Role of phospholipase a2 in orofacial pain and synaptic transmission 2

Discussion This part of the study aimed to examine sPLA2-III expression profile in the rat CNS and its effects on exocytosis to elucidate its role in nociceptive transmission.. Expressio

3.4 Discussion

This part of the study aimed to examine sPLA2-III expression profile in the rat CNS and its effects on exocytosis to elucidate its role in nociceptive transmission Expression of sPLA2-III was analyzed in entire regions of the rat brain including olfactory bulb, striatum, cortex, hippocampus, thalamus/hypothalamus, cerebellum, brainstem and cervical, thoracic and lumbar spinal segments using real-time RT-PCR Among these regions, sPLA2-III showed the highest level of mRNA expression in the brainstem and cervical, thoracic and lumbar spinal segments suggesting that this sPLA2-III plays a role in the ascending pain pathway Similarly, using Western blot analysis, protein expression sPLA2-III was observed to be at the highest level in homogenates of the spinal segments, consistent with the findings in the Chapter 2 The immunoreactivity of sPLA2-III was also observed to be high in the spinal trigeminal nucleus and dorsal horn of the spinal segments This protein was localized to dendrites, the postsynaptic terminal in neurons, reflecting the possible role of sPLA2-III in nociception These findings are supported by previous studies which showed that sPLA2-III was expressed in the neuronal cells, such as peripheral neuronal fibres, spinal dorsal root ganglia neurons and cerebellar Purkinje cells (Masuda et al 2008)

The findings above supported the results in the Chapter 2 which showed

an increased sPLA2-III mRNA expression in the CM region after peripheral inflammation induced by facial CA injection, indicating the role of this isozyme in nociception sPLA2-III has also been associated to various diseases such as

atherosclerosis and cancer (Murakami et al 2005; Sato et al 2008) thus leading

it to be a likely target for drugs sPLA2-III could regulate nociception in mammals

as subcutaneous injection of PLA2-related peptide isolated from the bee venom led to nociceptive paw flinches (Chen et al 2006; Chen and Lariviere 2010) Both sPLA2-III that was expressed in mouse skin and Tg mice overexpressing human

sPLA2-III could lead to spontaneous development of inflammation in the skin This was accompanied by entry of neutrophils and macrophages and augmented levels of pro-inflammatory cytokines, chemokines and PGE2 were observed (Sato

et al 2009) Studies have also shown the association of sPLA2-III with microvascular endothelium in human tissues after inflammation and ischemic injury as sPLA2-III expression is induced by pro-inflammatory cytokines (Murakami et al 2005) Moreover, the enzyme transcription was upregulated by the production of cytokines such as IL-1 α, TNF- α and interferon-γ by immune cells at the site of inflammation (Farroqui et al 2002) sPLA2-III when injected into cervical dorsolateral funiculus, it showed dose-dependent demyelination, and axonopathy, thus indicating its role in pain transmission (Titsworth et al 2007)

After the N- and C-terminal of sPLA2-III are proteolytically cleaved, it allows for the production of sPLA2 only domain in almost all cell types (Murakami

et al 2005) sPLA2 domain alone is adequate to conduct catalytic activity and generate PGE2 in various cell types (Murakami et al 2005), contributing to inflammatory pathway However, the mechanism by which this enzyme is proteolytically processed in cells is still not known (Murakami and Kudo 2004) Even so, it has been suggested that the proteolytic cleavage of the enzyme is

likely to occur prior to the secretion of the sPLA2 domain of the sPLA2-III into the extracellular space (Murakami et al 2005) The presence of either C or N or both terminals of the sPLA2-III results in the cytoplasmic localization of the enzyme, whereas in the absence of both, the enzyme was found to be distributed mainly

on the plasma membrane of the cell (Murakami et al 2003)

Upon the release of the enzyme into the extracellular space, the sPLA2-III will act upon plasma membrane of neighboring cells, giving rise to various catalytic products of membrane phospholipids, possibly suggesting its function in nociceptive transmission In this study, it was observed in PC-12 cells that there was an increase in capacitance measurement indicating exocytosis, under voltage clamp conditions after addition of sPLA2-III in this study Exocytosis induced by sPLA2-III was attenuated by pretreatment with MBCD suggesting dependence on integrity of lipid rafts on the cell membrane which are membrane domains in which neurotransmitter signaling could take place via clustering of receptors and components of receptor-activated signaling cascade (Allen et al 2007) Moreover, treatment of cells with thapsigargin and recording in zero Ca2+ conditions, or treatment of cells with lanthanum chloride and recording in external solution containing Ca2+, resulted in attenuation of sPLA2-III induced exocytosis.sPLA2-III also induced rise in [Ca2+]i, and this effect was abolished in cells which had been pre-incubated with MBCD, or cells that were pre-treated with lanthanum chloride and recorded in external solution containing Ca2+ Together, the results indicated that neurotransmitter release triggered by sPLA2 -III could possibly be dependent on the integrity of cholesterol rich lipid domains

on cellular membranes and a rise in [Ca2+]i concentration, through influx via Ca2+ channels

