Role of phospholipase a2 in orofacial pain and synaptic transmission 1

ROLE OF PHOSPHOLIPASE A2 IN OROFACIAL PAIN AND SYNAPTIC TRANSMISSION MA MAY THU (B.Sc. (Hons), NUS) SUPERVISOR: ASSOCIATE PROFESSOR YEO JIN FEI A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF ORAL AND MAXILOFACIAL SURGERY FACULTY OF DENTISTRY NATIONAL UNIVERSITY OF SINGAPORE 2011 Acknowledgements ACKNOWLEDGEMENTS With thanks to my supervisor, Associate Professor Yeo Jin Fei, Head, Department of Oral and Maxillofacial, National University of Singapore, who extended utmost support to my entire project; to my co-supervisor, Associate Professor Ong Wei Yi, Department of Anatomy, National University of Singapore, who proposed the topic of my study, provided relentless guidance throughout my entire candidature and most importantly, influenced me greatly with his knowledge and passion for research. Numerous people contributed to the realization of this project: Tang Ning, for her indefatigable teachings and guidance in my study; Pan Ning, for her brilliant technical support; Jinatta Jittiwat, Nuntiya Sompran, Chan Yee Gek and Wu Ya Jun, for their assistance in Electron Microscopy; Chew Wee Siong, Chia Wan Jie, Ee Sze Min, Guo Jing, Ho Mei Xuan, Kazuhiro Tanaka, Kim Ji Hyun, Lynette Lee Hui Wen, Lee Li Yen, Amy Lim Seok Wei, Loke Sau Yeen, Mary Ng Pei Ern, Poh Kay Wee, Tan Yan, Wong Li Ming, Wong Sin Kei, Yang Hui and Alicia Yap Mei Yi, for their selfless support of my interest in this study. Thanks to my mom and dad: Phyu Phyu Win and Htay Aung, who have always believed I could accomplish my dreams and to my beloved, Wei Ming, for his partnership and kindness in all things. II Table of Contents TABLE OF CONTENTS ACKNOWLEDGEMENTS …………………………………… …… .….……………II TABLE OF CONTENTS………………………………………… ……… ……… .III SUMMARY…………………………………………………….…………… .……VIII LIST OF TABLES………………………………………………………………… .XI LIST OF FIGURES……………………… ……………………………………… XII ABBREVIATIONS…………………………………….……………… ……… ….XIV PUBLICATIONS……………………………….…………… ……….….… .… XVIII SECTION I INTRODUCTION…………………………………………………… … .1 1. Phospholipase A2………………………………………………………… …….… 1.1. Cytosolic phospholipase A2 (cPLA2)……… .………… … ……… 1.2. Ca2+-independent phospholipase A2 (iPLA2)………… …… .….….7 1.3. Secretory phospholipase A2 (sPLA2)……………………… ……… .8 1.3.1. sPLA2 isozymes…………………………………………………9 1.3.1.1. sPLA2-IB…………………………………………………… … 1.3.1.2. sPLA2-IIA……………………………… .…………………… 10 1.3.1.3. sPLA2-IIC…………………… ……… ……… ………… .…11 1.3.1.4. sPLA2-IID……………… …………………………… … .… 12 1.3.1.5. sPLA2-IIE……………… ……….…….………… … .………12 1.3.1.6. sPLA2-IIF…………………………………………….… …… 13 1.3.1.7. sPLA2-III………….…………….….……………………….… .13 1.3.1.8. sPLA2-V……………………………………….… …….………14 III Table of Contents 1.3.1.9. sPLA2-X……… .…………………………………….…………15 1.3.2. sPLA2-XIIA…………………………… .………………………16 1.4. Arachidonic acid……………………………… .……………………18 1.5. Phospholipids and lysophospholipids……………… …… .………20 1.6. Exocytosis…………………………… ……………… …………….23 1.6.1. PLA2 and neurotransmission……………….……… .… … 25 1.6.2. Phospholipids and neurotransmission……………… … 27 1.6.3. Lysophospholipids and neurotransmission……………… 28 1.6.4. Factors affecting exocytosis-lipid rafts and Ca2+….… … 30 2. Pain…………………………………………………………… .………… .…… .32 2.1. Orofacial pain………………………………………………… ….….33 2.2. Nociception and nociceptors………………….……… …… … ….33 2.3. Pain models……………………………………….……… .…34 2.4. Pain pathways………………………………………………… …….36 3. PLA2 and inflammatory pain……………………………….……………… ……39 3.1. PLA2 and receptors………………………….……………………… .45 SECTION II Experimental studies…………………………………………….…… 48 CHAPTER Changes in brain lipids contents after carrageenan-induced orofacial pain…………………………………………………………….…………… 49 1.1. Introduction……………………………………………… ……………50 1.2. Materials and methods……………………………………….……….52 1.2.1. Time course study of pain responses after facial CA injection…………………………………………………………… .52 IV Table of Contents 1.2.2. Assessment of responses to mechanical stimulation………53 1.2.3. Lipidomics analyses……………………………………………54 1.2.3.1. Internal standard…………………………………… 55 1.2.3.2. Lipid extraction……………………………………….55 1.2.3.3. Analysis of lipids using liquid chromatography/mass spectrometry……………………………………………………56 1.3. Results……………………………… .………………………… ……57 1.3.1. Time course study of pain responses after facial CA injection…………………………………………………………………57 1.3.2. Lipidomics analyses………………………………………… .58 1.4. Discussion…………………………………………… ……….