in hippocampus (Fig. 3A, B), where they were associated with pyramidal cells in CAl (Fig. 3C) to CA3, and with granule cells in dentate gyrus (not shown). We also determined the cellular distribution of 5-LO mRNA. Figure 3D and E show silver grains overlying neuronal elements within the hippocampal CAl field. In agreement with the histologic localization, the num- ber of silver grains associated with neurons was sig- nificantly higher in hippocampus than in neocortex or thalamus. We counted 16.2 ~ 1.7 grains per neuron (mean ±SEM, n = 53) in the hippocampal CAl field and 14.2 ± 1 .3 grains per neuron in the dentate gyrus. By contrast, we measured only 6.5 ± 0.6 grains per neuron in the neocortex and 5.5 ±0.9 grains per neuron in the thalamus (p < 0.05, ANOVA). A sense- oriented 5-LO probe revealed no specific labeling (1 .6 ±0.3 grains per neuron, n = 30 for all regions). The extensive overlap between the cellular distribution of 5-LO and that of FLAP suggested that the two proteins may be coexpressed in CNS neurons. To examine this possibility, we stained sections of hippo- FIG. 1. Histologic localization of 5-LO and FLAP mRNAs in para- 35S-labeled 5- campal tissue with FLAP antiserum and hybridized sagittalLO oligoprobesections(A), of rata 35S-labeledbrain. HybridizationFLAP oligoprobe with a (B), and a them with the 35S-labeled 5-LO probe. As shown in 35S-labeled control missense probe (C) (for sequence, see Ex- Fig. 3E, most neurons in the CAl pyramidal cell layer perimental Procedures). Highest labeling densities are observed in hippocampus (Hi) and cerebellum (Cb); moderate labeling is showed both immunostaining and hybridization sig- observed in neocortex (Cx), thalamus (Th), primary olfactory nals, confirming the colocalization of 5-LO and FLAP. cortex (P0), and choroid plexus (ChP). To determine if FLAP is functional in hippocampus, diffuse, nonspecific labeling was seen in adjacent sections treated with a control, 35S-labeled missense probe (Fig. 1C). We obtained equivalent results using a labeled 5-LO riboprobe (derived from human 5-LO eDNA, which displays 93% identity with its rat homo- logue) and two additional 35S-labeled FLAP probes (data not shown). RT-PCR analysis confirmed that 5-LO and FLAP mRNAs areexpressed in rat brain. Southern blot analysis revealed PCR products of expected size when poly (A) + RNA samples from various regions of the FIG. 2. Expression of 5-LO (A) and FLAP mRNA5 (B, C) in rat brain were subjected to RT-PCR using oligonucleo- various regions of the rat brain determined by RT-PCR followed tide primers selective for 5-LO or FLAP (Fig. 2). It by Southern blot analyses. Samples of poly(A)~RNA (1 jig) is important that, although 5-LO and FLAP appear to were subjected to RT-PCR, fractionated by agarose gel electro- be present throughout the brain, our RT-PCR condi- phoresis and hybridized with selective 32P-labeled oligonucleo- tions would not provide a quantitative assessment of tide probes. Two distinct probes, FLAP1 (B) and FLAP2 (C), were used to detect products of FLAP mRNA amplification (for their level of expression; i.e., more stringent quantita- sequences, see Experimental Procedures). Blots were exposed tive PCR analyses must be performed to determine to x-ray films for 1 h at -—80°C. Molecular sizes (in kb) of the these levels. PCR products obtained are indicated by the bars.

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A—C: FIG. 3. Cellular localization of355-labeled 5-LO and FLAPFLAPmRNA5oligoprobe,and visualizedFLAP immunoreactivityusing dark-fieldin illuminationthe hippocampal(A), andCAlFLAP field.polyclonalLocalizationantiserum ofvisualizedFLAP in usingsectionslight-fieldexposed illuminationto (B). Cisa light-field detail of A with greater magnification, to show the cellular distribution of the FLAP transcript. D: Localization of 5-LO mRNA in a section exposed to 35S-labeled 5-LO oligoprobe visualized using dark-field illumination. E: Colocalization of FLAP immunoreactivity and 5-LO mRNA. Note the silver grains (355-labeled 5-LO oligoprobe) overlying FLAP-immunoreactive neurons. Bars indicate 50 p.m (except in C, where it indicates 20 pm).

we assessed the ability of the selective FLAP inhibitor 14 ± 10% increase; Fig. 4B). When MK-886 was MK-886 to affect the somatostatin augmentation of 154. superfused first (protocol 2), seven of eight cells did

We recorded intracellularly from 18 CAl pyramidal not show any ‘M increase with subsequent somatostatin neurons with an average resting membrane potential addition (1 ±7to8±5% increase overall; Fig. 4C). of —69 ±0.6 mV (mean ±SEM) and action potential To rule out nonspecific effects of MK-886 on the M- amplitude of 106 ±1.3 mV. We used two drug admin- channel, we also superfused LTC istration protocols; in protocol I, we first superfused 4 in the presence somatostatin (I jiM, 2—4 mm) to demonstrate an 154 of MK-886; LTC4 (6—8 pM) still increased IM (two increase, then washed out the peptide while superfus- cells; Fig. 4A), suggesting that MK-886 prevents ing MK-886 (0.25—1 jiM, 18—25 mm), and finally the somatostatin response by inhibiting biosynthesis superfused somatostatin again (2—4 mm) in the contin- of LTC4. ued presence of MK-886. In protocol 2, to ensure that the somatostatin response was not reduced by desensi- DISCUSSION tization, we superfused MK-886 first and then applied somatostatin in the presence of MK-886. In this report we show that 5-LO and FLAP, which Our results showed that MK-886 inhibited com- play apivotal role in the cellular biosynthesis of leuko- pletely the effect of somatostatin on ‘M using either trienes in peripheral tissue, are coexpressed in discrete protocol (Fig. 4). Superfusion of somatostatin alone neuronal populations of the CNS. By combining photo-

