Role of phospholipase a2 in orofacial pain and synaptic transmission 4

Infusion of lysoPI in the external solution resulted in an immediate increase in catecholamine release from PC-12 cells 132.7 ± 32.94 spikes Fig.. Chapter 5 Role of lysophospholipids in

Chapter 5 Role of lysophospholipids in synaptic transmission

Fig.2.5.3 Capacitance measurements A: Treatment of cells with MBCD prior to addition of lysoPI resulted in attenuation of lysoPI induced exocytosis B: Cells pre-treated with thapsigargin and recorded in zero external Ca2+ solution showed no induction of exocytosis after addition of lysoPI

*: significant difference (P < 0.05, analyzed by Student’s t-test)

5.3.3 Amperometry measurements (Fig 2.5.4.)

Infusion of lysoPI in the external solution resulted in an immediate increase in catecholamine release from PC-12 cells (132.7 ± 32.94 spikes) (Fig 2.5.4A) This increase was markedly attenuated in cells that were pre-incubated with MBCD (3.5 ± 3.4 spikes) (Fig 2.5.4B) or pre-treated with thapsigargin and recorded in zero Ca2+ conditions (2 ± 0.19 spikes) (Fig 2.5.4C) These results

B

*

Chapter 5 Role of lysophospholipids in synaptic transmission

show that neurotransmitter release triggered by lysoPI was dependent on the integrity of cholesterol rich domains on the cell membrane and [Ca2+]i

80 60

40 20

80 60

40 20

LysoPI, 0mM Ca 2+ , EGTA, thapsigargin B

C

5s 4pA

-10

30 70 110

80 60

40 20

80 60

40 20

80 60

40 20

LysoPI, 0mM Ca 2+ , EGTA, thapsigargin B

C

5s

4pA 5s 4pA

-10

30 70 110

Fig.2.5.4 Amperometry measurements A: infusion of lysoPI in the external solution resulted in

an immediate increase in catecholamine release from PC-12 cells B, C: this increase was completely abolished in cells that had been pre-incubated with MBCD (B) or Ca2+ free external solution containing the Ca2+ chelator EGTA and thapsigargin (C) D: summary results of experiments (A–C) Events are selected with spikes >2 pA for the first 1 min application of lysoPI

*: significant difference (P < 0.05, analyzed by Student’s t-test).

Chapter 5 Role of lysophospholipids in synaptic transmission

5.3.4 Fura-2 measurements (Fig 2.5.5.)

LysoPI induced a sustained increase in [Ca2+]i in PC-12 cells of 1.06 ± 0.06 of normalized 340/380 ratio compared to the resting state The increase in [Ca2+]i was markedly attenuated in cells that were pre-incubated with MBCD (1.0

± 0.002 of normalized 340/380 ratio) or pre-treated with thapsigargin and recorded in zero Ca2+ conditions (1.0 ± 0.02 of normalized 340/380 ratio) (Fig 2.5.5)

Fig.2.5.5 Fura-2 imaging LysoPI induced a sustained increase in [Ca2+]i in PC-12 cells The lysoPI induced increase in [Ca2+]i was attenuated by pretreatment of cells with MBCD and by pre- incubation of cells with thapsigargin No change in [Ca2+]i concentration was detected after addition of ethanol (vehicle control) Arrow indicates time of addition of lysoPI.

Chapter 5 Role of lysophospholipids in synaptic transmission

5.4 Discussion

The present study demonstrated possible effects of lysophospholipids on exocytosis An increase in vesicle fusion, indicating exocytosis, was observed in PC-12 cells after external infusion of the lysoPI, but not lysoPC or lysoPS by TIRFM Similarly, external infusion of lysoPI, but not lysoPC or lysoPS induced significant increases in capacitance, or number of spikes detected by carbon fiber electrodes at amperometry, indicating exocytosis Depletion of cholesterol

by pre-incubation of cells with MBCD and depletion of Ca2+ by thapsigargin and incubation in zero external Ca2+ resulted in attenuation of lysoPI induced exocytosis, indicating that exocytosis was dependent on the integrity of lipid rafts and [Ca2+]i Moreover, lysoPI induced a rise in [Ca2+]i suggesting that this could

