NGF OR V-SRC DIFFERENTIATED PC12 CELLS--CA2+ CURRENTS

拟议研究的目标是定义多个 控制神经元钙通道的机制。药理学 神经细胞钙电流的敏感性提示存在 几种钙通道类型。拟议研究的目的是：1) 进一步了解生长因子和癌基因在肿瘤中的作用 神经元钙通道类型的发育过程，2)到 检测GTP结合蛋白在调节特异性GTP中的作用 钙通道类型，以及3)描绘第二信使系统 它调节每种钙通道类型，提供潜在的药理作用 靶向这些复杂的细胞内通路上的任何点 病理性侮辱或神经错乱的治疗。 相应地，需要检验的具体假设和方法是：1) 生长因子和癌基因激活诱导神经元分化 可区分的钙通道类型的表达 在内分泌细胞中发现的钙通道类型。这将通过以下方式进行测试 PCl2细胞钙电流特性的比较 (嗜铬细胞瘤肿瘤细胞)用神经治疗分化 生长因子(NGF)和逆转录病毒感染 敏感的病毒src癌基因和未分化的PC12细胞 保持内分泌表型(类似于肾上腺嗜铬细胞)。这个 电流激活、失活和单一的生物物理特性 将检查通道开放概率。2)明显的钙电流 类型可以通过激活GTP结合蛋白来调节。GTP绑定 在整个细胞膜片钳结构中，蛋白质将被激活 包括GTPGammaS，一种不可水解的三磷酸鸟苷类似物，在 贴片吸管和直接应用神经递质和 已知的与GTP结合蛋白偶联的多肽。3)钙电流类型 与特定的第二信使通路相连。这将通过以下方式进行测试 磷脂酶C活化产物的直接应用 二酰甘油类似物或磷脂酶A2激活的副产物 如花生四烯酸、前列腺素和白三烯，以电压钳制 以确定它们对特定钙电流的影响。

The objectives of the proposed research are to define the multiple mechanisms that control neuronal calcium channels. The pharmacological sensitivity of calcium currents in neuronal cells indicate the existence of several calcium channel types. The aims of the proposed research are 1) to further the understanding of the role of growth factors and oncogenes in the processes of development of neuronal calcium channel types, 2) to examine the role of GTP-binding proteins in the modulation of specific calcium channel types, and 3) to delineate the second messenger systems that modulate each calcium channel type providing potential pharmacological targeting at any point along these complex intracellular pathways for the treatment of pathological insults or nervous disorders. Accordingly, the specific hypothesis to be tested and methods are: 1) Neuronal differentiation induced by growth factors and oncogenes activate the expression of calcium channel types which can be distinguished from calcium channel types found in endocrine cells. This will be tested by comparing the properties of calcium currents in PCl2 cells (pheochromocytoma tumor cells) differentiated by treatment with nerve growth factor (NGF) and by retroviral infection with a temperature sensitive viral src oncogene and in undifferentiated PC12 cells which maintain an endocrine phenotype (resembling adrenal chromaffin cells). The biophysical properties of current activation, inactivation, and single channel open probability will be examined. 2) Distinct calcium current types can be modulated by activation of GTP-binding proteins. GTP-binding proteins will be activated in the whole cell patch clamp configuration by including GTPgammaS, a non-hydrolyzable guanosine triphosphate analog, in the patch pipette and by direct application of neurotransmitters and peptides known to couple to GTP-binding proteins. 3) Calcium current types are coupled to specific second messenger pathways. This will be tested by direct application of products of phospholipase C activation such as diacylglycerol analogs or by products of phospholipase A2 activation such as arachidonic acid, prostaglandins, and leukotrienes to voltage-clamped cells to determine their effects on specific calcium currents.