………64 CHAPTER Differential expression pattern of PLA2 isoforms in CNS after orofacial pain ………….………………………………………………… ………… 67 2.1. Introduction……………………………………………………… ……68 2.2. Materials and methods……………………………………………… 70 2.2.1. Real-time RT-PCR…………………………………………… 70 2.2.2. Western blot analysis………………………………………… 71 2.2.3. Immunohistochemistry…………… ………………………… 72 2.3. Results…………………… .…………………………………… ……74 2.3.1. mRNA expression of PLA2 isoforms in the medulla oblongata……………………………………………………………….74 2.3.2. sPLA2-III protein expression and localization in the CM… .76 2.4. Discussion………………………………………………… ……….…78 V Table of Contents CHAPTER Role of group III sPLA2 in nociception and synaptic transmission in the CNS…………………………………………………………………………………81 3.1. Introduction……………………………………………………… ……82 3.2. Materials and methods……………………………………………… 85 3.2.1. Real-time RT-PCR…………………………………………… 85 3.2.2. Western blot analysis………………………………………… 86 3.2.3. Immunohistochemistry…………….………………………… 87 3.2.4. Electron microscopy……………………………………………88 3.2.5. Capacitance measurement……………………………………88 3.2.6. Intracellular Ca2+ imaging………………… ………………….90 3.3. Results………………………… .………………………… …………92 3.3.1. Differential expression of sPLA2-III in rat CNS…….……… 92 3.3.2. Western blot analysis of sPLA2-III……………….……………93 3.3.3. Immunohistochemistry…………………………………………95 3.3.4. Electron microscopy……………………………………………97 3.3.5. Capacitance measurements……………………………… …98 3.3.6. Intracellular Ca2+ imaging…………………….…………… .99 3.4. Discussion……………………………………………………….……101 CHAPTER Role of group IIA sPLA2 in nociception………………… …….…105 4.1. Introduction……………………………………………………………106 4.2. Materials and methods………………………………………………109 4.2.1. Real-time RT-PCR……………………………………………109 4.2.2. Western blot analysis…………………………………………110 VI Table of Contents 4.2.3. Immunohistochemistry……………………………………….111 4.2.4. Electron microscopy………………………………………….112 4.3. Results…………… .…………………………………………………113 4.3.1. Real-time RT PCR……………………………………………113 4.3.2. Western blot analysis…………………………………………117 4.3.3. Immunohistochemistry……………………………………….118 4.3.4. Electron microscopy………………………………………….121 4.4. Discussion…………………………………………………………….123 CHAPTER Role of lysophospholipids in synaptic transmission…… ……….127 5.1. Introduction……………………………………………………………128 5.2. Materials and methods………………………………………………131 5.2.1. TIRFM………………………………………………………….131 5.2.2. Capacitance measurements…………………………………132 5.2.3. Amperometry measurements……………………………… 133 5.2.4. Intracellular Ca2+ imaging……………………….………… .134 5.3. Results…………………………… .…………………………………136 5.3.1. TIRFM………………………………………………………….136 5.3.2. Capacitance measurements…………………………………138 5.3.3. Amperometry measurements……………………………… 141 5.3.4. Intracellular Ca2+ imaging……………………….………… .143 5.4. Discussion…………………………………………………………….144 SECTION IV CONCLUSION……………………………………………………… 149 SECTION V REFERENCES……………………………………………………… 154 VII Summary SUMMARY Phospholipase A2 (PLA2, EC 3.1.1.4) are enzymes which hydrolyze the acyl ester bond at the sn-2 position to generate free fatty acids such as arachidonic acid (AA) and lysophospholipids, from membrane glycerophospholipids. PLA2 isoforms include secretory phospholipase A2 (sPLA2), cytosolic phospholipase A2 (cPLA2) and Ca2+-independent phospholipase A2 (iPLA2). Although emerging evidences have shown the roles of PLA2 isoforms in nociception, direct evidences which indicate altered brain PLA2 activity and expression during allodynia or hyperalgesia are lacking. The function of PLA2 during nociceptive transmission also needs to be explored. The present study elucidated changes in brain lipids in medulla oblongata after orofacial pain induced by facial carrageenan (CA) injection. The caudal medulla oblongata (CM) showed decreases in phospholipids including phosphatidylethanolamine and phosphatidylinositol (PI) and increases in their corresponding lysophospholipids, lysophosphatidylethanolamine and lysophosphatidylinositol (lysoPI). These results indicated an enhanced PLA2 activity in the CM and release of AA after peripheral inflammation of the face. This study further examined changes in expression level of PLA2 isoforms after nociception. mRNA expression of sPLA2-III was highly expressed in the CM and this expression was significantly increased in the CA-injected rats. However, no corresponding increase in sPLA2-III protein expression was detected. These changes possibly take place in spinal trigeminal