be the trigger for exocytosis It is possible that lysoPI exerts it effects through binding to a lysoPI specific receptor on the cell membrane or by its physical properties on the cell membrane A lysoPI specific receptor (GPR55) has been identified in the frontal cortex, striatum, hypothalamus, caudate putamen of the brain (Sawzdargo et al 1999; Johns et al 2007; Ryberg et al 2007) In addition, lysoPI has ‘‘detergent like’’ actions and could affect the functions of ion channels

or receptors on the cell membrane to cause an increase in [Ca2+]i concentration and exocytosis The present results are consistent with previous studies which showed that lysoPI causes Ca2+ influx via store operated Ca2+ entry channels (Singaravelu et al 2008) Moreover, lysoPI has been shown to mediate the release of insulin upon melittin (induced sPLA2) stimulation, a process which

Chapter 5 Role of lysophospholipids in synaptic transmission

exhibits the characteristics of physiologic exocytosis, i.e., it is reversible, saturable and does not influence subsequent islet functioning (Metz 1986)

Besides lysoPI, phosphatidylinositol 4,5-bisphosphate (PtdIns (4,5)P2), a phosphoinositide found mainly in the plasma membrane also regulates exocytosis and synaptic transmission (Valtorta and Meldolesi 1994; Holz et al 2000) PtdIns (4,5)P2 is thought to have multiple roles in exocytosis, including the establishment of secretory granule docking sites on the plasma membrane as well as participating in a step linked to fusion (Martin 2001) Moreover, glycosylphosphatidylinositol-anchored proteins in the lipid rafts of neurons are able to activate signaling pathways affecting exocytosis (Brown 2006) Previous studies have suggested a link between PLA2 and lysophospholipids in pancreatic secretion and mast cell degranulation PLA2 and acyltransferase activities were identified in membranes associated with purified pancreatic zymogen granules PLA2 activity was correlated to protein concentration and was Ca2+-dependent, consistent with a sPLA2 isoform The intact zymogen granules and granule membranes demonstrated reacylating activity which was related to the concentration of lysophospholipid (Rubin et al 1990) Similarly, PLA2 which prefers PI over PC as substrate has been detected in mast cell secretory granules As with pancreatic zymogen granules, mast cell granules contain an active acylating system which rapidly reacylate lysoPI to form PI These findings provide a basis for linking PLA2 activity and formation of lysophospholipids, to granule exocytosis (Chock et al 1991) Besides lysoPI, lysoPS also provoked degranulation and histamine release in mast cells (Smith et al 1979; Bigon et al

Chapter 5 Role of lysophospholipids in synaptic transmission

1980; Bruni et al 1984) by enhancing stimulus-dependent Ca2+ ion influx through

a specific membrane receptor (Inoue K et al 1989) LysoPS did not cause exocytosis in PC-12 cells however Another lysophospholipid, lysoPC also did not cause any exocytosis in PC-12 but it able to stimulate cell motility and releases pro-inflammatory cytokines LysoPC modulates ion channel permeability in various brain preparations through at least three different mechanisms By incorporating into neural membranes, it can perturb the orderly packing of phospholipid bilayers inducing alterations of the normal conformation

of integral membrane proteins such as ion channels Secondly, lysoPC can interact directly with ion channel proteins, and finally, lysoPC can modulate the ion channel by modulating signal transduction processes Thus, in neurons, lysoPC produces prolonged hyperpolarization of K+ channels (Maingret et al 2000) Under certain conditions, lysoPC also causes cell fusion Thus, lysoPC may be involved in cell-cell and membrane-membrane interactions and neurotransmitter release (Farooqui and Horrocks 2006)

The findings of a role of lysoPI in exocytosis may be important from the standpoint of the function of sPLA2 in neuroendocrine cells Previous studies showed that external application of sPLA2-IIA (crotoxin B or purified human synovial sPLA2) to PC-12 cells and cultured rat hippocampal neurons resulted in

an immediate increase in exocytosis and neurotransmitter release EGTA and a specific inhibitor of sPLA2 activity, 12-epi-scalaradial, completely abolished the increase in neurotransmitter release, indicating that the effect of sPLA2 was dependent on Ca2+ and sPLA2 enzymatic activity (Wei et al 2003) These

