Chapter 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). 121 Chapter Role of Group IIA sPLA2 in nociception ER A S D S AT D AT B C Fig. 2.4.5. Electron micrographs of sPLA2-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. 122 Chapter Role of Group IIA sPLA2 in nociception 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 123 Chapter Role of Group IIA sPLA2 in nociception 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 124 Chapter Role of Group IIA sPLA2 in nociception 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 125 Chapter Role of Group IIA sPLA2 in nociception 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 kainatereceptor 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). 126 Chapter Role of lysophospholipids in synaptic transmission CHAPTER ROLE OF LYSOPHOSPHOLIPIDS IN SYNAPTIC TRANSMISSION 127 Chapter Role of lysophospholipids in synaptic transmission 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 128 Chapter Role of lysophospholipids in synaptic transmission 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 dosedependent 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 129 Chapter Role of lysophospholipids in synaptic transmission (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. 130 Chapter Role of lysophospholipids in synaptic transmission 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 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, 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 or 0.2 Hz by a CCD camera with exposure time of 18 ms, and image stacks were analyzed using MetaMorph 131 Chapter Role of lysophospholipids in synaptic transmission 6.3 software (Universal Imaging, Downingtown, PA, USA). The number of subplasmalemmal vesicles was counted, and the means of 11–24 cells in each treatment calculated. 5.2.2. Capacitance measurements The effects of lysoPC, lysoPS and lysoPI 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. 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. Only cells that were stably voltage-clamped were analyzed. These showed fairly stable series resistance throughout the recording (10–12 MOhm) with no sudden changes of more than 10%. Capacitance values were recorded from each cell during the first 30 s after addition of lysoPC, lysoPS or lysoPI and divided by the value 132 Chapter Role of lysophospholipids in synaptic transmission immediately before addition of lysophospholipids, to yield a normalized value. This was to take into account slight differences in initial capacitance between cells of different sizes. A total of 10–12 cells were recorded in each group. Possible significant differences were analyzed by Student’s t test. P < 0.05 was considered significant. Experimental studies were also carried out by to explore possible factors that could affect exocytosis induced by lysoPI: (1) Cells were pre-treated with MBCD which disrupts the integrity of cholesterol rich domains or ‘‘lipid rafts’’ on the cell membrane (Ko et al. 2005; Sun et al. 2005; Shvartsman et al. 2006) to determine a possible role of cholesterol rich domains on the cell membrane in the effects of lysoPI. PC-12 cells were incubated with 10 mM MBCD (Sigma-Aldrich) for 10 at 37 oC, and washed twice with PBS before transfer to external solution and addition of lysoPI. (2) Cells were pretreated with thapsigargin to deplete [Ca2+]i stores, and recorded in zero Ca2+ conditions, to determine a possible role of [Ca2+]i in the effects of lysoPI. PC-12 cells were incubated with μM thapsigargin for 15 at room temperature and 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), before infusion of lysoPI. 5.2.3. Amperometry measurements The quantal release of catecholamines from PC-12 cells after external application of lysoPI was also analyzed using carbon fiber electrodes as previously described (Chow et al. 1992; Bruns et al. 2000). Amperometric 133 Chapter Role of lysophospholipids in synaptic transmission currents were recorded using highly sensitive low-noise carbon fiber electrodes (ALA Scientific Instruments) with an EPC-9 amplifier (HEKA Elektronik, Germany) with electrode voltage set to 780 mV. The tip of the electrode was cut with a surgical blade before recordings, to ensure cleanliness and sensitivity of its surface. The recordings were filtered at kHz, digitized at 10 kHz, and analyzed using custom software. Each treatment group consists of ten to twelve cells. Possible significant differences between the groups were analyzed using twotailed unpaired Student’s t-test. P < 0.05 was considered significant. 