行政院國家科學委員會專題研究計畫 成果報告
乙型雨傘節神經毒素促進神經傳遞物質釋放之分子機制與
其蛋白質結構間關聯性之研究
計畫類別： 個別型計畫 計畫編號： NSC93-2320-B-110-012- 執行期間： 93 年 08 月 01 日至 94 年 07 月 31 日 執行單位： 國立中山大學生物科學系(所) 計畫主持人： 劉昭成 計畫參與人員： 康凱翔、何士因 報告類型： 精簡報告 處理方式： 本計畫可公開查詢中 華 民 國 94 年 10 月 12 日
行政院國家科學委員會補助專題研究計畫
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乙型雨傘節神經毒素促進神經傳遞物質釋放之分子機制與其
蛋白質結構間關聯性之研究
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93-2320-B-110-012
執行期間： 93 年 8 月 1 日至 94 年 7 月 31 日
計畫主持人：劉昭成
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計畫參與人員： 康凱翔，何士因
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中 華 民 國 94 年 10 月 7 日
行政院國家科學委員會專題研究計畫成果報告
乙型雨傘節神經毒素促進神經傳遞物質釋放之分子機制與其蛋白質結構間
關聯性之研究
計畫編號：NSC
93-2320-B-110-012
執行期限：93 年 8 月 1 日至 94 年 7 月 31 日 主持人：劉昭成 中山大學生物科學系 計畫參與人員：康凱翔，何士因 中山大學生物科學系 中文摘要 β-bungarotoxin（β-BuTx）屬於突觸 前 具 有 磷 酸 脂 酶 A2 活 性 之 神 經 毒 素 （presynaptic PLA2 neurotoxin），結構上 β-BuTx 由次單元 A-chain 及 B chain 兩個 subunits 間以一雙硫鍵共價鍵結所組成。雖 然自發現已來，β-BuTx 在蛋白質結構，磷 酸脂酶酵素活性以及對神經活性的影響已 經有廣泛的研究，但是對其影響神經功能的 作用機轉仍不是十分清楚，而這有一大部分 導因為沒有適當的 animal model。有別於動 物 組 織 實 驗 的 局 限 性 ， 我 們 的 爪 蟾 （Xenopus）神經-肌細胞培養提供一個絕佳 的機會使我們可以領先其他科學家來發掘 β-BuTx 促進神經傳遞物質釋放作用的分子 訊息傳遞機制。在我們的試驗結果中我們發 現約經過十二～十八分鐘之後 1µg/ml 的 β−BuTx 在爪蟾神經-肌細胞培養中可以造 成非常顯著而持久的神經傳遞物質釋放現 象（可達數十倍之多）。另外我們也發現 β-BuTx 的作用不須要鈣離子流入神經的動 作，而是 mobilize 細胞內鈣離子儲存池的 鈣，而其相關訊息傳遞路徑則是與細胞內 IP3的鈣離子儲存池鈣的釋放、PI 3-kinase／ G protein／Pholipase A2 活性的啟動有關。 英文摘要The mechanism of action of β-bungarotoxin in the facilitation of spontaneous transmitter release at developing
β-bungarotoxin dose-dependently enhances the frequency of spontaneous synaptic currents (SSCs). Buffering the rise of intracellular Ca2+ with BAPTA-AM hampered the facilitation of SSC frequency induced by β-bungarotoxin. The β-bungarotoxin-enhanced SSC frequency was not abolished when pharmacological Ca2+ channel inhibitor Cd2+ was added in the culture medium, indicating that Ca2+ influxes through voltage-activated Ca2+ channels are not required. Application of membrane-permeable inhibitors of inositol 1,4,5-trisphosphate (IP3) but not ryanodine receptors effectively occluded the increase of SSC frequency elicited by RA. Treating cells with either wortmannin or LY294002, two structurally different inhibitors of phosphatidylinositol 3-kinase (PI3K) and with phospholipase C (PLC) inhibitor U73122, abolished RA-induced facilitation of synaptic transmission. The β-bungarotoxin-induced synaptic facilitation was completely abolished while there was presynaptic loading of the motoneuron with GDPβS, a non-hydrolyzable GDP analogue and inhibitor of G protein. Taken collectively, these results suggest that β-bungarotoxin elicits Ca2+
release from IP3 sensitive intracellular Ca2+ stores of the presynaptic nerve terminal. This is done via PLC/PI3K signaling cascades and G protein activation, leading to an enhancement of spontaneous transmitter release.