Chapter 5 Role of lysophospholipids in synaptic transmission

findings suggest that sPLA2 may have a role in exocytosis and neurotransmitter release in neuroendocrine cells and neurons In recent findings (in chapter 4), it showed high levels of sPLA2-IIA expression in the brainstem and spinal cord sPLA2-IIA protein was found in the brainstem, cervical, thoracic and lumbar spinal segments by Western blots The enzyme was localized by immunohistochemistry to neuronal cell bodies and dendrites in the spinal trigeminal nucleus and the dorsal and ventral horns of the spinal cord Electron microscopy of the spinal cord showed that sPLA2-IIA was localized in dendrites

or dendritic spines that were postsynaptic to unlabeled axon terminals sPLA2-IIA contains a strong secretory signal peptide, and it is probable that this isoform is actually secreted from neurons In view of the effects of lysoPI on exocytosis mentioned above, it is possible that sPLA2-mediated effects on exocytosis could

be partly due to the generation of lysoPI from hydrolysis of phosphatidylinositol which is located on the exofacial aspect of the cell membrane/or the inner leaflet

of synaptic vesicles

It is postulated that secretion of sPLA2 from neurons and action on neuronal membranes to generate lysoPI from PI may play a role in neural transmission If this is the case, a rapid reacylating enzyme such as lysoPI acyltransferase (Gijon et al 2008) might also be expected to be present in CNS regions with high levels of sPLA2, and future studies are needed to evaluate this possibility The contribution of another lysophospholipid, lysoPE to exocytosis could not be excluded, although these were not analyzed in this study due to

Chapter 5 Role of lysophospholipids in synaptic transmission

their insolubility in ethanol or aqueous solutions Further work is necessary to elucidate possible sPLA2 and/or lysophospholipid mediated signaling in the CNS

Section IV Conclusion

SECTION IV CONCLUSION

Section IV Conclusion

PLA2 isoforms play different roles in the CNS Currently, there is still a lack

of information regarding changes in PLA2 activity and expression after hyperalgesia and the role of PLA2 in the synaptic transmission This study therefore, elucidated the role of PLA2, sPLA2 in particular, after orofacial pain The changes in brain lipids indicating an increased PLA2 activity was accompanied by an increased expression of sPLA2-III The study also demonstrated sPLA2-III function in neurotransmission using PC-12 cells

In elucidating the changes in brain lipids, it was observed that there was a decrease in phospholipids including PE and PI species, and an increase in corresponding lysophospholipids which included lysoPE, lysoPI and lysoPS in the CM after facial CA injection These results indicated an elevated PLA2 activity and release of AA due to PLA2 enzymatic action on the phospholipids of the membrane AA and its metabolites such as PEG2 and leukotrienes are shown to

be biologically active and cause inflammation at nanomolar and micromolar concentrations (Saxena 2000; Mechiche et al 2003; Wise 2006) These findings indicated increased CNS PLA2 activity, resulting in generation of metabolites that contribute to allodynia after peripheral inflammation

Having shown in previous studies that PLA2 inhibitors reduced nociception, the changes in expression of PLA2 isoforms including cPLA2, iPLA2, sPLA2 (sPLA2-IB, -IIA, -IIC, -IID, -IIE, -IIF, -III, -V, -X, -XIIA) were elucidated after facial

CA injection These changes likely take place in the spinal trigeminal nucleus which relays nociceptive inputs which originate from the orofacial region, and occupies a large proportion of the CM in rats sPLA2-III mRNA expression was

