Identification of avian vasotocin receptor subtype-specific antagonists involved in the stress response of the chicken, Gallus gallus.

Vasotocin 1a and 1b receptors (V1aR and V1bR) have been shown to play important roles in the neuroendocrine regulation of stress responses via the anterior pituitary (AP) of birds. To identify effective subtype-specific antagonists for the chicken V1aR (cV1aR) and cV1bR, potential antagonists to the mammalian V1R were screened against the cV1aR and cV1bR 3D structural models by molecular docking analysis with determination of binding pocket/amino acid residues involved in the interaction. The antagonistic effects of the selected ligands were examined by measuring pro-opiomelanocortin (POMC) heteronuclear RNA (hnPOMC) levels following the in vitro stress administration to primary chicken AP cells. Results of in silico analysis showed that the Manning compound and several other antagonists were bound to cV1bR with higher affinity than the natural agonist, arginine vasotocin (AVT). Similarities and differences in the antagonist-receptor binding interface with receptors were characterized for each ligand. Non-peptide mammalian V1bR antagonists, SSR-149415 and L-368899, were shown to be effective and had an additive effect in blocking POMC hnRNA expression in pituitary cell culture studies. SR-49059 antagonized the effect(s) of AVT/CRH on the downregulation of the cV1aR and the upregulation of the cCRH-R2 expression but not the cV1bR and cCRH-R1. The Manning compound antagonized the downregulation of cV1aR, cV1bR and cCRH-R1 and the upregulation of cCRH-R2 expression. The specificity of antagonists apparently resulted from unique differences in the interacting residues and their binding affinities. Collectively, these results provide valuable leads for future development of novel compounds capable of blocking or attenuating the AP stress response of avian species and perhaps other non-mammalian vertebrates as well.