AP4A MODULATION PC12 CELL MONOAMINE OXIDASE ACTIVITY

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DESCRIPTION: (from Abstract) Most studies dealing with monoamine oxidase (MAO) have emphasized the development of inhibitors of this enzyme for controlling neurotransmitter metabolism in patients with diseases such as Parkinson's. The literature is replete with information on natural and man-made inhibitors of MAO activity. However, there is virtually no information on modulators of MAO that result in enhanced activity of this enzyme. The applicants' laboratory has recently demonstrated that diadenosine tetraphosphate, Ap4A, may be one, of only a few, endogenous extracellular stimulatory agents of MAO activity. They have undertaken a series of experiments aimed at understanding the signal transduction pathway associated with Ap4A receptor binding and the enhanced activation of MAO. An understanding of the transduction pathway(s) involved with Ap4A receptor activation may result in a better understanding of the signal transduction events associated with MAO activation. In addition, if the Ap4A activated transduction pathway(s) is a novel activation pathway(s) for MAO, knowledge of this pathway(s) may allow for the development of a new class of agonists, antagonists or pharmacological agents for controlling MAO activity. Abnormal or elevated level of Ap4A in the CNS and adrenal gland may lead to pathogenic conditions associated with depleted catecholamine stores, such as Parkinson's Disease. The potential importance of MAO in the catabolism of various xenobiotics suggests that Ap4A may have an important therapeutic role. The specific aims of this proposal are: (A) To determine the dosage effects of the extracellular diadenosine polyphosphates on MAO activity in PC12 cells and brain synaptosomes obtained form the caudate putamen of rat brains. Dopamine metabolites will be measured by using 1) HPLC with electrochemical detection and 2) radiochemical analyses. The temporal dynamics of the application of Ap4A will be determined to assess the effects of short term and chronic exposure of the cells and synaptosomes; (B) To determine the effects of extracellular Ap4A on adenylate cyclase activity and cAMP levels in PC12 cells. cAMP levels will be assayed by an enzyme linked immunoassay; adenylate cyclase activity will be determined using [a32P] ATP and chromatography; (C) To determine the role of G protein involvement in the Ap4A elicited response by measuring GTPase enzymatic activity; (D) To determine the involvement of tyrosine kinases and tyrosine phosphatases in the Ap4A initiated signal transduction pathway. SDS-PAGE and [32P]phosphate will be used to label cells and determine the extent of phosphorylation or dephosphorylation of specific proteins. (E) To determine the effects of Ap4A delivery to the caudate putamen of the conscious rat with in vivo microdialysis in conjunction with HPLC.