nucleus which communicates VIII Summary nociceptive input from orofacial region, indicating the nociceptive function of sPLA2-III. The expression profile of sPLA2-III in CNS and its effects on exocytosis in rat PC-12 cells further illustrated the role of sPLA2-III in pain transmission. Both sPLA2-III mRNA and protein expression were expressed at the highest levels in the brainstem and spinal segments. The enzyme was localized to dendrites in spinal trigeminal nucleus, supporting its role in ascending pain pathway. External application of sPLA2-III to PC-12 cells augmented capacitance measurement, indicating exocytosis and this was dependent on lipid rafts and external Ca2+. Moreover, sPLA2-III caused an increased in intracellular Ca2+ ([Ca2+]i), indicating that it could be a trigger for exocytosis. Moreover, sPLA2-IIA with a strong secretory signal, showed high levels of mRNA and protein in the brainstem and spinal segments. sPLA2-IIA was also localized to the dendrites in spinal trigeminal nucleus and dorsal horn of spinal cord. The expressions of sPLA2-IIA were supported by previous studies which also illustrated a significant function of CNS sPLA2 in nociceptive transmission. PLA2 participates in the synaptic transmission through its secretion, i.e. in sPLA2-III and -IIA, and also via its enzymatic product, lysophospholipids. When the effects of lysophospholipids on exocytosis in PC-12 cells were elucidated, external infusion of lysoPI augmented vesicle fusion, indicating exocytosis. Similarly, significant increase in capacitance measurement, or number of spikes detected at amperometry, indicating exocytosis was observed after external IX Summary application of lysoPI. This process was affected by the lipid rafts and [Ca2+]i. LysoPI also caused an elevated [Ca2+]i, implying its effect on exocytosis. In conclusion, this study demonstrated significantly increased PLA2 activity and expression upon orofacial pain. Moreover, due to their localization and roles in synaptic transmission, both sPLA2-III and sPLA2-IIA are found to be important isozymes in the ascending pain pathway. X Chapter Role of Group III sPLA2 in nociception and synaptic transmission in the CNS 3.2. Materials and methods 3.2.1. Real-time RT-PCR 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 parts of 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 °C till analyses. Total RNA was extracted and isolated using TRizol reagent (Invitrogen, CA, USA) according to the manufacturer's protocol. RNeasy® Mini Kit (Qiagen, Inc., CA, USA) was used to purify the RNA. The samples were reverse transcribed using High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems, CA, USA). Real-time PCR amplification was then carried out using TaqMan® Universal PCR Master Mix (Applied Biosystems) rat sPLA2-III (Rn01442985_m1), or rat β-actin probes (Applied Biosystems) according to the manufacturers’ 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 = 2–∆∆CT. The mean and standard deviation of the relative fold change of four rats from each treatment group were calculated. Possible 85 Chapter Role of Group III sPLA2 in nociception and synaptic transmission in the CNS significant differences in expression between olfactory bulb and other parts of the CNS were analyzed using two-tailed unpaired Student’s t-test. P < 0.05 was considered significant 3.2.2. Western blot analysis Three uninjected rats were used for this part of the study to elucidate the protein expression profile of sPLA2-III in the rat CNS. 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 harvested and homogenized in 10 volumes of ice-cold buffer containing 0.32 M sucrose, mM Tris-HCl, pH 7.4, mM EDTA, and 0.25 mM dithiothreitol. After centrifugation at 12,000 g for 30 min, the supernatant containing the protein was measured for protein concentration using the BioRad protein assay kit (Bio-Rad Laboratories). Total proteins (40 μg) 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 in TTBS for h. The PVDF membrane was incubated overnight at °C with goat polyclonal antibody to sPLA2-III (Santa Cruz, Delaware, CA, USA) diluted to 1:500 in 5% non-fat milk in TTBS. After washing with TTBS, the membrane was incubated with horseradish peroxidase conjugated anti-goat immunoglobulin IgG (Amersham, Pharmacia Biotech) for h at room temperature. The protein was visualized with 86 Chapter Role of