Orexin-A has recently been identified as a new hypothalamic peptide working as a mediator in the regulation of feeding behavior and sleep control. To determine the role of orexin-A in peripheral metabolic processes, we examined direct effects of orexin-A on catecholamine synthesis and secretion in cultured bovine adrenal medullary cells. Incubation of cells with orexin-A (100 pM) for 20 min caused a small but significant increase in C-14-catecholamine synthesis from [C-14] tyrosine, but not from L-3,4-dihydroxyphenyl[3-C-14]alanine. Orexin-A (100 pM) potentiated the stimulatory effects of acetylcholine (0.3 mm) on C-14-catecholamine synthesis. Orexin-A significantly increased tyrosine hydroxylase activity, which was evident at I pM and maximal at 100 pM. 4beta-Phorbol-12beta-myristate-13alpha-acetate, an activator of protein kinase C, did not enhance the stimulatory effects of orexin-A on tyrosine hydroxylase activity, while H-7 and staurosporine, inhibitors of protein kinase C, nullified the effects of orexin-A. Orexin-A had little effect on catecholamine secretion from the cells. Orexin receptor 1 (OX1R) but not orexin receptor 2 (OX2R) mRNA was detected in bovine adrenal medullary cells by reverse transcriptase-polymerase chain reaction. These findings suggest that orexin-A activates tyrosine hydroxylase and then stimulates catecholamine synthesis, probably via activation of the OX1R-protein kinase C pathway in adrenal medullary cells. (C) 2003 Elsevier Science Inc. All rights reserved.