Effects of dexamethasone and forskolin on neurotensin production in rat hypothalamic cultures.

In the present study, the effects of glucocorticoids and forskolin, an activator of adenylate cyclase, were examined on neurotensin (NT) production from rat hypothalamic neurons in primary culture. Treatment with dexamethasone induced a dose-dependent increase in NT content. The maximum was reached at 1 microM dexamethasone, which induced a 100% increase in NT levels. The effect of dexamethasone was mimicked by the glucocorticoid agonist RU28362 and blocked by the antiglucocorticoid RU38486, suggesting that this effect was mediated through the glucocorticoid receptor. The treatment with dexamethasone also enhanced the number of immunoreactive NTergic cells (92% increase). In contrast to dexamethasone, forskolin affected neither the NT content nor the number of immunoreactive NTergic cells. However, when cells were treated with both dexamethasone and forskolin, a 285% increase in NT content and a 430% increase in the number of immunoreactive NTergic cells were observed, representing 2.8- and 4.7-fold increases, respectively, compared to the effect of dexamethasone alone. Moreover, this combined treatment increased the accumulation of NT in the culture medium (160% increase) as well as the abundance of NT messenger RNA. We conclude from the present findings that dexamethasone and forskolin act synergistically to enhance NT production in hypothalamic neurons.

Treatment of PC12 cells by nerve growth factor, dexamethasone, and forskolin. Effects on cell morphology and expression of neurotensin and tyrosine hydroxylase.

Several lines of anatomical, neurochemical, electrophysiological, and behavioral evidence suggest the existence of physiological interactions between neurotensin (NT) and the brain dopaminergic systems. Thus, NT has been shown to exert a neuroleptic-like action and could be implicated in the pathogenesis and treatment of schizophrenia. It is thus of particular importance to develop in vitro cell culture systems as models to study such interactions. Rat adrenal pheochromocytoma PC12 cells, which expressed high levels of tyrosine hydroxylase, were used in the present study. In contrast to rat brain cells in primary cultures, PC12 cells did not express functional NT receptors. However, they were able to express both NTmRNA and NT in response to NGF, forskolin, and dexamethasone. Those neurochemical modifications furthermore may be related to changes in the morphology of the PC12 cells in response to NGF, forskolin, and dexamethasone alone or in combination. These data suggest that PC12 cells may provide a useful model to study in vitro the regulation of both catecholamine and neurotensin phenotypes.

Plasma neurotensin levels in humans: relation to hormone levels in diseases involving the hypothalamo-pituitary-thyroid axis.

This study was aimed to investigate, in humans, the possible relationship between plasma neurotensin (NT) levels and the activity of the hypothalamo-pituitary-thyroid axis. Neurotensin was measured by radioimmunoassay in 14 healthy adult volunteers and in 41 patients among whom 10 were considered as controls and 31 had thyroid dysfunction according to free thyroxine and thyrotropin plasma values. Basal NT levels were not significantly different in healthy adults and in control patients: 9.7 +/- 1.1 fmol/ml (mean +/- SEM) vs 13.3 +/- 2.9 fmol/ml, respectively. In patients with central hypothyroidism the NT level was significantly lower (5.7 +/- 1.2 vs healthy volunteers and controls; p < 0.05) and in patients with peripheral hypothyroidism and hyperthyroidism the NT level was significantly higher (35.9 +/- 12.8 and 29.9 +/- 9.5 fmol/ml, respectively, vs healthy adults (p < 0.01) and vs controls (p < 0.05)). After thyrotropin-releasing hormone (TRH) injection (250 micrograms iv) in nine subjects (two control patients, five patients with hypothyroidism and two patients with hyperthyroidism), NT levels decreased independently of the endocrine status from mean values of 13.4 +/- 8.4 at basal level to 7.3 +/- 0.8 fmol/ml 30 min after injection (p < 0.01 on paired percentage decrease values). These data suggest that plasma NT levels in humans depend upon the pituitary-thyroid status and indicate that TRH could exert a negative regulation on circulating NT levels.

Neurotensin receptor down-regulation induced by dexamethasone and forskolin in rat hypothalamic cultures is mediated by endogenous neurotensin.

