Ziolkowska Agnieszka, Spinazzi Raffaella, Albertin Giovanna, Nowak Magdalena, Malendowicz Ludwik K, Tortorella Cinzia, Nussdorfer Gastone G
Department of Histology and Embryology, Poznan School of Medicine, PL-60781 Poznan, Poland.
J Steroid Biochem Mol Biol. 2005 Sep;96(5):423-9. doi: 10.1016/j.jsbmb.2005.05.003.
Orexins A and B are hypothalamic peptides, that act via two subtypes of receptors, named OX1-R and OX2-R. Rat and human adrenal cortexes are provided with both OX1-R and OX2-R, and we have previously shown that orexin-A, but not orexin-B, enhances glucocorticoid secretion from dispersed adrenocortical cells. Since OX1-Rs preferentially bind orexin-A and OX2-Rs are non-selective for both orexins, the hypothesis has been advanced that the secretagogue effect of orexin-A is exclusively mediated by the OX1-R. Here, we aimed to verify this contention and to gain insight into the signaling mechanism(s) underlying the secretagogue effect of orexins using primary cultures of rat and human adrenocortical cells. Reverse transcription-polymerase chain reaction showed that cultured cells, as freshly dispersed cells, expressed both OX1-R and OX2-R mRNAs. Orexin-A, but not orexin-B, concentration-dependently increased corticosterone and cortisol secretion from cultured rat and human adrenocortical cells, respectively. The blockade of OX1-Rs by selective antibodies abrogated the secretagogue effect of orexin-A, while the immuno-blockade of OX2-Rs was ineffective. The glucocorticoid response of cultured cells to orexin-A was annulled by the adenylate cyclase and protein kinase (PK) A inhibitors SQ-22536 and H-89, and unaffected by the phospholipase C and PKC inhibitors U-73122 and calphostin-C. Orexin-A, but not orexin-B, enhanced cyclic-AMP production from cultured cells, and did not alter inositol-3-phosphate release. Collectively, our present results allow us to conclude that orexins stimulate glucocorticoid secretion from rat and human adrenocortical cells, exclusively acting through OX1-Rs coupled to the adenylate cyclase/PKA-dependent signaling cascade.
食欲素A和B是下丘脑肽，通过两种受体亚型（即OX1-R和OX2-R）发挥作用。大鼠和人类肾上腺皮质同时具有OX1-R和OX2-R，我们之前已经表明，食欲素A而非食欲素B可增强分散的肾上腺皮质细胞的糖皮质激素分泌。由于OX1-R优先结合食欲素A，而OX2-R对两种食欲素均无选择性，因此有人提出假设，食欲素A的促分泌作用仅由OX1-R介导。在此，我们旨在验证这一论点，并利用大鼠和人类肾上腺皮质细胞的原代培养物深入了解食欲素促分泌作用的信号传导机制。逆转录-聚合酶链反应表明，培养的细胞，如同新鲜分散的细胞一样，表达OX1-R和OX2-R的mRNA。食欲素A而非食欲素B分别浓度依赖性地增加培养的大鼠和人类肾上腺皮质细胞的皮质酮和皮质醇分泌。选择性抗体对OX1-R的阻断消除了食欲素A的促分泌作用，而对OX2-R的免疫阻断则无效。培养细胞对食欲素A的糖皮质激素反应被腺苷酸环化酶和蛋白激酶（PK）A抑制剂SQ-22536和H-89消除，且不受磷脂酶C和PKC抑制剂U-73122和钙泊三醇-C的影响。食欲素A而非食欲素B增强了培养细胞的环磷酸腺苷生成，且未改变肌醇-3-磷酸释放。总体而言，我们目前的结果使我们能够得出结论，食欲素刺激大鼠和人类肾上腺皮质细胞的糖皮质激素分泌，仅通过与腺苷酸环化酶/蛋白激酶A依赖性信号级联偶联的OX1-R起作用。
