Immunocytochemical localization of dopamine-β-hydroxylase in adrenal chromaffin granules

Abstract

Electron microscopic localization of dopamine-β-hydroxylase (DBH) was carried out in bovine adrenal medullary chromaffin granule fractions and in intact bovine adrenal medulla by means of a peroxidase-antiperoxidase unlabelled antibody technique according to Sternberger, Hardy, Cuculis and Meyer (1970). Adrenal medullary chromaffin granule fractions showed retention of DBH despite successive fractionation, fixation and prolonged washing procedures, indicating that a portion of the DBH in the chromaffin granule is quite tightly bound. Under these circumstances the electron-opaque granular matrix was absent, implying loss of the more soluble constituents of the chromaffin granules, including matrix-associated DBH. Intact bovine adrenal medulla which was fixed, sectioned and subjected to immunocytochemical localization of DBH showed the enzyme to be present throughout the matrix of the chromaffin granule and demonstrated that, under these less vigorous conditions, the electron-opaque granule matrix was not removed. It would therefore appear to be feasible to differentiate directly between the “soluble” and “bound” forms of DBH by immunocytochemical procedures and to apply this technique to elucidate the process of exocytosis in adrenal medulla and adrenergic nerve terminals.