Immunohistochemical and immunocytochemical localization of myosin, chromogranin A and dopamine-?-hydroxylase in nerve cells in culture and in adrenal glands

Abstract

Dopamine-β-hydroxylase, chromogranin A and myosin were purified from bovine adrenal medulla and antibodies prepared against these proteins. Indirect immunocytochemical methods were used to localize dopamine-β-hydroxylase, chromogranin A and myosin in bovine adrenal medulla and myosin in rat adrenal glands and cells from rat C.N.S. maintained in primary culture. Dopamine-β-hydroxylase and chromogranin A were found in chromaffin granules, in agreement with biochemical data and, using electron microscopy, dopamine-β-hydroxylase was found within the matrix and in the surrounding membrane of the storage granule, whereas chromogranin A was confined to the granule matrix. Myosin was localized in the vascular system irrigating adrenal glands, fibroblasts lining the vessels and chromaffin cells. In chromaffin cells, staining was found at the cell boundaries and electron microscopy showed myosin to be associated with the plasma membrane. Faint immunocytochemical staining by antimyosin antibodies was observed around certain exocytotic profiles but particular association with such structures was not demonstrable. Myosin localization was also studied in bovine adrenal cortex, where it was found in vascular channels and faintly in adrenal cortical cells, as in rat adrenal cortex and medulla, where identical patterns were obtained. In neuronal and glial cells dissociated from 13 day rat embryo cerebral hemispheres and cultured for 48 h, localization of myosin was studied using immunohistochemistry. The neuritic expansions and growth cones of neurons were fluorescent, whereas in glial cells, filamentous networks were visualized enclosing the nucleus and as long fibres traversing the entire cytoplasm.