The SK‐N‐MC cell line expresses an orexin binding site different from recombinant orexin 1‐type receptor

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Abstract
Orexin A and B (also known as hypocretins), two recently discovered neuropeptides, play an important role in food intake, sleep/wake cycle and neuroendocrine functions. Orexins are endogenous ligands of two G‐protein‐coupled receptors, termed OX1 and OX2. This work presents the first short orexin A and B analogues, orexin A 23–33 and orexin B 18–28, with high affinity (119 ± 49 and 49 ± 23 nm) for OX1 receptors expressed on SK‐N‐MC cells and indicates the importance of the C‐terminal part of the orexin peptides for this ligand–receptor interaction. However, these C‐terminal fragments of orexin did not displace the 125I‐labelled orexin B from the recombinant orexin 1 receptor stably expressed in Chinese hamster ovary cells. To examine the role of the shortened orexin A 23–33 in feeding, its effects in mimicking or antagonizing the effects of orexin A were studied in rats after administration via the lateral hypothalamus. In contrast with orexin A, which potently induced feeding up to 4 h after administration, orexin A 23–33 neither induced feeding nor inhibited orexin A‐induced feeding. Modafinil (Vigil®), which was shown earlier to activate orexin neurons, displayed binding neither to the orexin receptor expressed on SK‐N‐MC cells nor to the recombinant orexin 1 receptor, which indicates that modafinil displays its antinarcoleptic action via another yet unknown mechanism. PCR and subsequent sequencing revealed expression of the full‐length orexin 1 receptor mRNA in SK‐N‐MC and NT‐2 cells. Interestingly, sequencing of several cDNA clones derived from RNA of both SK‐N‐MC and NT‐2 cells differed from the published nucleotide sequence at position 1375. Amino acid prediction of this A→G change results in an isoleucine→valine substitution at the protein level, which may provide evidence for an editing process.