Role of phospholipase a2 in orofacial pain and synaptic transmission 3

Discussion The present study elucidated the expression profile of multiple sPLA2 isoforms in the rat CNS with focus on sPLA2-IIA in the brainstem and spinal cord.. Of the isoforms, sPLA

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Chapter 4 Role of Group IIA sPLA2 in nociception

4.3.4 Electron microscopy

The electron microscopy showed sPLA2-IIA immunoreactivity in neuronal cell bodies and dendrites in the spinal cord Label was observed on the endoplasmic reticulum of neuronal cell bodies (Fig 2.4.5A), and dendrites or dendritic spines that were postsynaptic to unlabeled axon terminals (Fig 2.4.5B and C) The latter contained small round vesicles, typical of glutamatergic axon terminals (Edwards, 1995)

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Fig 2.4.5 Electron micrographs of sPLA 2 -IIA immunolabeled sections from the dorsal horn of the spinal cord of a normal rat (A) Section from the lumbar spinal segment Reaction product (arrows)

is associated with the endoplasmic reticulum (ER) of a neuronal cell body (B and C) Sections from a cervical (B) or lumbar (C) spinal segment Label is present in dendrites (D) that formed asymmetrical synapses (S) with unlabeled axon terminals containing small round vesicles (AT) Scale: A = 0.5 μm, B and C = 0.2 μm

AT

S

D

B

A

ER

AT

S

D

C

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4.4 Discussion

The present study elucidated the expression profile of multiple sPLA2

isoforms in the rat CNS with focus on sPLA2-IIA in the brainstem and spinal cord Expression of the sPLA2 isoforms sPLA2-IB, sPLA2-IIA, sPLA2-IIC, sPLA2-X (with secretory signals) and sPLA2-V (without secretory signal) were analysed in various regions of the rat brain including the olfactory bulb, cerebral neocortex, hippocampus, striatum, thalamus/ hypothalamus, cerebellum, brainstem and cervical, thoracic and lumbar spinal segments using real-time RT-PCR sPLA2-IB expression was low throughout the CNS, sPLA2-IIA expression was high in the brainstem and spinal cord, sPLA2-IIC expression was high in the cerebral neocortex, hippocampus and thalamus/ hypothalamus, sPLA2-V expression was high in the olfactory bulb and cerebellum, and sPLA2-X was expressed at very low levels in the normal CNS Of the isoforms, sPLA2-IIA mRNA expression was highest in the brainstem and spinal cord suggesting that this could be the most relevant isoform in the ascending pain pathway Western blots showed bands at

14 kDa of sPLA2-IIA in homogenates from brainstem and spinal cord and a second band at approximately 30 kDa in the spinal cord The second band could

be due to dimerization of sPLA2-IIA The expression of sPLA2-IIA in the spinal trigeminal nucleus and dorsal horn cervical spinal segments was further analysed

by immunohistochemistry, and shown to be localized to neuronal cell bodies and dendrites The findings were consistent with previous results showing sPLA2-IIA protein and activity in spinal cord homogenates of normal rats (Svensson et al 2005) In contrast to the spinal cord, sPLA2-IIA mRNA level was not detected by

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ribonuclease protection assay in non-treated dorsal root ganglion neurons (Morioka et al 2002)

sPLA2-IIA is able to induce Ca2+ influx via L-type voltage sensitive Ca2+ channels (L-VSCCs) in rat cortical neuronal cells (Yagami et al 2003b) External application of sPLA2-IIA caused an increase in exocytosis and neurotransmitter release from PC-12 cells as well as in the hippocampal neurons This action of sPLA2-IIA was completely abolished when the cells were treated with EGTA to deplete Ca2+ (Wei et al 2003) sPLA2 binds to presynaptic membrane after it is released to enter the lumen of the synaptic vesicle and hydrolyze phospholipids

of the inner leaflet of synaptic vesicles This process alters the phospholipid composition of vesicles, and has been proposed to be critical for presynaptic neurotransmission (Moskowitz et al 1986; Kim et al 1995b; Matsuzawa et al 1996) sPLA2-IIA could itself be released by rat brain synaptosomes upon stimulation by acetylcholine and glutamate receptors or via voltage dependent

Ca2+ channels through depolarization (Kim et al 1995b; Matsuzawa et al 1996)

