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

拟议研究的目标是确定多个 控制神经元钙通道的机制。药理 神经元细胞中钙电流的敏感性表明存在 几种钙通道类型。本研究的目的是：（1） 进一步了解生长因子和癌基因在 神经元钙通道类型的发展过程，2） 研究GTP结合蛋白在调节特异性 钙通道类型，和3）描绘第二信使系统 其调节每种钙通道类型， 靶向沿着这些复杂的细胞内途径的任何点， 病理损伤或神经紊乱的治疗。 因此，具体的假设和方法是：1） 生长因子和癌基因激活诱导的神经元分化 钙通道类型的表达，可以区分 内分泌细胞中发现的钙通道类型。这将由以下人员进行测试： 比较PCl 2细胞的钙电流特性 （嗜铬细胞瘤肿瘤细胞）通过神经处理分化 生长因子（NGF）和逆转录病毒感染的温度 敏感的病毒src癌基因和未分化的PC12细胞， 维持内分泌表型（类似肾上腺嗜铬细胞）。的 电流激活、失活和单电流的生物物理特性 将检查信道打开概率。2)特异性钙电流 类型可以通过激活GTP结合蛋白来调节。gtp结合 蛋白质将在全细胞膜片钳结构中被激活， 包括GTP γ S，一种不可水解的鸟苷三磷酸类似物， 贴片吸管和直接应用神经递质， 已知与GTP结合蛋白偶联的肽。3)钙电流类型 与特定的第二信使途径相连。这将由以下人员进行测试： 直接应用磷脂酶C活化产物， 二酰基甘油类似物或磷脂酶A2活化的副产物， 如花生四烯酸、洋地黄素和白三烯， 细胞，以确定其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.