Orexin-Induced Apoptosis: The Key Role of the Seven-Transmembrane Domain Orexin Type 2 Receptor

Orexin-A and orexin-B are regulatory peptides involved in the control of feeding, sleep-wakefulness, and exerting various endocrine and metabolic actions. Recently we demonstrated that orexins, acting at OX(1) receptor (OX(1)R), are proapoptotic peptides. The aim of this study was to investigate the role of the receptor subtype OX(2)R in the control of apoptosis. Orexins caused a caspase-dependent cell death by apoptosis and a drastic cell growth inhibition in Chinese hamster ovary cells transfected with OX(2)R cDNA. On addition of either orexin (10(-6) m) for 48 h, apoptosis was demonstrated by DNA fragmentation, chromatin condensation, annexin-V binding, and activation of caspase-3 and caspase-9. Orexins were active on apoptosis and cell growth inhibition in the range of concentrations between 10(-10) and 10(-5) m with an EC(50) of 5 x 10(-8) m peptides. No effect of orexins could be detected in parental Chinese hamster ovary cells. A rat pancreatic acinar cell line, AR42J, which expresses OX(2)R but not OX(1)R, also underwent growth suppression and apoptosis on treatment with orexins. Suppression of AR42J cell growth by 10(-6) m orexin was more than 75% after 24 h. Induction of annexin-V-labeled AR42J cell number was dose dependent, with EC(50) of 5.1 x 10(-8) m orexin-A and 9.8 x 10(-8) m orexin-B. The OX(2)R agonist [Ala (11), d-Leu (15)]orexin-B promoted effects on cell growth and apoptosis, which were similar to those elicited by orexins. The OX(1)R antagonist SB33487 did not alter orexin-induced inhibition of growth or orexin-induced stimulation of apoptosis in AR42J cells. For the first time, we provide functional and pharmacological evidence for a role of the OX(2)R in orexin-induced apoptosis.