Neurofilament Proteins in Cultured Chromaffin Cells

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Abstract
Antibodies were raised against the 200-kd [kilodalton] 145-kd, and 68-kd subunits of a rat neurofilament preparation. Immunoblots showed that each antibody was specific for its antigen and that it did not cross-react with any of the 2 other neurofilament polypeptides. Use of the 3 antibody preparations to stain bovine chromaffin cells in culture by the indirect immunofluorescence technique indicated that the 3 neurofilament polypeptides are present in chromaffin cells maintained in culture for 3 or 7 days. The 3 anti-neurofilament antibodies labeled the cells in a similar pattern. Very thin filaments specifically localized around the nucleus were observed whereas neurites and growth cones, developed by cultured chromaffin cells, were generally not stained. Some fibroblasts were present in the cultures but they were never stained by any of the neurofilament antibodies. This indicated that the antibodies used do not react with vimentin, the major intermediate filament protein found in fibroblasts. The 3 neurofilament antibodies were used to immunoprecipitate specifically 3 proteins of MW 210 kd, 160 kd, 70 kd from solubilized extracts of cultured chromaffin cells that were radiolabeled with [35S]methionine. These proteins correspond in MW to the neurofilament triplet found in bovine brain. The presence of neurofilaments in freshly isolated chromaffin cells was tested by immunoblotting using the 68-kd antibody. A 70-kd protein was specifically stained by this antibody, suggesting that neurofilaments are not only present in cultured chromaffin cells but also in the adrenal gland in vivo. Evidently, chromaffin cells contain completely assembled neurofilaments. Chromaffin cells are closely related to neurons and therefore represent an attractive model system for the study of functional aspects of adrenergic neurons.

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