Group III sPLA2 in nociception and synaptic transmission in the CNS an enhanced chemiluminescence kit (Pierce) according to the manufacturer’s instructions. The densities of the sPLA2-III bands were normalized against those of β-actin, and the mean ratios calculated. Possible significant differences in expression between olfactory bulb and other parts of the CNS were analyzed using two-tailed unpaired Student’s t-test. P < 0.05 was considered significant 3.2.3. Immunohistochemistry of sPLA2-III Another four uninjected male Wistar rats were used for this portion of the study. The rats were deeply anesthetized and perfused through the left ventricle with a solution of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The rat brains and spinal cords were removed and sectioned coronally at 100 μm using a Vibrating microtome. The sections were washed for h in PBS. They were then incubated overnight with goat polyclonal sPLA2-III antibody (Santa Cruz) diluted to 1:50 in 0.15 M NaCl in 0.05 M Tris buffer, pH 8.6, containing 1% bovine serum albumin. This was followed by washing in PBS times and incubated for h at room temperature in a 1:200 dilution of biotinylated horse anti-rabbit IgG (Vector, Burlingame, CA). The sections were reacted for h at room temperature with an avidin-biotinylated horseradish peroxidase complex, and visualized by treatment for in 0.05% DAB solution in TBS containing 0.05% hydrogen peroxide. The color reaction was stopped with several washes of TBS. Some sections were mounted on glass slides and lightly counterstained with methyl green before coverslipping. The remaining sections were processed for electron microscopy. 87 Chapter Role of Group III sPLA2 in nociception and synaptic transmission in the CNS 3.2.4. Electron microscopy Electron microscopy was carried out by subdissecting the immunolabeled spinal cord sections into smaller portions that included the dorsal or ventral horn. The sections were soaked in PBS overnight at oC before processing. Following which, the sections were post fixed in 1% osmium tetroxide, pH 7.4 for h at room temperature. This was followed by washing the sections with PBS two times for 5-10 at room temperature. The sections were then dehydrated through a series of ascending ethanol series at room temperature. Finally the sections were embedded in araldite and left to polymerise at 60 oC for 24 h. Semi-thin sections were obtained from the first µm of the sections, mounted on Formvar-coated copper grids, and stained with lead citrate. They were viewed using a Jeol 1010EX electron microscope. 3.2.5. Capacitance measurements The effects of sPLA2-III from bee venom on vesicle fusion during exocytosis were also quantified using capacitance measurement as previously described (Gillis 1995; Chen and Gillis 2000; Chen et al. 2001; Sun and Wu 2001; Zhang and Zhou 2002; Wei et al. 2003). PC-12 cells were cultured in RPMI Medium supplemented with 10% fetal bovine serum and 1% penicillin/ streptomycin (Gibco, Invitrogen). The cells were plated in 35 mm Petri dishes, and maintained in an incubator at 37 oC, 100% humidity, with 95% air and 5% CO2. Coverslips containing attached cells were transferred to a fresh 35 mm dish containing ml of external solution prior to the patch clamp experiments. The 88 Chapter Role of Group III sPLA2 in nociception and synaptic transmission in the CNS latter contained (in mM): 150 NaCl, 2.8 KCl, 10 CaCl2, MgCl2 and 10 HEPES and mg/ml glucose pH 7.2 (310 mOsm). The internal solution for patch pipettes contained (in mM): 130 K-gluconate, NaCl, 20 HEPES, MgCl2, Na2ATP, 0.4 NaGTP and EGTA. Capacitance measurements were carried out on PC-12 cells under whole-cell voltage clamp conditions, using 3-7 MOhm pipettes. The series resistance ranged from to 16 MOhms. Measurements were performed using an EPC-9 patch-clamp amplifier (HEKA Electronics, Germany) and the Lindau-Neher (‘sine + dc’) technique (Gillis 1995) implemented in Pulse software. This enabled long duration capacitance measurements in a single sweep. A 1000 Hz, 50 mV peak-to-peak sinusoidal voltage stimuli was superimposed onto a DC holding potential of -70 mV. Only stably voltage-clamped cells were analyzed. These showed fairly stable series resistance throughout the recording (10 to 12 MOhm) with no sudden changes of more than 10%. Capacitance values were recorded from each cell during the first 30 s after addition of sPLA2-III (Cayman, MI. USA) and divided by the value immediately before addition of sPLA2-III, to yield a normalized value. 