The presence and specific structures of the oligosaccharides on TSH have been shown to be important for its production and bioactivity. Since the carbohydrate structure of a protein reflects the glycosylation apparatus of the host cells in which the protein is expressed, we examined the biological activity and metabolic clearance of a preparation of purified recombinant human (rh) TSH derived from a stable transfectant of Chinese hamster ovary cells. Carbohydrate compositional analysis of this rTSH showed it to be more highly sialylated than a nonrecombinant, cadaver-derived pituitary hTSH. In addition, no N-acetyl galactosamine was detectable in rhTSH, which implies the absence of terminal sulfate moieties, both of which are present in pituitary-derived TSH. The immunologic activity and porcine TSH receptor-binding activity of the preparation of rhTSH were 3- to 4-fold lower than those of a standard pituitary hTSH. The rhTSH showed a maximum stimulatory activity similar to that of pituitary hTSH in two different in vitro bioassays. However, rhTSH elicited about 3-fold and 5-fold less cAMP than pituitary TSH after stimulation of adenylyl cyclase in bovine thyroid membranes and the rat FRTL-5 cell line, respectively. Removal of sialic acid did not alter the immunologic activity of rhTSH. However, the potencies of rhTSH in receptor-binding, adenylyl cyclase, and FRTL-5 assays were increased 2.4-, 2.6- and 26.7-fold, respectively after sialic acid removal. These data suggest that the in vitro biological activity of rhTSH is influenced by its highly sialylated oligosaccharide chains. The rhTSH had a 2-fold lower metabolic clearance rate than pituitary TSH, resulting in a greater than 10-fold higher serum concentration of rhTSH at 3 h as compared to pituitary hTSH. After sialic acid removal, the rhTSH was cleared faster (7.5-fold) than pituitary hTSH, showing that its longer plasma half-life was due to its higher sialylation. Biologically active rhTSH should be of clinical value in the diagnosis and treatment of patients with thyroid cancer and as a pure hTSH reference preparation.

Temporal alterations in plasma prolactin levels caused by the administration of 2-hydroxyestradiol and 2-hydroxyestrone (100 μg/kg) into the right atrium of freely-moving conscious male rats were examined. The catechol estrogens were given in a single bolus via an indwelling cannula and plasma prolactin concentration was monitored by taking blood samples every 2 min. A pulsatile elevation of plasma prolactin occurred approximately 4 h after the injection of 2-hydroxyestradiol and a small increase was also observed when it was administered to rats bearing a Silastic capsule containing estradiol. 2-Hydroxyestrone had no effect in untreated male rats but produced a 5- to 6-fold elevation in plasma prolactin level 4 h after its administration to rats implanted with estradiol. It is proposed that 2-hydroxyestrone suppresses the action of estradiol on prolactin secretion from the pituitary and that the accumulated hormone is released when the concentration of this catechol estrogen falls below a critical level. A longer latent period was required to produce an elevation in plasma prolactin levels by 2-hydroxyestradiol than by estradiol.

Basal plasma prolactin concentration is controlled by tonic inhibition. The major prolactin-inhibiting factor (PIF) is believed to be dopamine. Factors other than dopamine have also been suggested as possible physiological PIF. One of the major candidates for the nondopaminergic PIF is considered to be gamma-aminobutyric acid (GABA). We have carefully examined the possible physiological role of GABA by monitoring, at every 2 min, the circulating prolactin concentration after GABA administration, in conscious freely moving rats. GABA (0.1 or 1 g/kg) had no significant direct effect on plasma prolactin in rats in which the dopaminergic receptors were completely blocked by pimozide, nor in hypophysectomized rats in which a pituitary had been grafted under the kidney capsule and was therefore removed from any hypothalamic influence. The effects of bicuculline, a GABA-receptor-blocking agent, was examined in order to find out whether a tonic inhibition is exerted by GABA after elimination of tonic dopaminergic inhibition on prolactin secretion. The pimozide-treated rat in which the dopaminergic tone is completely eliminated did not show any prominent elevation of plasma prolactin concentration after bicuculline (300 µg/kg) administration. However, GABA did have an inhibitory effect in a primary pituitary cell monolayer culture system. Therefore, we conclude that GABA does not play a significant role as a physiological PIF and that the inhibitory effect of GABA in vitro is of a pharmacological nature.

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