The possible existence of extrapituitary melanocyte stimulating hormone (MSH) in various regions of the rat brain has been studied in intact and hypophysectomized rats. Using a sensitive and specific radioimmunoassay (RIA), αMSH has been found in a number of brain regions in intact rats. The standard curves of synthetic αMSH and the dilution curves for pars intermedia nervosa (PIN), pars distalis (PD), hypothalamus and thalamus extracts were strictly parallel. The αMSH concentrations were measured in PIN (6,225 ± 962 ng/mg wet tissue); PD (12.5 ± 1.41 ng/mg); pineal (380 ± 29 ng/g wet tissue); hypothalamus (645 ± 161 ng/g) and thalamus (33.3 ± 5.26 ng/g). In rats hypophysectomized for 1 or 2 months, the highest concentrations of immunoreactive αMSH were found in pineal (353 ± 140 ng/g wet tissue), hypothalamus (85.8 ±14.1 ng/g) and thalamus (39.8 ± 13.9 ng/g). Hypophysectomy significantly reduced hypothalamic MSH content and concentration but did not alter MSH concentration in pineal and thalamus. From these results, we conclude that hypothalamic αMSH is, in part, of hypophyseal origin while pineal and thalamus «MSH does not originate from the pituitary. After Sephadex G-25 gel filtration, synthetic αMSH and PIN extracts showed a single peak of both bioactive and immunoreactive αMSH. In the same conditions, extracts from the 5 brain regions studied in hypophysectomized rats chromatographed as a single peak of immunoreactive MSH but as 2 peaks of apparent bioactive MSH, 1 concident with synthetic αMSH and the other far after the salt volume. We conclude that αMSH is found in a number of brain areas and its presence after hypophysectomy would indicate synthesis within the central nervous system.

Argininosuccinate lyase (ASL), the fourth enzyme of the urea cycle, belongs to a group of liver enzymes appearing in the late foetal period in the rat. Several hormones, including glucocorticosteroids and insulin have been implicated in the control of the development of this enzyme activity. In this study, the cloned cDNA was used to measure the relative abundance of ASL mRNA in the livers of rats at various stages of perinatal development and in cultured foetal hepatocytes during hormonal manipulations.The ASL mRNA was first detectable on day 15.5 of gestation and increased in amount concomitantly with the rise in the enzyme activity, suggesting that the appearance of enzyme activity reflects the turning on of specific gene transcription. When foetal hepatocytes were exposed to dexamethasone, an increase in ASL mRNA was detected, which was completely abolished by addition of actinomycin D, suggesting a transcriptional effect of the steroid. In contrast, administration of cortisol to foetuses in utero had no effect on the mRNA level, suggesting that the steroid action is inhibited in the intra-uterine environment. Insulin might be the inhibiting factor since it completely repressed the dexamethasone-induced accumulation of ASL mRNA in foetal hepatocytes. These data were confirmed in vivo by experiments using streptozotocin, which produces insulin-depleted foetuses and causes the accumulation of ASL mRNA. This regulation of ASL mRNA by glucocorticoids and insulin could account for the modulation of the enzyme activity observed in vivo and in vitro.

In the present study we examined the in vivo effects of glucocorticosteroids and glucagon on the developmental formation of the individual urea cycle enzymes argininosuccinate synthetase, argininosuccinase, and arginase during the late fetal period. In particular, addition of exogenous glucagon caused a rise in argininosuccinase and arginase activities in the livers of rat fetuses at term but not at earlier stages. Glucagon produced a rise in argininosuccinase activity earlier if fetuses were previously treated with cortisol. When fetuses were deprived of corticosteroid (hypophysectomy in utero), glucagon no longer promoted the argninosuccinase activity, indicating that adrenal glucocorticoids are required for normal enhancement of the enzyme activity by glucagon. Dibutyryl cAMP was still effective in hypophysectomized fetuses. Results obtained by injected combinations of inducers indicated that a glucocorticosteroid-glucagon interaction might be involved in the regulation of argininosuccinate synthetase and argininosuccinase. No synergistic action was found on arginase activity in vivo.

Foetal-rat hepatocytes were cultured in primary monolayer culture, and activity changes of argininosuccinate synthetase (ASS, EC 6.3.4.5) and argininosuccinase (ASL, EC 4.3.2.1) were followed under defined hormone conditions. In hormone-free medium, cultured cells maintained the enzyme activities at values equal to those of freshly isolated cells for at least 3 days. Continuous addition of dexamethasone produced the development of the two enzyme activities, but only after the first 20h of culture. Under these conditions, urea production by the foetal hepatocytes was concomitantly increased in the culture medium. Pretreatment with dexamethasone for 20h was sufficient to produce the development of ASL activity within the 2 following days. Introduced alone, glucagon induced an increase of ASL activity, but did not affect the ASS activity. The most powerful stimulation of ASS and ASL could be observed in cultured hepatocytes if glucagon and dexamethasone were added simultaneously or sequentially. These results indicated that the development of the receptor complex for the induction of urea-cycle enzymes appears early before birth and established that glucocorticoids amplify the glucagon stimulation of these enzyme activities during foetal life.