Section IV Conclusion

increased in the medulla of CA-injected rats, although no upregulation in III protein expression was detected This was supported by previous studies which showed increased activity of cPLA2 but no change in its mRNA or protein expression in the spinal cord after hyperalgesia induced by CA injection to the hind paw (Lucas et al 2005) The changes in lipids as detected by lipidomics are therefore consistent with an increase in enzyme activity without any changes in enzyme protein expression Together, these findings showed enhanced PLA2 activity in the CM after inflammatory orofacial pain

sPLA2-Further study on the expression profile of sPLA2-III in the CNS showed that the enzyme was expressed at the highest level in the medulla oblongata Both mRNA and protein expression of sPLA2-III in normal rat CNS were at the highest level in the brainstem and spinal cord It was localized in the spinal trigeminal nucleus, further supporting its role in the nociceptive pathway Functionally, sPLA2-III was observed to be involved in pain transmission as the external application of purified form of sPLA2-III, the bee venom, showed an increased membrane capacitance indicating exocytosis sPLA2-III induced exocytosis could be affected by external Ca2+ and presumably by the integrity of lipid rafts External application of sPLA2-III also resulted in an increase in [Ca2+]i, suggesting that this could be the trigger for exocytosis These findings are supported by evidence from previous studies that subcutaneous injection of PLA2-related peptide isolated from the bee venom induced nociceptive paw flinches

Section IV Conclusion

Besides sPLA2-III, another sPLA2 isozyme, sPLA2-IIA was also observed

to be highly involved in nociception Using sPLA2-V as a comparison, the expression profiles of multiple sPLA2 isoforms with the strong secretory signals which included sPLA2-IB, -IIA, -IIC and –X, showed that both mRNA and protein expression of sPLA2-IIA were at high levels in the brainstem and spinal cord Moreover, sPLA2-IIA was also localized in the spinal trigeminal nucleus of the brainstem and the dorsal horn of cervical spinal cord which indicates its role in the nociceptive pathway The mechanism by which sPLA2-IIA was secreted could

be via kainate-receptor binding sPLA2-IIA was released upon stimulation by kainate and this effect was abolished after treatment with a kainate receptor antagonist (Than et al 2011) In addition to this, there are evidences that sPLA2-IIA could also be released by rat brain synaptosomes upon stimulation by acetylcholine and glutamate receptors or via depolarization of voltage dependent

Ca2+ channels (Kim et al 1995b; Matsuzawa et al 1996) In view of its postsynaptic localization and the fact that sPLA2-IIA is a secreted protein (Komada et al 1990), it can be postulated that sPLA2 might be released from dendrites upon depolarization, resulting in axonal exocytosis These evidences supported that sPLA2-IIA plays an active role in the synaptic transmission External application of sPLA2-IIA to cultured hippocampal neurons and PC-12 cells resulted in an instantaneous increase in exocytosis and neurotransmitter release (Wei et al 2003) As sPLA2-III has also been shown to be localized in the dendrites, it can be postulated that this enzyme will be secreted in a similar manner to induce exocytosis

Section IV Conclusion

PLA2 also participates in the synaptic transmission not only via the secretion of itself, i.e in sPLA2-III and –IIA, but also through its enzymatic product such as lysophospholipids External infusion of lysoPI showed an increased rate of membrane fusion indicating exocytosis using TIRFM Similarly, significant increases in capacitance and the number of spikes detected by amperometry, indicating exocytosis were observed after external application of lysoPI LysoPI induced exocytosis was found to be influenced by the structure of lipid rafts and [Ca2+]i Moreover, lysoPI induced an increase in [Ca2+]i which could trigger the exocytotic process It is therefore postulated that lysoPI may be an active participant in sPLA2-mediated exocytosis in the CNS In view of the effects

of lysoPI on exocytosis mentioned above, it is possible that sPLA2-mediated effects on exocytosis could be partly due to the generation of lysoPI from hydrolysis of phosphatidylinositol which is located on the exofacial aspect of the cell membrane/or the inner leaflet of synaptic vesicles

In conclusion, this study has shown that both PLA2 activity and expression were significantly elevated upon orofacial pain induced by facial CA injection Both sPLA2-III and sPLA2-IIA were observed to be the isozymes which play critical roles in the ascending pain pathway Moreover, evidence from this study has shown that sPLA2 could participate in pain transmission via the release of itself or through its enzymatic product, lysophospholipid This study therefore has provided a better understanding of this enzyme, PLA2, in the pain transmission pathway and could possibly contribute towards formulating drug targets which may alleviate pain symptoms in various diseases

Section V References

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