SYNTHESIS OF DBH, CHROMOGRANINS AND CHROMAFFIN VESICLES

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The long range objectives of this project are to obtain an understanding of the factors regulating the synthesis of dopamine- beta-hydroxylase (DBH) and chromogranin A (CgA) and their incorporation into chromaffin vesicles. Primary cultures of bovine adrenal medullary cells will be used to pursue the following specific objectives: 1) to define the conditions for the synthesis of membrane-bound DBH, soluble DBH and CgA and their incorporation into chromaffin vesicles; 2) to examine the effects of cholinergic stimulation on DBH and CgA synthesis and their incorporation into chromaffin vesicles; 3) to determine whether chromaffin vesicle membranes are reutilized; 4) to determine whether growth factors, cortical cells or altered levels of protein kinase activities affect DBH and CgA synthesis and/or their incorporation into chromaffin vesicles; 5) to examine the role of glycosylation in the synthesis of DBH and CgA and their incorporation into chromaffin vesicles. Cell cultures will be incubated with (35S)methionine and its incorporation into DBH and CgA will be quantified by immunoprecipitation with monovalent polyclonal anti-sera. The radiopurity and quantitation of the immunoprecipitates will be verified by gel electrophoresis, autoradiography and densitometric scanning. Incorporation of newly synthesized DBH and CgA into chromaffin vesicles will be determined by subcellular fractionation procedures and purification of chromaffin vesicles by centrifugation through continuous density gradients. Since adrenal medullary cells have been shown to be good biochemical and functional models of peripheral and CNS catecholaminergic neurons, the information to be gained from these studies will be relevant to these tissues as well. CgA has recently been shown to be widely distributed in non-catecholamine neuroendocrine tissues and knowledge of its synthesis and packaging into chromaffin vesicles may also be relevant to other neuroendocrine tissues.