Molecular cloning and functional expression of the α1A-adrenoceptor of Medaka fish, Oryzias latipes

Abstract

A genomic DNA encoding a subtype adrenoceptor (AR) was cloned from Medaka fish, Oryzias latipes, using an oligonucleotide probe corresponding to the consensus sequence of mammalian α-AR and β-AR. The gene spans at least 9 kbp, and the coding region consists of two exons split by an intron of 7.2 kbp located at the same position as those of mammalian α1B-AR genes. The gene encodes 470 amino acid residues, the sequence of which shows the highest similarity to that of mammalian α1A-AR (61%) and significant but lower similarities to other α-AR and β-AR proteins (31-45%), indicating that the gene encodes a Medaka homolog of α1A-AR. To characterize the encoded protein, the mRNA was synthesized in vitro and injected into Xenopus oocytes. As a result, the oocytes responded to 100 nM epinephrine evoking a Ca2+-dependent Cl- current in the order of microamperes, which was not observed for oocytes injected with water alone. The response was reversibly inhibited by an α1-selective antagonist, WB4101 (2-([2,6-dimethoxyphenoxyethyl]aminomethyl)-1,4-benzodioxane). Similar experiments using several adrenegic agonists revealed that Medaka α1A-AR responds to the following agonists in the order: epinephrine ≥ (-)norepinephrine > oxymetazoline ≥ methoxamine, which is similar to the responses of rat α1A receptor expressed in COS cells. The results indicate that fish contains adrenoceptor systems similar to those of mammals in terms of primary structure and pharmacological properties.