Molecular cloning and characterization of V2-type receptor in two ray-finned fish, gray bichir, Polypterus senegalus and medaka, Oryzias latipes

In tetrapods, vasopressin (VP) and vasotocin (VT) are involved in various aspects of physiology and behavior including osmoregulation, cardiovascular function, reproduction, stress response and social behavior. Pharmacological and molecular studies have identified three types of VP/VT receptors, V1a-type (V1aR), V1b-type (V1bR) and V2-type (V2R). On the other hand, only V1aR has so far been identified in teleosts. In the present study, we successfully cloned V2Rs from two ray-finned fish, gray bichir and medaka. Phylogenetic analysis showed that the cloned receptors belong to the V2R group of lobe-finned fish and tetrapods. The amino acid sequences of bichir V2R and medaka V2R were high identity (60-65.5% and 53.2-80.9%, respectively) with other known V2R members, respectively. Reverse transcriptase PCR revealed that ray-finned fish V2R transcripts have been detected in various tissues including brain, gill, heart, liver, kidney and reproductive organs, suggesting that ray-finned fish V2R might mediate multiple functions of VT. In functional analysis, the cells transfected with the cloned receptors responded with the accumulation of intracellular cAMP in a concentration-dependent manner following VT stimulation, but not respond with [Ca²⁺]i. Furthermore, pretreatment with mammalian V2R antagonist (OPC-31260) to the cells transfected with medaka V2R significantly inhibited an increase of the VT-induced intracellular cAMP. These results suggest that ray-finned fish possess a functional V2R linked to adenylate cyclase and the cAMP signaling pathway as well as V2Rs of lobe-finned fish and tetrapods. Thus, the present study suggests that functional V2R evolved prior to the divergence of the ray- and lobe-finned fish lineages.