multicinctus (Taiwan banded krait) and
pharmacologically characterized as a 21.8 kDa-phospholipase A2 (PLA2) neurotroxin (Chang and Lee, 1963). A rich history of research dating back to the time of CY Lee and CC Chang suggests that this toxin consists of two dissimilar polypeptide subunits, a structurally homologous PLA2 subunit named A chain, and a non-PLA2 subunits B chain which is long thought to act as an affinity probe to guide the neurotoxin to its target on nerve terminal (Chang, 1985). The A chain and B chain are covalently linked by one disulfide bridge and modern chromatographic techniques and amino acid analysis have revealed that there consists three main isoforms of β-BuTx which share a common PLA2 subunit but possess distinct subunits B (Chu et al., 1995). Envenoming bites by kraits are associated with an acute onset of neuromuscular paralysis and followed by a prolonged, wide-spread neuronal cell death throughout the mammalian and avian CNS (Rowan, 2001). Upon systemic administration to the chick embryo (Hirokawa, 1977; Zilles et al., 1982) and stereotaxic administration to rat brain, β-BuTx causes a widespread degeneration of neurons (Hanley & Emson, 1979; Ciutat et al., 1996). Results from several studies performed on frog sartorius and mouse phrenic nerve-diaphragm preparation have suggested that following exposure of isolated nerve-muscle preparations to β-BuTx, the development of neuromuscular transmission failure undergoes a triphasic change. An initial phase of weak reduction of spontaneous acetylcholine release is followed by a second prolonged phase of facilitated release, and then by a third phase of progressive decline of spontaneous neurotransmission (Su and Chang, 1984). It have been shown that β-BuTx causes massive Ca2+ influx through NMDA and L-type Ca2+ channels that induced production of reactive oxygen species (ROS) and disturbed mitochondrial function, resulting in collapse of mitochondrial membrane potential and ATP depletion (Tseng and Lin-Shiau, 2002). Furthermore, results from recent studies using cultured cerebellar granule neurons and rat phrenic nerve-diaphragm preparation have indicated that β-BuTx-induced depletion of synaptic vesicles
resulted in synaptic block.
The sophisticated of the anatomically fine structure of neuromuscular junction had limited the possibility of experimental manipulation of this tissue preparation while trying to understand the detailed signaling mechanisms that are responsible for β-BuTx-induced change on synaptic transmission. Most studies have conducted by employing in vitro twitch tension, central neuronal cell culture, or by using synaptic membranes from brain tissue and biochemical techniques to investigate the effects of β-BuTx. Notwithstanding the innumerable studies carried out with different experimental approaches have been made to reveal the molecular basis of presynaptic neurotoxicity of β-BuTx, we still do not known their underlying molecular mechanisms for the early changes in transmitter release and the subsequent block of transmitter release induced by β-BuTx. Therefore, the searching for an ideal in vitro model more amenable to direct evaluate the β-BuTx-induced activation of signaling cascades in related to change of synaptic transmission is required. We have previous shown that in Xenopus motoneuron-muscle cell culture, β-BuTx cause a dramatic change in frequency of spontaneous neurotransmitter release. In this study, we asked how the β-BuTx causes the facilitation in spontaneous transmitter release. As a result of our current investigations we show for the first time that the facilitation on spontaneous synaptic transmission induced by β-BuTx was resulted from triggering the liberation of Ca2+ from internal store, which is the result of pleiotropic convergent signaling pathways involving PLCγ, phosphatidylinositol 3-kinase (PI3K) and G protein activation. Moreover, our results also indicate that Xenopus nerve–muscle cultures are a useful model system for investigation in the signal transduction pathway responsible for β-BuTx-induced synaptic facilitation.
結果與討論
β-BuTx facilitation of spontaneous neurotransmitter release
In 1-day-old Xenopus nerve-muscle cultures, numerous of neuromuscular synapse are built by spontaneously contacted the myocyte with co-cultured motoneuron (Xie and Poo, 1986). The spontaneous synaptic currents (SSCs) are readily detectable from the innervated muscle cell with the whole-cell voltage-clamp recordings and unaffected by tetrodotoxin, which blocks action potentials in neurons (Xie and Poo, 1986). Addition of the competitive skeletal muscle nicotinic ACh receptor antagonist (+)-tubocurarine and subsequent abolition of the SSCs suggest that these currents were resulted from spontaneous ACh secretion from the motoneuron. Bath application of the cultures to 47.6 nM β-BuTx slightly, yet insignificantly, inhibit the SSC frequency at initial stage (phase I) followed by a dramatic enhancement on spontaneous transmitter release (phase II), as evidenced by a marked increase in the frequency of spontaneous synaptic events. In general, there is a lag period of between 12 and 18 minutes before the increase in SSC frequency reached its plateau after bath application of β-BuTx, and the effect persisted for more than 30 minutes. The β-BuTx-induced increase in the frequency of spontaneous ACh secretion showed a steep dependence on the concentration of b-BuTx between 4.8 and 157.1 nM.
