The effects of in vivo treatment with estradiol-17β and pimozide on the in vitro incorporation of radioactive amino acids into prolactin were determined. The synthesis of anterior pituitary proteins was examined in a double label experiment in which pituitaries from ovariectomized, female rats were incubated with [14C]leucine and pituitaries from rats receiving estradiol injections were incubated with [3H]leucine. Electrophoresis of the combined samples demonstrated that estradiol specifically increased incorporation into prolactin, but not other pituitary proteins. The quantitation of prolactin synthesis was compared using gel electrophoresis and immunoprecipitation. The methods yielded results which were indistinguishable quantitatively and both methods demonstrated the ability of estradiol to stimulate prolactin synthesis. Anterior pituitaries were able to incorporate [3H]leucine into total protein, prolactin and growth hormone for at least 6 h in vitro. Differences between pituitaries from control and estradiol-treated rats were maintained for the 6 h in vitro. When daily estradiol injections were given to ovariectomized female and intact male rats, the rate of [3H]leucine incorporation into prolactin was increased by 50% on day one and reached maximal levels at 3 to 7 days. Incorporation into growth hormone was not increased, but showed a slight decline. In this experiment the pituitary content of protein and DNA did not change; however, it was demonstrated that estradiol did stimulate the incorporation of [3H]thymidine into DNA. A single injection of pimozide into ovariectomized female rats was also shown to stimulate prolactin synthesis. In females estradiol plus pimozide produced no greater effect than did estradiol alone, while in males the effects of pimozide and estradiol seemed to be additive.
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