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 stimulator of food consumption and gastric acid secretion. In contrast, orexin-B, a peptide containing 28 amino acids without disulfide bond, which has no stimulatory action of gastric acid. The objective of the present study was to characterize the receptor-mediated mechanism of orexin-A-induced stimulation of gastric acid secretion using orexin-A-related peptides with modification of disulfide bonds. Intracisternal injection of orexin-A, but not orexin-B or orexin-A (15-33), that does not contain both disulfide bonds stimulated gastric acid secretion in pylorus-ligated conscious rats. The ability of the stimulation of gastric acid output was less in three alanine-substituted 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 increase in [Ca(2+)]i in CHO-K1/OX1R cells in a dose-dependent manner. EC50 values for OX1R of orexin-A, orexin-B, or orexin-A (15-33) was 0.068, 0.69 or 4.1 nM, respectively, suggesting that peptides containing no disulfide bonds have lower potency for the receptor. Agonistic activity for OX1R of the three orexin-A analogues with modification of one or both disulfide bonds was significantly 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 orexin-A (15-33) and three alanine substituted orexin-A was less than that of orexin-A. A significant inverse relationship between gastric acid output and EC50 values for OX1R, but not OX2R, was observed. These results suggested that the orexin-A-induced acid stimulation requires OX1R activation and that disulfide bonds in orexin-A may have a key role in the receptor activation.
Okumura
T
T
Third Department of Internal Medicine, Asahikawa Medical College, Asahikawa, Japan. okumurat@asahikawa-med.ac.jp
Takeuchi
S
S
Motomura
W
W
Yamada
H
H
Egashira Si
S
S
Asahi
S
S
Kanatani
A
A
Ihara
M
M
Kohgo
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism / Q000494:pharmacology*; D002417:Cattle; D006224:Cricetinae; D004220:Disulfides; D004305:Dose-Response Relationship, Drug; D005744:Gastric Acid / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D008024:Ligands; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D009479:Neuropeptides / Q000378:metabolism / Q000494:pharmacology*; D064446:Orexin Receptors; D000068797:Orexins; D051381:Rats; D017207:Rats, Sprague-Dawley; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide / Q000378:metabolism*
D002352:Carrier Proteins; D004220:Disulfides; C578627:Hcrtr1 protein, rat; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D009479:Neuropeptides; D064446:Orexin Receptors; D000068797:Orexins; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide; D002118:Calcium