500ng/ml of sPLA2 III was used in the present study (Kolko et al. 1996; Wang et al. 2010; Yang et al. 2010). Ten cells were recorded in each group. Possible significant differences were analyzed by two-tailed unpaired Student’s t-test. P < 0.05 was considered significant. Experiments were also carried out to show the possible factors influencing exocytosis sPLA2-III induced exocytosis: (1) MBCD was used to pre-treat the cells to establish the function of cholesterol rich domains on the cell membrane after addition of sPLA2-III (Ko et al. 2005; Sun et al. 2005; Shvartsman et al. 89 Chapter Role of Group III sPLA2 in nociception and synaptic transmission in the CNS 2006). PC-12 cells were pre-incubated with 10 mM MBCD (Sigma–Aldrich, St. Louis, MI) for 10 at 37 oC, and washed twice with PBS before transferring to external solution and application of sPLA2-III. (2) Thapsigargin was used to deplete intracellular calcium ([Ca2+]i) stores (Nathan et al. 1988), and cells were recorded in the presence of zero extracellular Ca2+ conditions, to determine the role of [Ca2+]i in the sPLA2-III-induced exocytosis. PC-12 cells were preincubated with μM thapsigargin for 15 at room temperature and were transferred to external solution containing EGTA (Sigma–Aldrich) dissolved in (mM) 150 NaCl, 2.8 KCl, 10 EDTA, MgCl2 and 10 HEPES and mg/ml glucose pH 7.2 (310 mOsm), prior to addition of sPLA2-III. (3) Cells were pre-treated with thapsigargin and recorded in external solution containing Ca2+ to determine a possible role of [Ca2+]i in the effects of sPLA2-III. PC-12 cells were incubated with μM thapsigargin for 15 at room temperature and transferred to external solution containing 10 mM Ca2+ before addition of sPLA2-III. (4) Cells were preincubated with lanthanum chloride (Sigma–Aldrich) to block Ca2+ channels (Shen et al. 2009) and recorded in external solution containing Ca2+ to establish the effect of Ca2+ influx on sPLA2-III-induced exocytosis. PC-12 cells were incubated with 10 μM lanthanum chloride for 30 at room temperature and transferred to external solution containing 10 mM Ca2+ prior to addition of sPLA2-III. 3.2.6. Intracellular calcium imaging [Ca2+]i concentration of PC-12 cells was analyzed after external infusion of sPLA2-III as described previously (Pal et al. 1999; Raza et al. 2001). PC-12 cells 90 Chapter Role of Group III sPLA2 in nociception and synaptic transmission in the CNS were cultured on glass bottom culture dishes, and loaded with acetoxymethyl form of the membrane-permeable ratiometric fluorescent Ca2+ indicator Fura-2 (5 µM, Invitrogen) in HEPES buffer (20 mM HEPES, 115 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgCl2, 13.8 mM glucose, pH 7.4) for h at 37 oC. Loaded cells were washed two times with the buffer and incubated for an additional 30 to allow activation of cellular esterase cleavage of the acetoxymethyl moiety and intracellular trapping of the free acid Fura-2 indicator. The fluorescence source was a 150W Xenon arc lamp (OptoSource Arc Lamp, Cairn Research Limited, Faversham, UK). Alternating excitation wavelengths of 340 and 380 nm were generated using an OptoScan Monochromator (Cairn Research Limited) and 510-nm emissions acquired through a Fura filter cube with a dichroic at 400 nm (Omega Optical Inc. XF-04-2, Brattleboro, USA), and a CoolSNAP HQ2 digital CCD camera (Photometrics, Tucson, AZ). Images were acquired and processed using MetaMorph 7.0.4 software (Molecular Devices, Sunnyvale, CA, USA). Background autofluorescence values for both 340- and 380-nm excitations were obtained and subtracted from experimental 340/380-nm excitation-emission values. Regions of interest corresponding to PC-12 cells were selected, and the ratio of 340/380-nm excitation-emissions in these regions calculated to give an indication of [Ca2+]i levels. At least ten cells were measured from each treatment group. 