In view of its postsynaptic localization and the fact that sPLA2-IIA is a secreted protein with a 21 amino acid signal peptide (Komada et al 1990), it could be postulated that sPLA2-IIA might be released from dendrites upon depolarization, resulting in axonal exocytosis This, postulated role of sPLA2-IIA as a secreted neuromodulator during synaptic transmission seems consistent with previous findings that sPLA2 plays role in membrane depolarization and Ca2+ entry which

is necessary for maintenance of healthy neurons: cerebellar granule neurons

require membrane depolarization and neurotrophic factors to survive in vitro, and

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sPLA2 was shown to protect these neurons from apoptosis caused by K+ deprivation Moreover, the ability of sPLA2 to promote neuronal survival is inhibited when extracellular Ca2+ is depleted or when L-type Ca2+ channel is blocked by nicardipine (Arioka et al 2005)

The contribution of sPLA2 in inflammation has been extensively studied Recent findings showed that sPLA2 induced expression of pro-inflammatory cytokines including TNF-α and IL-1β in the injured spinal cord (Liu et al 2006) sPLA2 also provoked AA release and COX-2 expression in cultured neurons independent of other cytokines (Kolko et al 2003) Conversely, there is strong evidence to support that sPLA2-IIA in vitro and in vivo can be upregulated by

cytokines and also by TNF-α and IL-1α/β after transient focal cerebral ischemia

in rats (Adibhatla and Hatcher 2007) This induction of sPLA2-IIA expression is suppressed by anti-inflammatory glucocorticoids and downregulated by transforming growth factor and platelet-derived growth factor (Murakami et al., 1997; Kudo and Murakami, 2002) Moreover, astrocytes isolated from mice deficient of sPLA2-IIA gene were less sensitive to cytokines to produce PGE2

than those astrocytes which expressed sPLA2-IIA (Xu et al 2003a) In addition, IL-1β and TNFα activate COX-2 to maintain the pro-inflammatory pathways (Kuwata et al 1998; Murakami et al 1999; Sawada et al 1999; Morioka et al 2000a; Morioka et al 2002), affirming the role of sPLA2 in inflammation

The present finding of dense immunolabeling of sPLA2-IIA in the spinal trigeminal nucleus of the brainstem and dorsal horn of the cervical spinal cord is consistent with previous findings of an important role of sPLA2 in nociceptive

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transmission, both from the orofacial region (Yeo et al 2004) and the rest of the body IT injection of sPLA2 inhibitor, LY311727 significantly reduced nociception after inflammation-induced hyperalgesia sPLA2 inhibition results in attenuation of hypersensitivity, even after direct activation of second order dorsal horn neurons (Svensson et al 2005) In conclusion, the above findings in this study support an important role of sPLA2-IIA in nociceptive transmission in the brainstem and spinal cord The mechanism by which sPLA2-IIA is secreted could be via kainate- receptor binding sPLA2-IIA is released upon stimulation by kainate and this effect is abolished by addition of UBP 302, a GluR5 specific kainate receptor antagonist (Than et al 2011) This evidence suggests that sPLA2-III could be released via the similar mechanism as this enzyme is also localized in the dendrites The evidence of sPLA2-IIA localization and release supported that it plays an active role in the synaptic transmission Moreover, it has been shown that after external application of sPLA2-IIA to cultured hippocampal neurons and PC-12 cells, it resulted in an instantaneous increase in exocytosis (Wei et al 2003)

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Chapter 5 Role of lysophospholipids in synaptic transmission

CHAPTER 5 ROLE OF LYSOPHOSPHOLIPIDS IN SYNAPTIC TRANSMISSION

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5.1 Introduction

Exocytosis is the release of neurotransmitters from synaptic vesicles via targeting and fusion events which are similar to the release of secretory proteins

In resting neurons some synaptic vesicles filled with neurotransmitters are

‘‘docked’’ at the plasma membrane while others are in reserve near the plasma membrane at the synaptic cleft Synaptic vesicle membranes contain Ca2+

-binding proteins such as synaptotagmin that can detect cytosolic Ca2+ increase after the arrival of an action potential, to trigger rapid fusion of docked vesicles with the synaptic membrane and release of neurotransmitters (Lodish H 2007)

PLA2 differ in structure, enzymatic properties, subcellular localization and cellular functions and include sPLA2, cPLA2 and iPLA2 isoforms (Farooqui et al 2006) sPLA2 has been shown to be associated with synaptosomes and synaptic vesicle fractions (Kim et al 1995b; Matsuzawa et al 1996) PLA2 is able to disrupt the synaptic vesicle integrity in a Ca2+- dependent manner (O'Regan et al 1995a; O'Regan et al 1995b; O'Regan et al 1996), and stimulation of PLA2 in synaptic vesicles correlates with induction of vesicle-vesicle aggregation and alterations in vesicle permeability sPLA2 binds to the presynaptic membrane, enters the lumen of the synaptic vesicle during the vesicle’s retrieval from the plasma membrane, and hydrolyzes phospholipids of the inner leaflet of synaptic vesicles (Matsuzawa et al 1996; Wei et al 2003) SPANs hydrolyze phospholipids of cultured neurons with generation of a lysophospholipid, lysoPC and fatty acids (Rigoni et al 2005) This leads to massive release of synaptic vesicles, with their incorporation into the presynaptic plasma membrane and

