| 摘要: | 蛋白激.C (Protein kinase C; PKC)以高濃度存在於神經組織(Tanaka and Nishizuka, 1994)，且已知與許多神經功能的調控有關：活化PKC可調節離子通道(Shearman et al 1989)，去敏感化接受體(Huganir Greengard 1990)，增加神經傳遞物質的釋放(Robinson 1992)。此外，近來的研究也顯示PKC的活性與抽搐現象的產生有關。PKC活化劑12-myristate,13-acetate (PMA, 10 fM-10 nM) 及 phorbol 12,13-dibutyrate (PDBu, 0.2-495 nM)注射至小鼠腦室(intracerebroventricular; i.c.v.)會引發抽搐現象(Smith and Meldrum, 1992)。從一個第一期臨床試驗顯示：為治療癌症服用PKC活化劑12-O-tetradecanoylphorbol-13-acetate (TPA)會產生包括大發作等之嚴重不良反應(Schaar et al., 2006)。此外，Pentylenetetrazol (PTZ)是一個熟知的致抽搐劑，有報導指出，PTZ引發小鼠抽搐現象時，可量測到腦內海馬回的PKCγ有增加之情形(Chen, 1994)，這些證據顯示PKC的活化與可能抽搐現象的產生有關 ，然而，其機轉至今仍不十分清楚。動物產生抽搐現象時，可於中樞神經元記錄到動作電位猝發現象(bursts of action potentials) (Jefferys, 1994; Jefferys and Traub, 1998; McCormick and Contreras, 2001)。蝸牛神經節含有神經傳遞物質及接受體，其上的神經元常用來作為研究的材料。在脊椎動物誘發產生抽搐現象之藥物例如：PTZ，可以在哺乳類動物引發大腦皮質神經元產生動作電位的猝發現象(Sugaya et al., 1964)，也可在蝸牛神經元誘導神經元產生動作電位的猝發現象(Ferrendelli and Kinscherf, 1977; Sugaya and Onozuka, 1978a; Sugaya and Onozuka, 1978b; Onozuka et al., 1983; Onozuka et al., 1986; Sugaya et al., 1987; Onozuka et al., 1991a; Onozuka et al., 1991b; Chen and Tsai, 1996)，且其猝發現象與PTZ在大腦皮質細胞引起之猝發現象十分相似。(±)3,4-Methylenedioxyamphetamine (MDA)及(±)3,4-methylene-dioxymethamphetamine (MDMA)是環狀取代(ring-substituted)的安非他命類藥物，高劑量也會引起動物及人產生抽搐現象(Cooper and Egleston, 1997; Hanson et al., 1999; Holmes et al., 1999)，我們最近的研究顯示MDMA及MDA也會在非洲大蝸牛(Achatina fulica)中樞RP4神經元引發動作電位的猝發現象，此一MDMA或MDA引起之猝發現象會被PKC抑制劑抑制，且會被PKC活化劑所促進。另而PKC的活化劑phorbol 12,13-dibutyrate (PDBu) 及1-oleoyl-2-acety-sn-glycerol (OAG; a membrane-permeant DAG analog)在高濃度時，也會造成細胞膜電位上下振盪並有輕微的動作電位猝發現象(Tsai and Chen, 2006; unpublished data)，然而，此一PKC引起之作用還未被深入研究。而本計劃即是要利用一般藥理學及電生理學的方法，探討PKC引發動作電位猝發現象之機轉，及PKC在CNS興奮劑如MDMA、MDA、PTZ引起動作電位猝發現象及抽搐現象所扮演的角色，本計劃將以3個實驗模型進行，分別是體外蝸牛神經元，大鼠腦切片，及活體大鼠中。在蝸牛神經細胞，將以雙電極膜電位箝制的方法，詳盡的研究PKC活化對於蝸牛RP4神經細胞興奮性的影響，包括對各種離子電流之作用，及PKC與MDMA、MDA及PTZ之交互作用。此外，也將量測神經元PKC的活性以瞭解神經元PKC之活性受到這些藥物影響之情形。在哺乳類動物神經元，近幾十年來有關癲癇之機轉之研究主要在三個腦部構造，即大腦皮質、海馬回及視丘(McCormick and Contreras, 2001)。而本計劃將以大鼠腦切片為材料，並以全細胞膜片箝制的方法研究測試MDMA、MDA是否會在毒性濃度引發大腦皮質、海馬回或視丘神經元產生動作電位猝發現象，並且我們也會測試PKC的活性是否與MDMA、MDA及PTZ所引起的動作電位改變及離子電流的改變有關。在活體大鼠，擬將MDMA、MDA注射至大鼠腦室(i.c.v.)或以腹腔注射PTZ引起抽搐現象，並同時注射PKC的活化劑及抑制劑觀察變化，同時也將測試單胺類神經傳遞物質受體抑制劑以釐清抽搐現象之機轉是否與單胺類神經傳遞物質有關，計畫中也涵蓋對於新上市抗癲癇藥物對於MDMA引起抽搐現象之臨床前藥效測試。本計劃之完成，將使我們更加瞭解PKC如何影響神經元之功能及其致病之機轉，這些結果可作為抽搐現象治療藥物設計之依據。
Protein kinase C (PKC) is present in high concentrations in neuronal tissues and has been implicated in a broad spectrum of neuronal functions (Tanaka and Nishizuka, 1994). Activation of this enzyme in nerve cells is frequently associated with the modulation of ion channels (Shearman et al 1989), desensitization of receptors (Huganir Greengard 1990), and enhancement of neurotransmitter release (Robinson 1992). Moreover, recent studies show that PKC activity in the brain is involved in the mechanisms of epileptogenesis. Intracerebroventricular (ICV) administration of the phorbol ester, phorbol 12-myristate,13-acetate (PMA, 10 fmol-10 nmol) or phorbol 12,13-dibutyrate (PDBu, 0.2-495 nmol), an activator of PKC, has been shown to induce seizures in mice (Smith and Meldrum, 1992). In addition, in a phase I dose escalation trial, administration of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) in patients with relapsed or refractory malignancies was associated with serious adverse events, including a grand mal seizure (Schaar et al., 2006). Pentylenetetrazol (PTZ) is a well-known convulsant. An increase in content of the PKCγ isoform was observed in the mouse hippocampus after PTZ-induced chemoshock (Chen, 1994). These data suggest that PKC activity in the brain is involved in the epileptogenesis. However, the mechanisms whereby PKC activation induces seizures remain unclear. Bursts of action potentials are considered to be an identifiable unit of epileptiform activity in neurons (Jefferys, 1994; Jefferys and Traub, 1998; McCormick and Contreras, 