Neuromuscular synaptic transmission involves several distinct steps and each step is susceptible to change under the presence of neurotoxin. The observed increase in the frequency of SSC events after β-BuTx treatment could be due to an increased presynaptic release of neurotransmitter or to an increased postsynaptic sensitivity to the neurotransmitter. Increased postsynaptic ACh sensitivity could explain the increase in the SSC frequency, because previously undetectable small ACh quanta may emerge after exposure to the factor. We found no detectable change in the amplitude distribution of the SSC amplitude, indicating the sensitivity of endplates toward acetylcholine remains unchanged. Thus, the facilitation of
Ca2+-dependent facilitation of spontaneous ACh secretion by β-BuTx
How does β-BuTx come into play in enhancing presynaptic efficacy? It is well-known that the [Ca2+]i level in the nerve terminal exerts a dominant effect on the rate of spontaneous transmitter release, we set out to examine whether a rise in [Ca2+]i is required for the RA-induced SSC frequency facilitation (Augustine et al., 1987). The membrane-permeable Ca2+ chelator BAPTA-AM has been widely used as a probe to test the role of Ca2+ in a large variety of cell functions (Liou et al., 2003). The SSC frequency facilitation by β-BuTx was completely blocked by buffering the [Ca2+]i rise with 60-80 min pretreatment of 30 µM BAPTA-AM, suggesting that the effect of β-BuTx on synaptic transmission requires an increase in [Ca2+]i in the presynaptic neurons. However, what is the source of Ca2+ involved in synaptic facilitation induced by β-BuTx? The rise in [Ca2+]i may be the consequence of Ca2+ influx from the extracellular fluid or liberation of Ca2+ from intracellular stores. It has been suggested that calcium ion is requisite for neurotoxic activity of β-BuTx (Rowan, 2001), thus it was difficult to make direct measurements of the role of [Ca2+]o on β-BuTx-induced facilitatory effect. We thus examined whether the Ca2+ released from intracellular stores is responsible for β-BuTx-induced synaptic facilitation. To approach this problem, the Ca2+-ATPase inhibitor thapsigargin was initially used to deplete intracellular Ca2+ stores. We have previously shown that bath-application of thapsigargin (2 µM) elicited an increase in SSC frequency, which returned to control levels within 40–80 min (Liou et al., 2003). Exposure of the cultures to β-BuTx no longer elicited any changes in SSC frequency in thapsigargin-pretreated synapses. These results suggest that Ca2+ released from intracellular stores was responsible for synaptic potentiation induced by β-BuTx.
ryanodine-sensitive Ca2+ stores (Berridge, 1998). We next investigate the routes of Ca2+ released from intracellular stores that resulted in RA-induced SSC frequency facilitation. Pretreatment of the culture 15-20 minutes with membrane-permeable inhibitors of IP3 receptor XeC (1 µM) or 2-APB (50 µM) effectively ceased the increase of SSC frequency elicited by β-BuTx. The release of Ca2+ from IP3 receptors may further trigger Ca2+-induced Ca2+ release from ryanodine receptors. Treating the cells with β-BuTx still elicited an increase in SSC frequency under the presence of ryanodine receptor antagonist TMB-8 (3 µM) or ruthenium red (10 µM). Thus, an intracellular liberation of Ca2+ from IP3-sensitive pools, rather than an influx of extracellular Ca2+, is mostly like the cause of β-BuTx -induced synaptic facilitation.
Signaling pathways of β-BuTx action
How might β-BuTx participate in mobilizing intracellular Ca2+ store? To approach this enigma, we investigated the signaling cascade that is responsible for the action of β-BuTx in Xenopus neuromuscular synapses. PLC activation is an attractive candidate involving in this synaptic facilitation because its activation would result in intracellular Ca2+ release via the second messenger IP3. To evaluate whether the PLC is part of the β-BuTx signaling mechanism in facilitating neurotransmitter release, we set out to examine the effect of PLC inhibitor on β-BuTx’s action. Pretreatment of cells with PLC inhibitor U73122 (5 µM) prior to β-BuTx treatment completely hampered the β-BuTx -induced increase in SSC frequency, indicating that PLC activity is required for β-BuTx-induced SSC frequency facilitation. Given the suggestion that apart from IP3, DAG and subsequently activated PKC is another signaling molecule initiated by membrane hydrolytic activity of PLC. We test the possible participation of PKC in β-BuTx-induced SSC frequency facilitation. Bath application of a protein kianse C inhibitor H-9 (100 µM) failed to blunt the β-BuTx facilitating effect, suggesting that
PKC is exclusive in the signaling pathway of β-BuTx -induced synaptic facilitation.