(protocol 1) increased ‘M amplitude by 45 ±6to75 graphic emulsion and immunohistochemical tech- ± 16% in eight of 10 cells, with recovery to control niques, we also demonstrate that 5-LO mRNA and levels after washout (15—25 mm). In the eight re- FLAP protein are expressed in the same neurons, as sponding cells, somatostatin showed very little effect suggested by their concerted role in leukotriene biosyn- when reapplied in the presence of MK-886 (8 ±9to thesis. Our findings, together with those of Nishiyama and coworkers (1993), establish that mammalian CNS

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ies have demonstrated that the IM-augmenting effect of somatostatin in these neurons are prevented by inhibiting PLA2 or 5-LO activities and mimicked by exogenous LTC4 (Schweitzer et al., 1990, 1993). Thus, leukotrienes may serve, in CNS neurons, a signaling function similar to that proposed for I2-lipoxygenase metabolites in Aplysia. Our results raise several questions. We do not know yet whether somatostatin stimulates formation of leukotrienes in hippocampal neurons, and, if so, through what mechanism. The discovery that a somatostatin receptor (SSTR4) highly expressed in hippocampus is linked to PLA2 activation and release of arachidonic acid suggests the involvement of this transmembrane signaling pathway (Bito et a]., 1994; Sakanaka et al., 1994). Further, the mechanism of action of LTC4 in regulating M-channel function is still unknown. LTC4 may act on intracellular targets (e.g., the M-channel), as shown for I 2-lipoxygenase metabolites in Aplysia sensory neurons (Belardetti et al., 1989). Alterna- tively, LTC4 may exit the cell of origin and act by binding to membrane receptors on adjacent neurons, a hypothesis supported by theoccurrence of high-affinity binding sites for LTC4 in neural tissue (Schalling et al., 1986). Interest in this question lies in the possibility that LTC4 may provide a diffusible message, of the type postulated to operate in synaptic plasticity and development, linking intracellular to extracellular sig- FIG. 4. Neutralization of the somatostatin effect on ‘M by the naling (Harish and Poo, 1992). Finally, we have found FLAP inhibitor MK886. A: ‘M recording of a CAl neuron held at —46 mV (resting potential was —70 my); ‘M appears as a slow other brain regions, besides hippocampus, that express inward current ‘relaxation” after the instantaneous inward cur- relatively high levels of 5-LO and FLAP mRNAs. Fur- rent drop. Somatostatin was applied at 1 pMand LTC 4 at 8 jiM. ther experiments are necessary to demonstrate the cel- B: Average of‘M amplitudes in eight neurons tested with protocol lular localization of these transcripts in such regions, 1 [somatostatin first (A); see text]. C: Average of I~amplitudes for instance, in the cerebellum, and to determine in eight neurons tested with protocol 2, i.e., initial exposure to whether 5-LO metabolites serve signaling functions MK-886 (0) followed by somatostatin application (V) in thecon- tinuous presence of MK-886. similar to that proposed here for hippocampal CAl neurons.

Acknowledgment: We thank Dr. A. W. Ford-Hutchinson neurons possess the enzymatic complement necessary (Merck-Frosst, Canada) for many of the tools used in to produce both 5- and 12-lipoxygenase metabolites. this study, D. Vial for technical help, Dr. J.-C. Schwartz for In further support of this conclusion, formation of leu- support, and Drs. F. E. Bloom and J. Diaz for suggestions. kotrienes was demonstrated in a preparation of purified This study was supported in part by a grant from pinched-off synaptic terminals (synaptosomes), which NARSAD (D.P.), NIMH (MH44346), NIAAA (AA06420, contain little or no astrocyte contamination (Yates et AA07456), and NATO (P.S. and G.R.S.). C.-H.L. was the recipient of a fellowship from the Deutsche Forschungsge- al., 1990). By contrast, embryonic neurons cultured in meinschaft. serum-free medium do not produce eicosanoids, a re- sult that is likely explained by the culture conditions REFERENCES used in those experiments (Oomagari et al., 1991). Arachidonic acid metabolites produced via the 12- Abramovitz M., Wong E., Cox M. E., Richardson C. D., Li C., and lipoxygenase pathway have been proposed to act as Vickers P. J. (1993) 5-Lipoxygenase-activating protein stimu- intracellular second messengers in invertebrate and lates the utilization of arachidonic acid by 5-lipoxygenase. Eur. J. I3iochem. 215, 105—111. vertebrate neurons (Piomelli et al., 1987, 1989; Belar- Adesuyi S. A., Cockrell C. S., Gamache D. A., and Ellis E. F. detti et al., 1989; Dorman et al., 1992). Several find- (1985) Lipoxygenase metabolism of arachidonic acid in brain. ings support the possibility that leukotrienes play a J. Neurochern. 45, 770—776. similarrole. Here,we show that MK-886, a leukotriene Balcarek J. M., Theisen T. W., Cook M. N., Varrichio A., Hwang biosynthesis inhibitor that acts by binding to FLAP, S. M., Stroh.sacker M. W., and Crooke S. T. (1988) Isolation and characterization of a cDNA clone encoding rat 5-lipoxygen- prevents somatostatin enhancement of the K + M-cur- ase. J. Biol. Chem. 263, 13937—13941. rent in hippocampal pyramidal neurons. Previous stud- Belardetti F., Campbell W. B., Faick J. R., Demontis G., and Roso-

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