The mechanism of action of notexin in the facilitation of spontaneous transmitter release at neuromuscular synapse was investigated in Xenopus cell culture by using whole-cell patch clamp recording. Exposure of the culture to notexin dose-dependently enhanced the frequency of spontaneous synaptic currents (SSCs). Either buffering of intracellular Ca2+ rise by BAPTA or replacing culture medium with Ca2+-free Ringer¡¦s solution effectively hampered notexin effect, suggesting Ca2+ influx is requisite for this facilitation. Pretreatment of the cultures with a TRP channel inhibitor SKF96365, instead of voltage-dependent L-type Ca2+ channel blockers nifedipine or verapamil, significantly abolished the SSC facilitating effect of notexin. Furthermore, knockdown the expression of TRPC channels by TRPC-specific morpholino abruptly abolished notexin effect, suggesting that TRPC channel is the major entranceway of extracellular Ca2+. The notexin-enhanced SSC frequency was also obviously reduced under intracellular Ca2+ store depletion by pretreatment of the cultures with pharmacological Ca2+-ATPase inhibitor thapsigargin or CPA. Bath application of membrane-permeable inositol 1,4,5-triphosphate (IP3) inhibitor, XeC, effectively occluded the facilitation of SSC frequency elicited by notexin. Furthermore, notexin-induced SSC frequency facilitation was blocked in the presence of phospholipase C (PLC) inhibitor, U73122. Taken collectively, these results suggest that notexin elicits a Ca2+ release from the IP3-sensitive intracellular Ca2+ store which resulted in further store depletion-operated Ca2+ influx through membrane TRPC channels of the presynaptic nerve terminal. This is done via PLC signaling cascade, leading to an enhancement of spontaneous transmitter release.
Notexin-induced synaptic facilitation is potentially reduced while structural modification with phenylglyoxal. In addition, bath application of PLA2 inhibitor either aristolochic acid or glycyhirrzin effectively abolished notexin effect, suggesting the PLA2 activity is involved in notexin-induced SSC frequency facilitation. Previously it has been suggested local accumulation of PLA2-induced lipid metabolites myristoyl lysophosphatidylcholine (mLPC) and oleic acid (OA) promotes the fusion of the hemifused synaptic vesicles with plasma membrane, hence facilitating the neurotransmitter release in cultured cerebella granular neurons and thus resulted in bulges formation along the neurite. Our real-time morphometric analysis and synaptic activity assays showed that bulges formation along the neurite appeared significantly earlier and was induced at lower notexin concentrations than synaptic activity facilitation. Bath application of either mLPC, OA alone or their mixtures failed to mimic the notexin-induced facilitation in spontaneous transmitter release. Attenuation of PLA2 activity by chemical modification (notexin-80) resulted in correlated decrease of notexin-induced synaptic facilitation but not the degenerative morphological sign. Moreover, PLP-notexin, a site-specific modification of notexin with full intact PLA2 activity, shows significant loss the ability of notexin-induced neurite degeneration. Overall, results from our studies suggest the morphologic changes and synaptic facilitating effect induced by notexin are resulted from different cellular mechanisms.
| Identifer | oai:union.ndltd.org:NSYSU/oai:NSYSU:etd-0811109-175515 |
| Date | 11 August 2009 |
| Creators | Hsu, Fang-jui |
| Contributors | Ing-Jun Chen, Jau-Cheng Liou, Bin-Nan Wu, L.S. Chang |
| Publisher | NSYSU |
| Source Sets | NSYSU Electronic Thesis and Dissertation Archive |
| Language | Cholon |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.lib.nsysu.edu.tw/ETD-db/ETD-search/view_etd?URN=etd-0811109-175515 |
| Rights | not_available, Copyright information available at source archive |
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