91 Chapter Role of Group III sPLA2 in nociception and synaptic transmission in the CNS 3.3. Results 3.3.1. Differential expression of sPLA2-III in rat CNS (Fig. 2.3.2.) The expression of various sPLA2-III was normalized to the lowest level of message in the different brain regions, i.e. the value for sPLA2-III in the olfactory bulb, to give an indication of its relative expression in different parts of the brain and spinal segments. sPLA2-III showed the highest level of mRNA expression in the brainstem and spinal segments. In contrast, low levels of sPLA2-III expression were observed in various parts of the forebrain (Fig. 2.3.2.). Relative fold change 60 40 20 Lu m ba r Th or ac ic ic al er v Th al am H C or te x ip po ca us m /H pu yp s ot la m us C er eb el lu m B in st em um br al C ia t C er e St r O l fa ct or y B ul b Fig.2.3.2. Real-time RT-PCR analysis of sPLA2-III in the various regions of CNS. The values were normalized to the lowest level of message in the different brain regions, i.e., the value for sPLA2-II in the olfactory bulb, to give an indication of relative expression in different parts of the brain and spinal segments. sPLA2-III expression was low throughout the forebrain but showed greatest expression in the hindbrain. Data represent the mean and standard deviation of four rats. 92 Chapter Role of Group III sPLA2 in nociception and synaptic transmission in the CNS 3.3.2. Western blot analysis of sPLA2-III (Fig. 2.3.3.) The antibody to sPLA2-III detects a band at ~19 kDa in homogenates from various parts of the CNS was consistent with the expected molecular weight of the active sPLA2 domain of sPLA2-III. High levels of expression were detected in the cervical, thoracic and lumbar spinal segments whilst low level of expression was found in the olfactory bulb, cortex, hippocampus, striatum, thalamus and hypothalamus and cerebellum (Fig. 2.3.3A, B). 93 Chapter Role of Group III sPLA2 in nociception and synaptic transmission in the CNS β-actin sPLA2-III A 10 sPLA2-III Density 0.8 0.6 0.4 0.2 Lu m ba r Th or ac ic ic al er v C ex ip po ca m us pu /h s yp ot la m us C er eb el lu m B in st em C tu m ria or t H Th al am B St O l fa ct or y bu lb Fig.2.3.3. A: Western blot analyses of sPLA2-III protein expression in different parts of the rat CNS. A: The antibody to sPLA2-III detects a band at 19 kDa in homogenates from various parts of the CNS was consistent with the expected molecular weight of the active form of sPLA2-III. High level of expression was detected in the cervical (lane 8), thoracic (lane 9) and lumbar (lane 10) spinal segments. In contrast, low level of expression was found in the olfactory bulb (lane 1), striatum (lane 2), cerebral neocortex (lane 3), hippocampus (lane 4), thalamus and hypothalamus (lane 5), cerebellum (lane 6) and brainstem (lane 7). B: Quantification of Western blots. sPLA2-III was normalized to β-actin. High level of protein was expressed in different parts of the spinal cord. Analyzed by two-tailed unpaired Student’s t-test. Asterisks indicate significant difference (P < 0.05). 3.3.3. Immunohistochemistry (Fig. 2.3.4, 2.3.5, 2.3.6) 94 Chapter Role of Group III sPLA2 in nociception and synaptic transmission in the CNS Sections incubated with sPLA2-III showed very little labeling in the forebrain including the cerebral cortex and striatum (Fig. 2.3.4A and B). ST CX A B Fig.2.3.4. Light micrographs of sPLA2-IIA immunolabeled sections from a normal rat CNS. Very little labeling is observed in the cerebral cortex (A) and striatum (B). Abbreviations: CX, cerebral cortex; ST, striatum. Scale: 200 μm. In contrast to the forebrain, dense labeling for sPLA2-III was detected in the cerebellar cortex (Fig. 2.3.5A), in neurons of periaqueductal grey (Fig. 2.3.5B) and spinal trigeminal nucleus of the brainstem (Fig. 2.3.5C) and dorsal horn (Fig. 2.3.5E) spinal segments. Control section incubated with antigen-absorbed antibody of sPLA2-III, was absent of labeling (Fig. 2.3.5D and F). 95 Chapter Role of Group III sPLA2 in nociception and synaptic transmission in the CNS * CCX PAG * B A * C V * V(C) D DH DH(C) * E * F Fig.2.3.5. Dense staining is observed in the cerebellar cortex (A, asterisk), periaqueductal grey (B, asterisk), spinal trigeminal nucleus (C, asterisk), control section of spinal trigeminal nucleus incubated with antigen-absorbed antibody, showing background labeling (D, asterisk), dorsal horn (E, asterisk) and control section of dorsal horn incubated with antigen-absorbed antibody, showing background labeling (F, asterisk). Abbreviations: CCX, cerebellar cortex; PAG, periaqueductal grey; V, spinal trigeminal nucleus; V(C), control section; DH, dorsal horn of spinal cord; DH (C), control section. Scale: 200 μm. 96 