The sciatic nerve, as a part of the peripheral nervous system (PNS), has been used to study axonal transport for decades. It contains motor, sensory as well as autonomic axons. The present study has concentrated on the axonal transport of the synaptic vesicle acetylcholine transporter (VAChT), using the "stop–flow\erve crush” method. After blocking fast axonal transport by means of a crush, distinct accumulations of various synaptic vesicle proteins, including VAChT, and peptides developed during the first hour after crush–operation and marked increases were observed up to 8 h post–operative. Semiquantitative analysis, using cytofluorimetric scanning (CFS) of immuno–incubated sections, revealed a rapid rate of accumulation proximal to the crush, and that the ratio between distal accumulations (organelles in retrograde transport) and proximal accumulations (organelles in anterograde transport) was about 40%. Most synaptic vesicle proteins were colocalized in the axons proximal to the crush. VAChT–immu–noreactive axons were also immunoreactive for choline acetyltransferase (ChAT). Autonomic axons with VAChT also contained VIP–LI.
Efficient cholinergic transmission requires accurate targeting of vesicular acetylcholine transporter (VAChT) to synaptic vesicles (SVs). However, the signals that regulate this vesicular targeting are not well characterized. Although previous studies suggest that the C-terminus of the transporter is required for its SV targeting, it is not clear whether this region is sufficient for this process. Furthermore, a synaptic vesicle-targeting motif (SVTM) within this sequence remains to be identified. Here we use a chimeric protein, TacA, between an unrelated plasma membrane protein, Tac, and the C-terminus of VAChT to demonstrate the sufficiency of the C-terminus for targeting to synaptic vesicle-like vesicles (SVLVs) in PC12 cells. TacA shows colocalization and cosedimentation with the SV marker synaptophysin. Deletion mutation analysis of TacA demonstrates that a short, dileucine motif-containing sequence is required and sufficient to direct this targeting. Dialanine mutation analysis within this sequence suggests indistinguishable signals for both internalization and SV sorting. Using additional chimeras as controls, we confirm the specificity of this region for SVLVs targeting. Therefore, we suggest that the dileucine-containing motif is sufficient as a dual signal for both internalization and SV targeting during VAChT trafficking. 相似文献
Neurotransmission depends on the regulated release of chemical transmitter molecules. This requires the packaging of these
substances into the specialized secretory vesicles of neurons and neuroendocrine cells, a process mediated by specific vesicular
transporters. The family of genes encoding the vesicular transporters for biogenic amines and acetylcholine have recently
been cloned. Direct comparison of their transport characteristics and pharmacology provides information about vesicular transport
bioenergetics, substrate feature recognition by each transporter, and the role of vesicular amine storage in the mechanism
of action of psychopharmacologic and neurotoxic agents. Regulation of vesicular transport activity may affect levels of neurotransmitter
available for neurosecretion and be an important site for the regulation of synaptic function. Gene knockout studies have
determined vesicular transport function is critical for survival and have enabled further evaluation of the role of vesicular
neurotransmitter transporters in behavior and neurotoxicity. Molecular analysis is beginning to reveal the sites involved
in vesicular transporter function and the sites that determine substrate specificity. In addition, the molecular basis for
the selective targeting of these transporters to specific vesicle populations and the biogenesis of monoaminergic and cholinergic
synaptic vesicles are areas of research that are currently being explored. This information provides new insights into the
pharmacology and physiology of biogenic amine and acetylcholine vesicular storage in cardiovascular, endocrine, and central
nervous system function and has important implications for neurodegenerative disease. 相似文献
1. Synaptic vesicles (SVs) mediate fast regulated secretion of classical neurotransmitters. In order to perform their task SVs rely on a restrict set of membrane proteins. The mechanisms responsible for targeting these proteins to the SV membrane are still poorly understood.2. Likewise, little is known about the intracellular routes taken by these proteins in their way to SV membrane. Recently, several domains and motifs necessary for correct localization of SV proteins have been identified.3. In this review we summarize the sequence motifs that have been identified in the cytoplasmic domains of SV proteins that are involved in endocytosis and targeting of SVs. We suggest that the vesicular acetylcholine transporter, a protein found predominantly in synaptic vesicles, is perhaps a model protein to understand the pathways and interactions that are used for synaptic vesicle targeting. 相似文献
The feline gastrointestinal (GI) tract is an important model for GI physiology but no immunohistochemical assessment of interstitial
cells of Cajal (ICC) has been performed because of the lack of suitable antibodies. The aim of the present study was to investigate
the various types of ICC and associated nerve structures in the pyloric sphincter region, by using immunohistochemistry and
electron microscopy to complement functional studies. In the sphincter, ICC associated with Auerbach’s plexus (ICC-AP) were
markedly decreased within a region of 6–8 mm in length, thereby forming an interruption in this network of ICC-AP, which is
otherwise continuous from corpus to distal ileum. In contrast, intramuscular ICC (ICC-IM) were abundant within the pylorus,
especially at the inner edge of the circular muscle adjacent to the submucosa. Similar distribution patterns of nerves positive
for vesicular acetylcholine transporter (VAChT), nitric oxide synthase (NOS) and substance P (SP) were encountered. Quantification
showed a significantly higher number of ICC-IM and the various types of nerves in the pylorus compared with the circular muscle
layers in the adjacent antrum and duodenum. Electron-microscopic studies demonstrated that ICC-IM were closely associated
with enteric nerves through synapse-like junctions and with smooth muscle cells through gap junctions. Thus, for the first
time, immunohistochemical studies have been successful in documenting the unique distribution of ICC in the feline pylorus.
A lack of ICC-AP guarantees the distinct properties of antral and duodenal pacemaker activities. ICC-IM are associated with
enteric nerves, which are concentrated in the inner portion of the circular muscle layer, being part of a unique innervation
pattern of the sphincter.
This study was supported by operating grants from the Canadian Institutes of Health Research (to J.D.H. and N.E.D.) and from
the Canadian Association of Gastroenterology (to L.W.C.L.). 相似文献
The choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) are fundamental to neurophysiological functions of the central cholinergic system. We confirmed and quantified the presence of extracellular ChAT protein in human plasma and also characterized ChAT and VAChT polymorphisms, protein and activity levels in plasma of Alzheimer''s disease patients (AD; N = 112) and in cognitively healthy controls (EC; N = 118). We found no significant differences in plasma levels of ChAT activity and protein between AD and EC groups. Although no differences were observed in plasma ChAT activity and protein concentration among ChEI-treated and untreated AD patients, ChAT activity and protein levels variance in plasma were higher among the rivastigmine-treated group (ChAT protein: p = 0.005; ChAT activity: p = 0.0002). Moreover, AD patients homozygous for SNP rs1880676 A allele exhibited higher levels of ChAT activity. Considering this is the first study to report the influence of genetic variability of CHAT locus over ChAT activity in AD patients plasma, it opens a new set of important questions on peripheral cholinergic signaling in AD. 相似文献