Immunofluorescent patterns of clathrin and dopamine beta-hydroxylase in chromaffin cells in culture

Abstract

Bovine chromaffin cells maintained in culture for eight days were loaded with [3H]noradrenaline and then stimulated by a depolarizing concentration (56 mM) of K+. Control and stimulated cells were fixed in 3.7% formaldehyde, treated with acetone or Triton X-100, and then exposed to antibodies raised against dopamine beta-hydroxylase (a secretory granule marker) and clathrin, and purified by affinity chromatography. The cellular distribution of the correspondent antigens was investigated by indirect immunofluorescence. Cells treated with anti-dopamine beta-hydroxylase exhibited a granular pattern of fluorescence in the cytosol of the cell body, neurites, and terminal cones. Chromaffin cells exposed to anti-clathrin also showed a punctate pattern of fluorescence staining. However, in this case, the fluorescent dots were smaller than those observed with anti-dopamine beta-hydroxylase, and they were differently distributed. The speckled anti-clathrin fluorescence was preferentially condensed in the juxtanuclear region of the cell bodies, suggesting the possibility that clathrin was concentrated at the level of the Golgi apparatus. The stimulation of cultured chromaffin cells by 10 pulses of 56 mM K+ produced 91 +/- 2% (n = 5) depletion in the [3H]noradrenaline cell content and a concomitant displacement of the dopamine beta-hydroxylase fluorescence to the periphery of the cells. Four days after cell stimulation the dopamine beta-hydroxylase fluorescence was similar to that observed in control cells.(ABSTRACT TRUNCATED AT 250 WORDS)