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

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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.