2001). Snail ganglia contain many identifiable neurotransmitters and receptors, and their neurons are used for biological studies. Convulsants such as PTZ induce bursts of action potential in snail central neurons (Ferrendelli and Kinscherf, 1977; Sugaya and Onozuka, 1978a; Sugaya and Onozuka, 1978b; Onozuka et al., 1983; Onozuka et al., 1986; Sugaya et al., 1987; Onozuka et al., 1991a; Onozuka et al., 1991b; Chen and Tsai, 1996). This response strongly resembles the PTZ-induced seizure changes observed in cerebral cortical neurons of mammals (Sugaya et al., 1964). (±)3,4-Methylenedioxyamphetamine (MDA) and (±)3,4-methylene-dioxymethamphetamine (MDMA) are ring-substituted amphetamines and induce seizures in animals and humans at high doses (Cooper and Egleston, 1997; Hanson et al., 1999; Holmes et al., 1999). Our recent studies revealed that MDMA and MDA elicited abnormal bursts of action potentials in a central RP4 neuron of the African snail, Achatina fulica Ferussac (Tsai and Chen, 2006; unpublished data). The MDMA- and MDA-elicited action potential bursts are prevented by PKC inhibitors and facilitated by PKC activators. Specifically, administration of PKC activators (i.e. phorbol 12,13-dibutyrate [PDBu]) and 1-oleoyl-2-acety-sn-glycerol [OAG; a membrane-permeant DAG analog]) at high concentrations elicited membrane potential oscillations and slight action potential bursts (Tsai and Chen, 2006). PKC-elicited action potential bursts have not been studied extensively. Accordingly, the present study aims to investigate the mechanism whereby PKC elicits action potential bursts and explore the role of PKC in the action potential bursts and seizures induced by CNS stimulants, i.e. MDMA, MDA and PTZ, using general pharmacological and electrophysiological methods. The studies will be performed in three models, i.e. in vitro snail neurons and rat brain slices, and in vivo rats. In snail neurons, we will use the two-electrode voltage-clamp method to closely investigate the effects of PKC activation on the electrophysiological behavior of the snail neuron, as well as the effects of PKC activation on various ionic currents and the interaction of PKC with MDMA, MDA and PTZ. In addition, PKC assay will be performed to test whether these agents affect the PKC activity of the neuron. In mammalian central neurons, the investigation of the cellular and network mechanisms of epilepsy over the last several decades has focused largely on three structures; namely, the cerebral cortex, the hippocampus, and the thalamus (McCormick and Contreras, 2001). In the present study, we will use the whole-cell patch clamp method to test the hypothesis that MDMA and MDA at toxic concentrations induce a bursting firing of action potentials in the cerebral cortex, hippocampus, and thalamus of young rat brain slices. Further, we will test whether PKC activity is involved in the generation of action potential changes or ionic current changes elicited by MDMA, MDA and PTZ. In rats in vivo, seizures will be induced in rats by intracerebroventricular (ICV) injection of MDMA and MDA (Hanson et al., 1999) or by intraperitoneal injection of PTZ. PKC inhibitors and activators will be co-administered by ICV injections to test the role of PKC in the seizure-generating mechanism. In addition, we will also test the effects of monoamine receptor antagonists, to clarify whether the monoamine-releasing effect is involved in the generation of seizures induced by MDMA or MDA. The proposed study will also include a preclinical evaluation of newly approved antiepileptic drugs against MDMA-induced seizures. The completion of this proposed study may enhance our knowledge as to how PKC activity affects neuronal activity and causes pathogenesis. These results would permit a rational approach to the design of novel therapeutic agents for the treatment of seizures. |
