Requirement of intact disulfide bonds in orexin-A-induced stimulation of gastric acid secretion that is mediated by OX1 receptor activation

Orexin-A is a neuropeptide consisting of 33 amino acids with two intrachain disulfide bonds, namely Cys6-Cys12, and Cys7-Cys14, and is a potent stimul ator of food consumption and gastric acid secretion. In contrast, orexin-B, a peptide containing 28 amino acids without disulfide bond, which has no s timulatory action of gastric acid. The objective of the present study was t o characterize the receptor-mediated mechanism of orexin-A-induced stimulat ion of gastric acid secretion using orexin-A-related peptides with modifica tion of disulfide bonds. Intracisternal injection of orexin-A, but not orex in-B or orexin-A (15-33), that does not contain both disulfide bonds stimul ated gastric acid secretion in pylorus-ligated conscious rats. The ability of the stimulation of gastric acid output was less in three alanine-substit uted orexin-A, [Ala(6,12)]orexin-A, [Ala(7,14)]orexin-A, and [Ala(6,7,12,14 )]orexin-A, than orexin-A. Orexins-induced calcium increase was measured in CHO-K1 cells expressing OX1R or OX2R. Orexin-A induced a transient increas e in [Ca2+]i in CHO-K1/OX1R cells in a dose-dependent manner. EC50 values f or OX1R of orexin-A, orexin-B, or orexin-A (15-33) was 0.068, 0.69 or 4.1 n M, respectively, suggesting that peptides containing no disulfide bonds hav e lower potency for the receptor. Agonistic activity for OX1R of the three orexin-A analogues with modification of one or both disulfide bonds was sig nificantly reduced as compared with that of orexin-A. EC50 values for OX2R of orexin-A and orexin-B was almost equal but potency for the receptor of o rexin-A (15-33) and three alanine substituted orexin-A was less than that o f orexin-A A significant inverse relationship between gastric acid output a nd EC50 values for OX1R, but not OX2R, was observed. These results suggeste d that the orexin-A-induced acid stimulation requires OX1R activation and t hat disulfide bonds in orexin-A may have a key role in the receptor activat ion. (C) 2001 Academic Press.