5.2.4. Intracellular calcium imaging [Ca2+]i concentration of PC-12 cells was analyzed after external infusion of lysoPI as previously described (Pal et al. 1999; Raza et al. 2001). PC-12 cells 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 for h at 37 °C. Loaded cells were washed two times with buffer and incubated for an additional 30 to allow for cellular esterase cleavage of the acetoxymethyl moiety and intracellular trapping of the free acid Fura-2 indicator. 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 processing using MetaMorph 7.0.4 software (Molecular Devices, Sunnyvale, CA, 134 Chapter Role of lysophospholipids in synaptic transmission 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. Each treatment group measured 10-12 cells. 135 Chapter Role of lysophospholipids in synaptic transmission 5.3. Results 5.3.1. TIRFM (Fig. 2.5.1.) A pronounced increase in the rate of membrane fusion indicating exocytosis was observed after external infusion of lysoPI (Fig. 2.5.1.). The vesicles were depleted by 58.3 ± 5.6% after lysoPI treatment for min. In comparison, vesicles were depleted by 48.3 ± 10.7%, 25.5 ± 6.6% and 25.2 ± 11.0% after lysoPC, lysoPS or ethanol treatment for min, respectively (Fig. 2.5.1C). LysoPC, lysoPS and ethanol (vehicle control) were also less effective or ineffective in inducing vesicle fusion, compared to lysoPI (Fig. 2.5.1D). LysoPI increased vesicle delivery to the subplasmalemmal membrane (53.86 ± 4.40 vesicles per min), while lysoPC and lysoPS had no effect compared to vehicle treated controls (lysoPC, 18.63 ± 5.17 vesicles; lysoPS, 13 ± 1.73 vesicles; ethanol, 8.17 ± 1.80 vesicles). 136 Chapter Role of lysophospholipids in synaptic transmission Fig.2.5.1. TIRFM imaging of vesicles footprints and fusion events happened at subplasmalemmal region. A: An example recording of secretory vesicles reduction before (left) and ~2 after (right) lysoPI stimulation. Predocking vesicles were labeled with circles, which represented vesicles appearing on the first plane of a stack; new-delivered vesicles were labeled with squares. B: Representative process of a fusion event captured at Hz. 137 Chapter Role of lysophospholipids in synaptic transmission C D Ethanol LysoPS LysoPC LysoPI Lyso PI Lyso PC Lyso PS Ethanol Fig.2.5.1. TIRFM imaging of vesicles footprints and fusion events happened at subplasmalemmal region. C: Lysophospholipids triggered exocytosis in PC-12 cells. LysoPI was most effective in depleting vesicles while the lysoPC and lysoPS which have less significant vesicle reduction ratio as compared to vehicle control ethanol. D: LysoPI increased vesicles delivery to the subplasmalemmal membrane while lysoPC and lysoPS had no significant effect in new vesicle delivering compared to vehicle control. *: significant difference compared to ethanol (vehicle control) (P < 0.05 analyzed by Student’s t-test). 5.3.2. Capacitance measurements (Fig. 2.5.2., 2.5.3.) There was significant increase in membrane capacitance by 1.04 ± 0.02% compared to the resting state, after addition of lysoPI for 30 s. This indicates significant induction of exocytosis after addition of lysoPI. In contrast, no significant change in capacitance was observed after addition of lysoPC (0.97 ± 0.02%), lysoPS (1.01 ± 0.01%) or ethanol (vehicle control) (1.01 ± 0.01%) (Fig. 2.5.2.). Pre-incubation of cells with MBCD, resulted in attenuation of lysoPI 138 Chapter Role of lysophospholipids in synaptic transmission induced exocytosis (1.01 ± 0.01%) (Fig. 2.5.3.). Cells that were pre-treated with thapsigargin and recorded in zero Ca2+ conditions also showed no induction of exocytosis after addition of lysoPI (0.99 ± 0.01%) (Fig. 2.5.3.). 139 Chapter Role of lysophospholipids in synaptic transmission Ethanol LysoPC Normalized Capacitance(%) 1.08 1.04 1.00 0.96 30 Time(s) A Ethanol LysoPI Normalized Capacitance(%) 1.08 * 1.04 1.00 0.96 30 Time(s) B Ethanol LysoPS Normalized Capacitance(%) 1.08 1.04 1.00 0.96 C 30 Time(s) Fig.2.5.2. Capacitance measurements. There was a significant increase in membrane capacitance indicating exocytosis after addition of lysoPI. In contrast, no significant change in capacitance was observed after addition of lysoPC and lysoPS. *: significant difference compared to ethanol (vehicle control) (P < 0.05 analyzed by Student’s t-test). 140 [...]