Secretion of corticotropin-releasing factor from cultured rat hypothalamic cells: effects of catecholamines.

An understanding of the regulation of CRF secretion in rats is currently incomplete, in part due to the lack of sensitive in vitro models available for studying this neuropeptide. In particular, the effects of catecholamines on CRF secretion, and the receptor subtypes mediating these actions have long been the subject of much debate. A cultured cell model has been adapted for studying secretory responses of hypothalamic cells of 1-week-old rats. Between 7-16 days in monolayer culture the cells secreted detectable levels of immunoreactive CRF, and this release was paralleled by the appearance of punctate bead-like regions of immunoreactivity along fine cellular processes. CRF secretion was increased up to 4-fold by norepinephrine (EC50, approximately 0.5 microM). The increase in CRF secretion produced by norepinephrine was blocked by the beta-receptor antagonist propranolol, but not by the alpha-antagonist prazosin. Moreover, the beta-receptor agonist isoproterenol significantly elevated CRF secretion, whereas the alpha-agonist phenylephrine was without effect, except at high concentrations. Addition of phenylephrine, however, potentiated the effect of isoproterenol, but this response was still significantly less than that produced by norepinephrine. Forskolin (EC50, approximately 0.7 microM) and the active phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (EC50, approximately 40 nM) also increased CRF secretion by 3- to 4-fold. Inactive phorbol derivatives had no effect on CRF release from these cultures. The results indicate that cultured neonatal rat hypothalamic cells are a powerful model for the study of CRF release in vitro, and that norepinephrine acts directly at the isolated cell level to stimulate secretion of this peptide, primarily by activating beta-adrenoceptors. The results also suggest that at least two functional second messenger systems (adenylate cyclase and protein kinase-C) are involved in CRF secretion and are already functional in the neonatal hypothalamus.