Neurotensin (NT) has been shown to be involved in neuroendocrine regulation, and the presence of both the peptide and its receptors has been demonstrated in the hypothalamus. In the present study, we show that hypothalamic neurons in primary cultures express the neurotensin receptor (NTR) and we examined a possible regulation of this receptor by glucocorticoids and activators of adenylate cyclase. In the hypothalamic cultures, 125I-NT bound to a single class of binding sites, presenting a selectivity similar to that observed for the high-affinity NTR previously described in the adult rat brain. Radioautographic studies demonstrated that these 125I-NT binding sites were present on 3% of the neurons. A 48-h treatment with forskolin (fsk) decreased 125I-NT binding by 30%. No effect of dexamethasone (dex) alone was found on that parameter. However, a combined treatment with both agents led to a 40% decrease in 125I-NT binding, corresponding to a reduced number of binding sites, and to a 68% decrease in the amount of NTR mRNA. In parallel, the dex plus forsk treatment increased NT release in the incubation medium. Moreover, the decreases in 125I-NT binding and NTR mRNA induced by this treatment were abolished in the presence of an anti-NT antibody or SR 48692, a non-peptidic antagonist of NTR, suggesting that the down-regulation of NTR observed after dex plus fsk treatment was mediated by the release of endogenous NT. Agonist-induced down-regulation of the NTR in this system was confirmed by the application of an exogenous NT analogue, JMV 449. The present findings indicate that, in hypothalamic cultures, dex and fsk indirectly down-regulate NTR expression via the release of endogenous NT.

Evidence for neurotensin autoreceptors and relationship of neurotensin and its receptors with tyrosine hydroxylase-positive neurons in rat primary hypothalamic cultures.

Neurotensin is present in high quantity in the hypothalamus, where it regulates pituitary hormone secretion. A relationship between dopaminergic and neurotensinergic systems has been suggested in the hypothalamus in studies showing an effect of neurotensin on tuberoinfundibular dopaminergic neurons. In order to determine the anatomical basis of such interactions, primary cultures of rat hypothalamic neurons were used. Tyrosine hydroxylase and neurotensin containing cells were identified by immunocytochemistry and neurotensin binding sites by [125I]Tyr3-neurotensin autoradiography. Colocalization studies showed that neurotensin immunoreactivity was present in 16% of tyrosine hydroxylase-positive cells, and that these neurotensin/tyrosine hydroxylase neurons represented more than half (58%) of the neurotensinergic population. Five percent of the tyrosine hydroxylase-positive cells had neurotensin binding sites, suggesting that only a restricted number of hypothalamic dopaminergic neurons is responsive to neurotensin. Neurotensin binding sites were also found on some neurotensin-positive cells, demonstrating for the first time the presence of autoreceptors for this peptide on neurons. These results in primary cultures provide a cellular basis for direct effects of neurotensin on a subpopulation of hypothalamic dopaminergic cells, and support the possibility of an autocrine action of neurotensin in the hypothalamus.

Steroid effects on brain functions: an example of the action of glucocorticoids on central dopaminergic and neurotensinergic systems.

It is now clearly established that steroid hormones released from peripheral endocrine glands may, through specific receptors in the brain, directly regulate brain function. These effects may be rapid or involve long-term modifications at the genomic level. Concerning the glucocorticoids, their receptors are found in most neuronal cells, an observation which can be related to their widespread effects on neuronal metabolism. Furthermore, glucocorticoids are often related to stress. We have previously demonstrated that neonatal handling of the rat prevented excessive endocrine response to stress. In adults, this action appeared to protect the animal from potential damaging effects of glucocorticoids and from related impairment of cognitive functions. The effects of glucocorticoids are thought to involve an interaction of several central neurotransmitter systems. One such neurotransmitter is neurotensin, a neuropeptide which was reported to be closely related to central dopaminergic system regulation. This paper presents a rapid overview of the central effects of glucocorticoids and possible evidence for the interrelationship between these steroids, dopamine and neurotensin systems in the regulation of the hypothalamo-pituitary-adrenal axis. It provides a new way to approach stress responses and to develop new substances that may become potential drugs in the treatment of some psychiatric disorders.