INCREASED SYNTHESIS OF DOPAMINE β‐HYDROXYLASE IN CULTURED RAT ADRENAL EDULLAE AFTER IN VIVO ADMINISTRATION OF RESERPINE

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Abstract
Abstract— The present experiments were designed to provide direct evidence that the increase in dopamine β‐hydroxylase (DBH) activity after treatment with reserpme results from an augmented synthesis of new enzyme protein. After in vivo experiments had shown that DBH could not be labelled to a sufficient extent even after administration of high doses (8.3 mCi/kg) of [3H]leucine we took advantage of earlier observations that neuronally‐mediated enzyme induction initiated in vivo progresses in organ cultures of adrenal medullae in a similar manner as in vivo. With that system it was possible to achieve a sufficient labelling of DBH molecules and to provide evidence that the increase in DBH activity produced by reserpine was really due to a specific increase in DBH synthesis. The fact that the half‐life of DBH was identical in organ cultures of controls and reserpine‐treated animals eliminated the possibility that the increased labelling of DBH after reserpine results from a slower rate of degradation.Both the experiments in vivo and in organ culture showed very clearly that the immunoprecipitation of labelled molecules has to be followed by additional purification by gel electrophoresis since in spite of the use of monospecific antibodies and careful washing of the immunoprecipitates contaminating coprecipitated labelled molecules accounted for up to 96% of the total precipitated radioactivity in whole adrenals (in vivo experiments) and up to 80% in adrenal medulla (organ culture experiments). The coprecipitation of contaminants most probably results from the addition of carrier proteins, used in order to make the precipitation of the small amounts of labelled proteins visible.
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