Chapter Role of Group III sPLA2 in nociception and synaptic transmission in the CNS 3.3.4. Electron microscopy Electron microscopy showed sPLA2-III immunoreactivity in the dendrites of neurons in the spinal cord (Fig. 2.3.6.). Label was observed on the dendrites that were postsynaptic to unlabelled axon terminals. The latter contained small round vesicles, typical of glutamatergic axon terminals (Edwards 1995). S AT D AT S D A 0.1μm B Fig.2.3.6. Electron micrographs of sPLA2-III immunolabeled sections from the spinal cord of a normal rat. (A and B). Section from the lumbar spinal segment. Label is present in dendrites (D) with unlabeled axon terminals containing small round vesicles (AT). Scale: A, B = 0.1 μm. 97 Chapter Role of Group III sPLA2 in nociception and synaptic transmission in the CNS 3.3.5. Capacitance measurements (Fig. 2.3.7.) Significant increase in membrane capacitance by 1.03±0.01% compared to the resting state (Fig. 2.3.7.), indicating exocytosis was detected after external infusion of sPLA2-III for 30s. Pre-incubation of cells with MBCD resulted in no significant increase in capacitance (1.0±0.01%) after addition of sPLA2-III. Cells that were pre-treated with thapsigargin and recorded in zero external Ca2+ conditions (1.0±0.01%), and cells pre-treated with lanthanum chloride and recorded in external solution containing Ca2+ (1.0±0.01%), also abolished increased in capacitance after addition of sPLA2-III. However, cells pre-treated with thapsigargin and recorded in external solution containing Ca2+ continued to show significant increase in sPLA2-III-induced capacitance (1.03±0.01%) (Fig. 2.3.7.). 98 Chapter Role of Group III sPLA2 in nociception and synaptic transmission in the CNS 0s 1.07 Normalized Capacitance (%) 30s * 1.05 * 1.03 1.01 0.99 0.97 control LaCl3+sPLA2-III 0Ca+sPLA2-III TSG+sPLA2-III MBCD+sPLA2-III sPLA2-III Fig.2.3.7. Increase in membrane capacitance in a PC-12 cell indicating exocytosis, after addition of sPLA2-III. Normalized membrane capacitance in PC-12 cells after addition of sPLA2-III. Significant increase in capacitance was observed after addition of sPLA2-III. Exocytosis induced by sPLA2-III was not observed in cells that were pretreated with MBCD; thapsigargin and recording in zero Ca2+ conditions; or thapsigargin (TSG) or lanthanum chloride (LaCl3) and recording in external solution containing Ca2+. *Significant increase in capacitance after addition of sPLA2-III (P < 0.05, analyzed by Student’s t-test). Ten to twelve cells were recorded in each group. 3.3.6. Intracellular calcium imaging (Fig. 2.3.8.) External infusion of sPLA2-III in the external solution resulted in an immediate and sustained increase in [Ca2+]i in PC-12 cells 1.20 ± 0.26 of normalized 340/380 ratio compared to the resting state (Fig. 2.3.8.). However, cells that had been pre-incubated with MBCD (1.0 ± 0.004 of normalized 340/380 ratio), or cells that were pre-treated with lanthanum chloride and recorded in 99 Chapter Role of Group III sPLA2 in nociception and synaptic transmission in the CNS external solution containing Ca2+ (1.0 ± 0.01 of normalized 340/380 ratio) showed no significant increase in [Ca2+]i after addition of sPLA2-III (Fig. 2.3.8.). sPLA2-III MBCD+sPLA2-III LaCl3+sPLA2-III Control Normalized 340/380 1.38 1.28 1.18 1.08 1600 1520 1440 1360 1280 1200 1120 1040 960 880 800 720 640 560 480 400 320 240 160 80 0.98 Time(s) Fig.2.3.8. Fura-2 imaging of [Ca2+]i concentrations in PC-12 cells. A rise in [Ca2+]i is observed after treatment with sPLA2-III alone. No rise in Ca2+ was observed in cells that were pre-treated with MBCD, thapsigargin and recorded in zero Ca2+ conditions, or pre-treated with lanthanum chloride (LaCl3) and recorded in external solution containing Ca2+, before addition of sPLA2-III. Arrow indicates time of sPLA2-III treatment. Treatment with water (vehicle control) resulted in no change in [Ca2+]i. Plots indicate the means of ten to twelve cells. Abbreviations as in Fig.2.3.7. Arrow indicates time of addition of sPLA2-III. 100 [...]... (2 011 ) Systems wide analyses of lipids in the brainstem during inflammatory orofacial pain – evidence for increased phospholipase A2 activity Eur J Pain 16 :38-48 6 Yang H, Ma MT, Siddiqi NJ, Alhomida AS, Ong WY (2 011 ) Enriched expression profile of sPLA2-III in rat CNS and its effects on exocytosis in PC12 cells Neuroscience (submitted) XVIII Section I Introduction SECTION I INTRODUCTION 1 Section I Introduction... al 2006), thus indicating its role in synaptic transmission 1. 