Two possible mechanisms are indicated for PLC activation: PLCβ by G protein coupled receptor activation and PLCγ via kinase-mediated phosphorylation. PLCγ is phosphorylated by diverse receptor tyrosine kinases and nonreceptor protein tyrosine kinases through a high affinity interaction with the SH2 domain of PLCγ (Rhee, 2001). Also, it has been shown that the binding of the PH
domain of PLCγ to phosphatidylinositol-3,4,5-trisphosphate
(PI-3,4,5-P3) present in the membrane as a result of PI3K activation leads to the activation of PLCγ (Bae et al., 1998; Falasca et al., 1998). To go further into the mechanisms that relay β-BuTx’s facilitatory effect, we first addressed whether the inhibition of PI3K by its specific inhibitor LY294002 would impair β-BuTx-induced SSC frequency facilitation. The β-BuTx-induced SSC frequency facilitation was abolished in the presence of PI3K inhibitor. Pretreatment with another PI3K inhibitor, wortmannin (100 nM), also prevented the β-BuTx-induced increase in SSC frequency. We further evaluate the possible involvement of tyrosine kinases activity in β-BuTx-induced SSC frequency facilitation. The facilitating effect on SSC frequency induced by β-BuTx was unaffected under the presence of genistein (100 µM), a broad-spectrum tyrosine kainse inhibitor.
Next, we evaluate a possible participation of G protein in the β-BuTx-induced facilitating effects, GDPβS (a non-hydrolyzable GDP analogue and inhibitor of G protein) were used. Loading presynaptic neuron with G protein inhibitor GDPβS (5 mM) prior to β-BuTx treatment effectively blunted the β-BuTx-induced SSC frequency facilitation. These results suggest that the facilitating effects of β-BuTx on the spontaneous transmitter release are dependent of a G protein serving as a signal transducer in the signaling pathway.
How β-BuTx, a presynaptic neurotoxin endowed with PLA2 activity, come into play in turning on this sophisticated signalling cascade? It has been suggested that the effects on neurotransmitter release by PLA2 neurotoxins are correlated with their enzymatic activities (Strong et al. 1976; Chang et al. 1977), while other studies indicated a dissociation between neurotoxicity and phospholipids hydrolysis (Rowan & Harvey, 1988; Rosenberg et al., 1989). Here, the effect of 250 µM glycyrrhizin and 100 µM aristolochic acid, two PLA2 inhibitors, on β-BuTx-induced synaptic facilitation was investigated to determine the role of PLA2 activity. The β-BuTx-induced SSC frequency facilitation was abolished either in the presence of glycyrrhizin or aristolochic acid, suggesting the association of PLA2 activity with β-BuTx-induced SSC frequency facilitation. To further establish the essential role of PLA2, we evaluated pancreatic PLA2, a non-neurotoxic phospholipase, in facilitating spontaneous transmitter release. However, pancreatic PLA2 at a 100-fold higher concentration than b-BuTx has no distinguishable effect on SSC frequency.
The PLA2 hydrolyze the ester bond in
sn-2 position of
1,2-diacyl-3-sn-phosphoglycerides, liberating fatty acids and lysophospholipids. Given the suggestion that fatty acid metabolites (prostaglandin and leukotriene, for example) are important mediators in many physiological and pathological conditions, we thus examine if any fatty acid metabolite production might mediate or regulate the facilitating action of β-BuTx. Pretreatment of the cultures with either nordihydroguaiaretic acid (NDGA) or indomethacin, inhibitor of cyclooxygenase and lipoxygenase, had no effect on the synaptic facilitation induced by β-BuTx. This indicates that fatty acid metabolites might not be the candidates that are not responsible for the action of β-BuTx.
計畫成果自評
Ca2+ stores via IP3 receptor activation, β-BuTx induced a facilitation of spontaneous transmitter release in the Xenopus neuromuscular synapses. The paper work is under preparation and will submit this article to Molecular Pharmacology.
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