The Journal of Steroid Biochemistry and Molecular Biology
Orexins stimulate glucocorticoid secretion from cultured rat and human adrenocortical cells, exclusively acting via the OX1 receptor
Introduction
Orexins A and B are two hypothalamic peptides involved in the central control of food intake [1], [2] and sleep regulation [3], [4]. They originate from the post-translational proteolytic processing of the prepro-orexin, and act via two subtypes of G protein-coupled receptors, referred to as OX1-R and OX2-R. The OX1-R preferentially binds orexin A, while the OX2-R is non-selective [5], [6].
In recent years evidence has accumulated that orexins also play a role as neuroendocrine regulators (for review, see [7], [8]). Accordingly, orexins, like other peptides involved in the central regulation of feeding (e.g. leptin and neuropeptide Y) [9], [10], [11], [12], have been shown to influence the hypothalamic–pituitary–adrenal (HPA) axis (for review, see [13]). Moreover, orexins have been reported to act not only centrally in the HPA axis, but also peripherally, i.e. directly on the adrenal cortex.
Consistent with this view, OX1-R and OX2-R expression has been detected in rat, pig and human adrenal cortex [13], [14], [15], [16], [17], [18], [19], [20], and orexins have been found to stimulate glucocorticoid secretion from dispersed human and rat or cultured pig adrenocortical cells [20], [21]. However, conflicting findings have been reported on the receptor subtype involved in the secretagogue action of orexins and their signaling mechanism: both OX1-R coupled to adenylate cyclase [17], [20], [21], and OX2-R coupled to both adenylate cyclase and inositol triphosphate (IP3) have been proposed as the major pathways [14], [15], [18].
It therefore seemed worthwhile to investigate the effects of orexins A and B on steroid secretion from rat and human adrenocortical cells in primary culture in vitro, and to try to address the subtype of receptor involved in the orexin action by selective immuno-blockade of OX1-R or OX2-R.
Section snippets
Reagents and adrenals
Orexins A and B were purchased from Bachem (Bubendorf, Switzerland) and goat polyclonal anti-OX1-R (C-19) and anti-OX2-R (C-20) antibodies from Santa Cruz, (Santa Cruz, CA). The adenylate cyclase inhibitor SQ-22536, the phospholipase C (PLC) inhibitor U-73122, the protein kinase (PK) A inhibitor H-89 and the PKC inhibitor calphostin-C (for references, see [22]) were obtained from BIOMOL Research Laboratories (Milan, Italy). Dulbecco's modified minimum essential medium (DMEM), fetal calf serum
Results and discussion
RT-PCR showed expression of OX1-R and OX2-R mRNA in both rat and human cultured adrenocortical cells, as well as in freshly dispersed cell preparations (Fig. 1). Real time-PCR did not reveal major differences in the level of expression of both receptors between dispersed and cultured cells (Table 1). These findings confirm that rat and human adrenocortical cells express both orexin-receptor subtypes [16], [17], [33], and not predominantly OX2-R, as previously suggested [13], [14], [15], [18].
Acknowledgments
We thank Miss Alberta Coi for her invaluable help in the search and delivery of bibliographic items.
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