Signal Transduction in Cultured Neural Cells

中枢神经系统内的信号转导涉及特定受体对神经递质或神经调节剂的识别，以及随后细胞事件的激活(或抑制)。这些细胞事件(离子转移、神经递质释放等)通常由受体占位引起的第二信使介导。Rasenick博士将确定神经元内的许多成分是如何相对于腺苷环化酶第二信使系统进行整合的。培养的神经胶质细胞、神经元细胞或混合细胞将被皂苷渗透，腺苷环化酶系统的组成将被原位探测。Rasenick博士的实验室已经开发出一种在渗透性细胞中检测腺苷环化酶(而不是cAMP积累)的方法；C6 NG.108.15和NCB.20细胞将用于拟议的实验。首先，Rasenick博士将研究受体通过刺激性GTP结合蛋白(GNS)激活腺苷环化酶系统的动力学。用GTP光亲和探针研究激动剂对GNS的激活作用。这项研究还将通过动力学实验和光亲和实验研究最近发现的GNS与抑制性GTP结合蛋白(GNI)之间的相互作用。Rasenick博士已经证明，GTP类似物可以在突触膜上的GNS和GNI(以及微管蛋白和GNI)之间交换；他进一步打算研究受体在核苷酸交换过程中的可能作用。通透性细胞为这类实验提供了理想的环境；许多可能协同作用以改变受体反应的元素保留在这个系统中。由于皂素处理的细胞有足够大的孔来允许蛋白质和Fab片段通过，计划进行各种实验来探索细胞骨架的作用，以及新发现的32 KDa GTP结合蛋白的作用。最后，对形态分化的影响进行了考察。这项研究为在与完整神经元相似的环境中研究细胞对神经递质的反应提供了令人兴奋的可能性。

Signal transduction within the central nervous system involves the recognition of a neurotransmitter or neuromodulator by a specific receptor and the subsequent activation (or inhibition) of a cellular event. These cellular events (ion translocation, neurotransmitter release, etc.) are often mediated by second messengers evoked as a result of receptor occupancy. Dr. Rasenick will determine how a number of components within the neuron are integrated with respect to the adenylate cyclase second messenger system. Cultured glial, neuronal or hybrid cells will be permeabilized with saponin, and the components of the adenylate cyclase system will be probed, in situ. Dr. Rasenick's laboratory has developed an assay for adenylate cyclase (as opposed to cAMP accumulation) in permeabilized cells; C6 NG.108.15 and NCB.20 cells will be used for the proposed experiments. Initially, Dr. Rasenick will study the kinetics for the receptor mediated activation of the adenylate cyclase system through the stimulatory GTP binding protein (GNs). The agonist catalyzed activation of GNs will be studied with a GTP.photoaffinity probe. This study also will examine the recently discovered interaction between GNs and the inhibitory GTP binding protein (GNi) both through kinetic experiments and by photoaffinity experiments. Dr. Rasenick has demonstrated that GTP analogues may be exchanged between GNs and GNi (as well as tubulin and GNi) in synaptic membranes; he further intends to examine the possible role of receptors in the nucleotide exchange process. The permeabilized cells provide the ideal milieu for such experiments; many of the elements which may act in concert to modify receptor responsiveness are retained in this system. As the saponin treated cells have holes sufficiently large to allow the passage of proteins and Fab fragments, a variety of experiments to probe the role of the cytoskeleton, as well as that of a newly discovered 32KDa GTP binding protein, are planned. Lastly, effects of morphological differentiation will be examined. This research provides the exciting possibility to investigate the cellular response to neurotransmitters in a environment resembling that of the intact neuron.