Various studies have also shown the involvement of bee venom in exocytosis in tissues and PC-12 cells (Kurihara et al 1986; Ray et al 1997; You

et al 2008) Evidence from previous study showed that application of PLA2

activators, melittin and mastoparan on rat anterior pituitary gland cells induced hormonal release (Kurihara et al 1986) The bee venom test, using bee venom

or components of bee venom is also a well established experimental animal model for pain to show its role in nociceptive transmission (Mogil et al 1999; Lariviere and Melzack 2000; Lariviere et al 2002) Peripheral bee venom injection induced changes in synaptic transmission of the anterior cingulate cortex which plays an important role in the affective dimension of pain (Gong et

al 2010), indicating the important role of bee venom in nociceptive transmission

The findings in this section of the study, together with the results from the Chapter 2, it suggests the role of sPLA2-III in nociceptive transmission Besides sPLA2-III, sPLA2-IIA also participated in neurotransmission in the hippocampal cultured neurons and PC-12 cells (Wei et al 2003) Therefore in the next chapter, the role of sPLA2-IIA in nociception by elucidating its expression and localization

in the CNS will be further explored

CHAPTER 4

4.1 Introduction

Mammalian sPLA2 consists of enzymes which have low molecular masses (13–19 kDa) (Yang et al 2009) and their require mM range of Ca2+ for their activities (Six and Dennis 2000; Valentin and Lambeau 2000; Farooqui and Horrocks 2004) These isozymes include sPLA2-IB, IIA, IIC, V and X and are involved in multiple physiological and pathological processes via release of AA from membrane phospholipids or specific binding to membrane receptors (Suzuki

et al 2000) Total sPLA2 activity is highest in the medulla oblongata, pons, and hippocampus, moderate in the hypothalamus, thalamus, and cerebral cortex, and low in the cerebellum and olfactory bulb (Thwin et al 2003)

sPLA2 has a well-established role in inflammation and inflammatory diseases (Nevalainen et al 2000) and thus, inhibition of sPLA2 would prevent the formation of inflammatory eicosanoids prior to the COX reaction in which PLA2 is the rate limiting precursor in AA production (Schaefers et al 1996) Therefore its blockade should eliminate the need for COX-1 versus COX-2 specificity in anti-inflammatory therapeutics sPLA2 activity is elevated in several body fluids of patients with acute pancreatitis (Makela et al 1990) Synovial fluid from arthritic joints of rheumatic patients contains sPLA2-IIA (Kramer et al 1989; Seilhamer et

al 1989) while the total PLA2 activity and sPLA2-IIA is also enhanced in bronchoalveolar lavage fluids from patients with adult respiratory distress syndrome (Kim et al 1995a) TNFα, IL-1, and LPS were shown to induce sPLA2 -IIA production in cultured astrocytes and direct injection of LPS into brain increased sPLA2-IIA mRNA (Oka and Arita 1991)

sPLA2-IB mRNA is present in the human brain and its distribution is mainly neuronal It is highly expressed in the cerebral cortex and hippocampus sPLA2

-IB mRNA has been identified in rat cerebral neurons, cells of neurodermal origin (Kolko et al 2005; Kolko et al 2007) and the normal spinal cord by quantitative PCR (Lucas et al 2005) sPLA2-IIA is ubiquitously expressed in the rat brain (Molloy et al 1998) and spinal cord (Lucas et al 2005) sPLA2-IIA is associated with the endoplasmic reticulum in perinuclear regions of Purkinje cell somata (Shirai and Ito 2004) Increased sPLA2-IIA mRNA and immunoreactivity is present in the rat brain after cerebral ischemia and in Alzheimer’s disease (Lauritzen et al 1994; Lin et al 2004; Moses et al 2006; Adibhatla and Hatcher 2007) Moreover, sPLA2-IIA is upregulated by cytokines including TNF-α and IL-1α/β (Adibhatla and Hatcher 2007) sPLA2-IIC mRNA expression is low in peripheral tissues but is found in all parts of the brain (Molloy et al 1998) and spinal cord (Lucas et al 2005) sPLA2-V mRNA is expressed in the cerebral cortex and hippocampus while present at low levels in most areas of the brain (Molloy et al 1998; Kolko et al 2006) and is also identified in the rat cerebellum

