Identification and characterization of mesotocin and V1a-like vasotocin receptors in a urodele amphibian, Taricha granulosa

Abstract

The cDNA sequences encoding the mesotocin receptor (MTR) and vasotocin 1a receptor (VTR-1a) were identified in a urodele amphibian, the rough-skinned newt, Taricha granulosa. Saturation binding of [ 3H]oxytocin (OT) to the Taricha MTR (tMTR) was best fit by a two-state model; a high affinity-low abundance site and a lower affinity-high abundance site. Competition-binding studies found the following rank-order affinities for the tMTR: mesotocin (MT) > OT ≈ vasotocin (VT) > vasopressin (VP) > isotocin (IT). Inositol phosphate (IP) accumulation studies demonstrated functional activity of both the tMTR and Taricha VTR-1a (tVTR-1a) in a heterologous cell culture system. The rank-order potencies for the tMTR were MT > OT > VT ≈ VP > IT. The combined binding and IP results indicate that VT may act as a partial agonist of the tMTR. Rank-order potencies for the tVTR-1a were VT > VP > MT ≈ OT > IT. For both receptors, stimulation of IP accumulation was blocked by d(CH 2) 5[Tyr(Me) 2]AVP (Manning compound) and d(CH 2) 5[Tyr(Me) 2,Thr 4,Tyr-NH 2]OVT (OTA). OTA was a more potent antagonist for the transiently expressed tMTR while Manning compound was relatively more potent at inhibiting IP accumulation in tVTR-1a expressing cells. In contradiction to earlier assumptions, the absolute IC 50 of Manning compound was lower for the tMTR (27 nM ± 13) than the tVTR-1a (586 nM ± 166) indicating its potential higher affinity for the tMTR, a finding with special relevance to interpretation of comparative studies investigating the behavioral and physiological actions of neurohypophysial peptides in non-mammalian species.