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consequent surface exposure of synaptic vesicle luminal epitopes (Rigoni et al 2005) LysoPC does not only facilitates synaptic vesicle fusion but it is also involved in dopamine turnover In endothelial cells, it is also involved in modulating Ca2+ signals and inhibits the phosphorylation of nitric oxide synthase and cPLA2 (Millanvoye-Van Brussel et al 2004) LysoPC is not simply a lipid metabolite producing neurotrophic and neurotoxic effects It participates in signal transduction processes LysoPC activates protein kinases such as PKC, PKA, and c-jun terminal kinase (Boggs et al 1995; Gomez-Munoz et al 1999) In addition, bee venom (melittin) mediated stimulation of PLA2 and generation of LysoPI in pancreatic islet cells promotes the release of insulin in a dose-dependent manner This effect is reversible, saturable and has no effect on subsequent islet cell functioning (Metz 1986)

Lysophospholipids are able to modify the function of membrane proteins including ion channels These alterations can take place through signal transduction pathways, for instance by binding to lysophospholipid receptors (Tachikawa et al 2009) or via ‘‘direct’’ effects on the cell membrane (Lundbaek and Andersen 1994) High concentrations of lysophospholipids may act as detergents to disrupt membrane structures (Weltzien 1979; Farooqui and Horrocks 2007), thus affecting the function of membrane proteins such as K+ channels (Kiyosue and Arita 1986), voltage-dependent Na+ channels (Burnashev

et al 1989) and K(ATP) channels The properties of lysophospholipids suggest that they may be active participants in sPLA2-mediated exocytosis (Amatore et al 2006) Different levels of sPLA2 activity are present in various parts of the CNS

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(Thwin et al 2003) and the sPLA2-IIA isoform has been shown to induce exocytosis in cultured hippocampal neurons (Wei et al 2003) However, until now, little is known about possible contributions of various lysophospholipid species to exocytosis in neurons or endocrine cells This study was therefore carried out using total internal reflection microscopy (TIRFM), capacitance measurements and amperometry, to examine the effects of several lysophospholipid species, lysoPC, lysoPS and lysoPI on exocytosis in a neuroendocrine cell, the rat PC-12 cells

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5.2 Materials and methods

5.2.1 TIRFM

The effect of external infusion of lysoPC, lysoPS (Avanti, Alabaster, Alabama) and lysoPI (from Sigma-Aldrich, St Louis, MI, USA) (200 nM, diluted from 200 μM stock in ethanol vehicle) on vesicle fusion in PC-12 cells was studied by TIRFM as previously described (Allersma et al 2004; Tang et al 2007; Zhang et al 2009) LysoPC, lysoPS and lysoPI were selected for study since they were readily soluble in a relatively non-toxic solvent, ethanol In contrast, lysoPE and lysoPA were much less soluble in ethanol or aqueous buffers, and were not further analyzed In brief, PC-12 cells were cultured in RPMI supplemented with 10% horse serum, 5% fetal bovine serum (both from Gibco, Invitrogen, Carlsbad, CA, USA) and 1% penicillin/streptomycin at 37 oC and 5%

CO2 Neuropeptide Y (NPY)-enhanced green fluorescence protein (EGFP) plasmid was a kind gift from Dr Wolf Almers (Vollum Institute, Oregon Health Sciences University) Cells were plated onto poly-L-lysine coated glass coverslips and transfected with 2 μg of NPYEGFP plasmid using FuGENE Transfection Reagent (FuGENE, Roche, USA), 1–2 days before the imaging experiments The cells were then transferred to buffer solution containing (in mM):

150 NaCl, 5.4 KCl, 2 MgCl2, 1.8 CaCl2 and 10 HEPES (pH 7.4) for TIRFM The measurements were carried out using a Zeiss Axiovert 200 inverted microscope EGFP was excited by 488 nm laser and the emission light was collected at 520

nm Time-lapse digital images were acquired at 1 or 0.2 Hz by a CCD camera with exposure time of 18 ms, and image stacks were analyzed using MetaMorph

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