... Chapter 5 Role of lysophospholipids in synaptic transmission USA) Background autofluorescence values for both 34 0- and 38 0-nm excitations were obtained and subtracted from experimental 34 0 /38 0-nm excitation-emission values Regions of interest corresponding to PC-12 cells were selected, and the ratio of 34 0 /38 0-nm excitation-emissions in these regions calculated to give an indication of [Ca2+]i levels... (Gillis 1995; Chen and Gillis 2000; Chen et al 2001; Sun and Wu 2001; Zhang and Zhou 2002; Wei et al 20 03) 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... transfer to external solution and addition of lysoPI (2) Cells were pretreated with thapsigargin to deplete [Ca2+]i stores, and recorded in zero Ca2+ conditions, to determine a possible role of [Ca2+]i in the effects of lysoPI PC-12 cells were incubated with 1 μM thapsigargin for 15 min at room temperature and transferred to external solution containing EGTA (Sigma-Aldrich) dissolved in (mM) 150 NaCl, 2.8... 10-12 cells 135 Chapter 5 Role of lysophospholipids in synaptic transmission 5 .3 Results 5 .3. 1 TIRFM (Fig 2.5.1.) A pronounced increase in the rate of membrane fusion indicating exocytosis was observed after external infusion of lysoPI (Fig 2.5.1.) The vesicles were depleted by 58 .3 ± 5.6% after lysoPI treatment for 2 min In comparison, vesicles were depleted by 48 .3 ± 10.7%, 25.5 ± 6.6% and 25.2 ± 11.0%... lysophospholipids in synaptic transmission induced exocytosis (1.01 ± 0.01%) (Fig 2.5 .3. ) Cells that were pre-treated with thapsigargin and recorded in zero Ca2+ conditions also showed no induction of exocytosis after addition of lysoPI (0.99 ± 0.01%) (Fig 2.5 .3. ) 139 Chapter 5 Role of lysophospholipids in synaptic transmission Ethanol LysoPC Normalized Capacitance(%) 1.08 1.04 1.00 0.96 5 30 Time(s) A... culture dishes, and loaded with acetoxymethyl form of the membrane-permeable ratiometric fluorescent Ca2+ indicator Fura-2 (5 µM, Invitrogen) in HEPES buffer for 1 h at 37 °C Loaded cells were washed two times with buffer and incubated for an additional 30 min to allow for cellular esterase cleavage of the acetoxymethyl moiety and intracellular trapping of the free acid Fura-2 indicator Alternating excitation... using MetaMorph 131 Chapter 5 Role of lysophospholipids in synaptic transmission 6 .3 software (Universal Imaging, Downingtown, PA, USA) The number of subplasmalemmal vesicles was counted, and the means of 11–24 cells in each treatment calculated 5.2.2 Capacitance measurements The effects of lysoPC, lysoPS and lysoPI on vesicle fusion during exocytosis were also quantified using capacitance measurement... vesicles) 136 Chapter 5 Role of lysophospholipids in synaptic transmission Fig.2.5.1 TIRFM imaging of vesicles footprints and fusion events happened at subplasmalemmal region A: An example recording of secretory vesicles reduction before (left) and ~2 min after (right) lysoPI stimulation Predocking vesicles were labeled with circles, which represented vesicles appearing on the first plane of a stack;... resting state, after addition of lysoPI for 30 s This indicates significant induction of exocytosis after addition of lysoPI In contrast, no significant change in capacitance was observed after addition of lysoPC (0.97 ± 0.02%), lysoPS (1.01 ± 0.01%) or ethanol (vehicle control) (1.01 ± 0.01%) (Fig 2.5.2.) Pre-incubation of cells with MBCD, resulted in attenuation of lysoPI 138 Chapter 5 Role of lysophospholipids... EDTA, 1 MgCl2 and 10 HEPES and 2 mg/ml glucose pH 7.2 (31 0 mOsm), before infusion of lysoPI 5.2 .3 Amperometry measurements The quantal release of catecholamines from PC-12 cells after external application of lysoPI was also analyzed using carbon fiber electrodes as previously described (Chow et al 1992; Bruns et al 2000) Amperometric 133 Chapter 5 Role of lysophospholipids in synaptic transmission currents . affirming the role of sPLA 2 in inflammation. The present finding of dense immunolabeling of sPLA 2 -IIA in the spinal trigeminal nucleus of the brainstem and dorsal horn of the cervical spinal. sPLA 2 in inflammation has been extensively studied. Recent findings showed that sPLA 2 induced expression of pro-inflammatory cytokines including TNF-α and IL-1β in the injured spinal cord. penicillin/streptomycin (Gibco, Invitrogen). The cells were plated in 35 mm Petri dishes, and maintained in an incubator at 37 o C, 100% humidity, with 95% air and 5% CO 2 . Coverslips containing