3 .1. 2 sPLA2-IIA Group II sPLA2, however, has a distinct structure and a widely known role in inflammatory pathway sPLA2-IIA is made up of disulfide linking residues 50 with residues at the C-terminus and contains a C-terminal extension of 7 amino acids in length which defines group II distinctively (Kramer et al 19 89) sPLA2-IIA potently... al 19 97) Both sPLA2-IIA mRNA expression and activity are upregulated by cytokines including tumor necrosis factor-α (TNF-α) and IL -1 α/β and endotoxins (Oka and Arita 19 91; Svensson et al 2005; Adibhatla and Hatcher 2007) Injection of this isozyme to the hind paw of rats with adjuvant arthritis (Murakami et al 19 90) exacerbated inflammatory responses, indicating the role of sPLA2 in inflammatory pain. .. studied 16 Section I Introduction Table 1. 1 Summary of differential mRNA and protein expression of PLA2 isoforms in the CNS and peripheral organs based on various studies done so far Differential levels of expression in various organs Molecular weight PLA2 Peripheral Brain Spinal cord (kDa) organs sPLA2 IB 13 -15 + ++ +++ IIA 13 -15 + ++ ++ IIC 15 +++ ++ + IID 14 -15 + + +++ IIE 14 -15 +++ + +++ IIF 16 -17 +... presence of sPLA2 activity in the neurons and in differentiated pheochromocytoma -12 (PC -12 ) cells (Matsuzawa et al 19 96) 1. 3 .1 sPLA2 isozymes 1. 3 .1. 1 sPLA2-IB sPLA2-IB has a distinct 5 amino acid extension known as pancreatic loop in the middle of the molecule with specific disulfide bond between residues 11 and 77, which defines group I (Six and Dennis 2000; Murakami and Kudo 2004) As the expression of. .. previous reports and current findings ……………………… … 11 6 XI List of Figures LIST OF FIGURES SECTION I Figure .1. 1 Site of action of phospholipase A1, A2, C and D on the phospholipid molecule………………………………………………………………………………….3 Figure .1. 4 .1 Chemical structure of AA …………………………………………… .19 Figure .1. 5 .1 Schematic diagrams of structures of lysophospholipids …….……22 Figure .1. 6 .1 Schematic diagram of neurotransmission... level of expression in the thalamus, cerebellum and brainstem compared to the rest of the brain regions (Molloy et al 19 98) Unlike sPLA2-IIA, sPLA2-IIC is less commonly known in inflammatory pathway 1. 3 .1. 4 sPLA2-IID The structure of sPLA2-IID most resembles to that of sPLA2-IIA Recombinant mouse and human of this sPLA2 isozyme are active against vesicles of PG, PE and PC with high substrate affinity... characteristics of group III sPLA2 purified from the bee venom, including 10 cysteines, the key residues of the Ca2+ loop and catalytic site (Valentin et al 2000) The sPLA2 domain of human sPLA2-III is 31% homologous to the bee venom sPLA2 and demonstrates similar features of group III sPLA2s (Valentin et al 2000) Although the residues surrounding the sPLA2 13 Section I Introduction domain suggests the presence of. ..List of Tables LIST OF TABLES SECTION I Table .1. 1 Summary of differential mRNA and protein expression of PLA2 isoforms in the CNS and peripheral organs based on various previous studies 17 SECTION II Table.2 .1. 1 Changes in selected lipids in the RM and CM after facial CA injection…….………………………………………………………………………………… 59 Table.2.4 .1 Comparison of sPLA2 isoforms mRNA and protein expression in rat CNS... cytosolic phospholipase A2 (cPLA2), and Ca2+-independent phospholipase A2 (iPLA2) (Balsinde and Dennis 19 96; Akiba and Sato 2004) These isoforms are differentiated according to their cellular localization, and activation pathways (Zhu et al 19 96) and they play different roles in the central nervous system (CNS) Studies have related the role of cPLA2 to modulating neuronal excitatory functions while sPLA2 . WY (2 011 ) Systems wide analyses of lipids in the brainstem during inflammatory orofacial pain – evidence for increased phospholipase A2 activity. Eur J Pain 16 :38-48 6. Yang H, Ma MT, Siddiqi. levels of mRNA and protein in the brainstem and spinal segments. sPLA 2 -IIA was also localized to the dendrites in spinal trigeminal nucleus and dorsal horn of spinal cord. The expressions of. dendrites in spinal trigeminal nucleus, supporting its role in ascending pain pathway. External application of sPLA 2 -III to PC -12 cells augmented capacitance measurement, indicating exocytosis and