In Vivo and In Vitro Effects of Acrylamide on Synaptosomal Neurotransmitter Uptake and Release

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• 作者：LoPachin ; Richard M. ; Schwarcz ; Aron I. ; Gaughan ; Christopher L. ; Mansukhani ; Shirley ; Das ; Soma
• 关键词：Acrylamide ; Distal axonopathy ; Synaptosomes ; Neurotransmitter release ; Uptake ; Protein thiol groups ; SNARE core complexes
• 刊名：Neurotoxicology
• 出版年：2004
• 期刊代码：35_0161813x
• 类别：pha
• 出版时间：March, 2004
• 卷：25
• 期：3
• 页码：349-363
• 文件大小：269 K

摘要

Evidence suggests acrylamide (ACR) neurotoxicity is mediated by impaired presynaptic transmission. To assess the effects of ACR on nerve terminal function, [³H]glutamate release and uptake were determined in brain synaptosomes isolated from intoxicated rats (50mg/kg per day×8 days, i.p. or 21mg/kg per day×21 days, p.o.). Regardless of ACR dose-rate, a significant reduction in synaptosomal K⁺-stimulated, Ca²⁺-dependent release was detected, whereas kinetic analysis of Na⁺-dependent uptake did not reveal consistent changes. Immunoblot analysis showed normal protein levels (e.g. SNAP-25) in dysfunctional synaptosomes isolated from ACR-intoxicated rats. This suggests that defective release does not involve changes in protein synthesis and/or anterograde delivery of presynaptic constituents. To identify potential targets, synaptosomes were exposed in vitro to [¹⁴C]-ACR and radiolabeled proteins were separated by gel electrophoresis and detected by autoradiography. [¹⁴C]-ACR labeling of distinct synaptosomal protein bands (10.5–154,000kDa) was blocked by the sulfhydryl alkylating agent, N-ethylmaleimide (NEM; 4mM) but not by the non-neurotoxic structural analog propionamide (10mM). In vitro characterization of synaptosomal [³H]glutamate uptake and release showed that ACR, NEM and iodoacetic acid (IAA) produced concentration-dependent decreases in each parameter that were highly correlated to reductions in free sulfhydryl content. All three chemicals were equiefficacious with respect to reducing sulfhydryl content and neurotransmitter uptake/release, although the relative potencies differed; NEM>IAA>ACR. Kinetic analysis of uptake showed that in vitro exposure to ACR, IAA or NEM at their respective IC₅₀’s caused similar reductions in V_max. These data suggest that ACR-induced synaptic dysfunction involves adduction of presynaptic protein thiol groups and subsequent reduction in neurotransmitter release.