Mechanisms of Norepinephrine Mediated Corticotropin-Releasing Factor-41 Release from Cultured Fetal Hypothalamic Cells

Abstract

Catecholamines have been shown to activate hypothalamic corticotropin-releasing factor-41 (CRF) synthesis and release. In order to study the mechanisms involved, fetal hypothalamic cells were cultured and CRF release was measured by radioimmunoassay. Norepinephrine (NE) induced CRF release in a dose-dependent manner. Further studies were performed with a protein kinase C inhibitor, H-7(l-(5-isoquinolinesulfonyl)-2-methylpiperazine) and a protein kinase A inhibitor, IP-20. NE-stimulated CRF release was reduced by H-7 (5 and 50 µM) in a dose-dependent fashion, while 5 µM IP-20 resulted in a small but significant inhibition. Pretreatment of the cells for 15 h with 20 and 200 nM 12-O-tetradecanoylphorbol-13-acetate, which down-regulates protein kinase C activity, blocked the release of CRF in response to NE (1 µM), further supporting protein kinase C as a mediator for NE-activated CRF release. Pretreatment with 50 and 500 ng/ml pertussis toxin (15 h) resulted in a dose-dependent inhibition of NE-activated CRF release. Both dexamethasone and aldosterone at the concentrations of 1 µM reduced NE-induced CRF release. These results sugget that CRF can be released from hypothalamic neurons in response to NE through both protein kinase C- and protein kinase A-dependent mechanisms, and that pertussis toxin-sensitive G-proteins are also involved in this response. Furthermore, glucocorticoids and mineralocorticoids can reduce NE-acivated CRF release from cultured hypothalamic cells.

Removal of Adrenal Steroids from the Medium Reverses the Stimulating Effect of Catecholamines on Corticotropin Releasing Hormone Neurons in Organotypic Cultures

“…These data also agree with those obtained from investigations of other in vitro systems [20,21,22,24], but not with those of Suda et al [25] who reported that NA inhibited CRH release from the hypothalamus in perifusion experiments. The present study also showed that CRH release increased both with increasing concentrations of NA for a fixed incubation time and increasing incubation times with a determined NA concentration.…”

“…We then studied the effect of nor adrenaline on short-term CRH release in cultured slices which has previously been reported as stimulatory in hypo thalamic explants [20] and in primary cultures of neonatal [21] or fetal [22] hypothalamic cells. Finally, we assessed the possible modulatory role of steroids on catecholamineinduced CRH release.…”

Increase of extracellular corticotropin-releasing factor-like immunoreactivity levels in the amygdala of awake rats during restraint stress and ethanol withdrawal as measured by microdialysis

“…A recent microdialysis experiment (Tanaka et al, 1991) showed that the temporal profile of norepinephrine release from the amygdala of rats exposed to 20 min restraint stress closely matches that of CRF-IR described in the present work. In vitro norepinephrine evokes CRF-IR release from hypothalamic neurons (Tsagarakis et al, 1988;Hu et al, 1992), while microdialysis experiments show that CRF produces norepinephrine release from nerve terminals in prefrontal cortex and hypothalamus (Lavicky and Dunn, 1993), suggesting the existence of a reciprocal stimulating interaction between CRF and norepinephrine. Although the relevance of central norepinephrine in anxiety is still under debate, some of the behavioral effects produced by stress and ethanol withdrawal involve adrenoreceptor mediation (Berridge and Dunn, 1989;Glue et al, 1989;Soderpalm and Engel, 1990).…”

“…In fact, 4-aminopyridine is known to release norepinephrine and acetylcholine in vitro (Drukarch et al, 1989;Heemskerk et al, 1990). Since norepinephrine or acetylcholine can produce CRF-IR release from the CNS neurons (Tsagarakis et al, 1988;Hu et al, 1992), it cannot be excluded that these or other neurotransmitters mediate the effects of 4-aminopyridine on CRF release within the amygdala.…”

Studies of the secretion of corticotropin-releasing factor and arginine vasopressin into the hypophysial-portal circulation of the conscious sheep. II. The central noradrenergic and neuropeptide Y pathways cause immediate and prolonged hypothalamic-pituitary-adrenal activation. Potential involvement in the pseudo-Cushing's syndrome of endogenous depression and anorexia nervosa.

“…The suggestion that the central noradrenergic pathways might stimulate CRF biosynthesis in vivo is supported by the observations that insulin-induced hypoglycemia increases both the turnover of NE and the level of CRF mRNA expression in the hypothalamus (35,57). NE also stimulates the in vitro release and biosynthesis of CRF in fetal rat hypothalamic cells, and this effect involves the activation of both the A and C protein kinases and is mimicked by the phorbol ester TPA (58,59). TPA activates protein kinase C and increases the expression of the two major constituents of the AP-1 transcription factor complex, namely the protooncogene products Fos and Jun (60).…”