by immunostaining and in situ hybridization histochemistry and localized in Bergmann glia cells (Shirai and Ito 2004) sPLA2-V is also present in the rat spinal cord as shown by quantitative PCR and Western blot analysis (Lucas et al 2005; Svensson et al 2005) sPLA2-X is expressed in the rat brain and in primary neuronal cell cultures, and is expressed at low levels in the cerebral cortex (Kolko et al 2006) Having shown in the previous chapters that sPLA2-III was the particular isozyme which was significantly increased after orofacial pain induced

by facial CA, the important role of sPLA2 isoforms with strong secretory signals in nociceptive transmission could not be neglected especially when it has been shown that addition of sPLA2-IIA which has strong secretory signal was able to induce exocytosis in hippocampal neurons (Wei et al 2003) In this part of the study the expression profile of multiple sPLA2 isoforms in the rat CNS was elucidated with focus on sPLA2-IIA in the brainstem and spinal cord

4.2 Materials and methods

4.2.1 Real-time RT-PCR

sPLA2 isoforms with strong secretory signal (P > 0.6), and which could be released as a neuromodulator were selected for analyses using SignalP 3.0 Server, which predicts the presence and location of signal peptide cleavage sites

in amino acid sequences from different organisms These included sPLA2-IB (P = 0.82), -IIA (P = 0.67), -IIC (P = 0.7), and -X (P = 0.63) sPLA2-V (P = 0.4) was also analysed for comparison Four uninjected adult male Wistar rats weighing approximately 200 g each were used for this portion of the study The rats were anesthetized with an intraperitoneal injection of ketamine and xylazine cocktail and killed by decapitation Adequate measures were taken to minimize pain and discomfort, and procedures involving rats were approved by the Institutional Animal Care and Use Committee

Various regions of the brain including olfactory bulb, cerebral neocortex, hippocampus, striatum, thalamus/hypothalamus, cerebellum, brainstem and cervical, thoracic and lumbar spinal segments were quickly removed and immersed in RNAlater (Ambion, TX,USA), snap frozen in liquid nitrogen and kept

at -80 oC till analyses Total RNA was extracted and isolated using TRizol reagent (Invitrogen, CA, USA) according to the manufacturer’s protocol RNeasy1 Mini Kit (Qiagen, Inc., CA, USA) was used to purify the RNA The samples were then reverse transcribed using High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems, CA, USA) Real-time PCR amplification was then carried out in the 7500 Real time PCR system using TaqMan1

Universal PCR Master Mix, and sPLA2-IB (Applied Biosystems ID Rn00580896_m1), sPLA2-IIA (Rn00580999_m1), sPLA2-IIC (Rn01520676_m1), sPLA2-V (Rn00567782_m1), sPLA2-X (Rn00691350_m1) or rat β-actin probes, according to the manufacturer’s instructions All reactions were carried out in triplicate The amplified transcripts were quantified using the comparative CT method (Livak and Schmittgen 2001), with the formula for relative fold change =

2ΔΔCT The fold change for each sPLA2 subgroup expression in different parts of the brain was observed

4.2.2 Western blot analysis

Four uninjected Wistar rats of 200 g each were used for this portion of the study The animals were anesthetized and killed as described above The olfactory bulb, cerebral neocortex, hippocampus, striatum, thalamus/hypothalamus, cerebellum, brainstem and cervical, thoracic, and lumbar spinal segments were dissected out and homogenized in 10 volumes of ice-cold buffer containing 0.32 M sucrose, 4 mM Tris–HCl, pH 7.4, 1 mM EDTA,

and 0.25 mM dithiothreitol After centrifugation at 1000 g for 30 min, the

supernatant was collected and protein concentrations in the preparation measured using the BioRadprotein assay kit (Bio-Rad Laboratories) Total proteins were resolved in 10% SDS polyacrylamide gels under reducing conditions and electrotransferred to a PVDF membrane (Amersham Pharmacia Biotech) Nonspecific binding sites on the PVDF membrane were blocked by incubation with 5% non-fat milk for 1 h The PVDF membrane was then