ISOLATION AND CHARACTERIZATION OF NEUROKININ-A RECEPTOR CDNAS FROM GUINEA-PIG LUNG AND RABBIT PULMONARY-ARTERY

cDNA clones for NK-2 receptors (NK-2R) were isolated from guinea-pig lung (GPl) and rabbit pulmonary artery (Rpa) using a polymerase chain reaction based methodology. The GPl NK-2R consists of 402 amino acids and encodes a protein with a relative molecular mass of 45,097. The Rpa NK-2R consists of 384 amino acids and encodes a protein with a relative molecular mass of 43,169. The GPl and Rpa NK-2Rs share significant amino acid sequence homology amongst themselves (90.1%), as well as with human, bovine, hamster and rat NK-2 receptors. The two receptors were stably transfected into mouse erythroleukemia cells, high-speed membranes were prepared from induced cells and their pharmacological properties examined utilizing [H-3]-NKA in a receptor-binding assay. [H-3]NKA bound to both NK-2Rs with high affinity (K-D = 2-7 nM) and saturable (B-max = 633 - 9000 fmol/me protein) manner which was inhibited by GTP analogs. Competition experiments with agonists demonstrated identical order of potency in both NK-2Rs; NKA > [Nle(10)]NKA(4-10) > [beta-Ala(8)]NKA(4-10) >> Substance P >>> Senktide. Similarly, an identical profile for both receptors was observed with selective NK-2 antagonists: SR48,968 > MEN10,376 >> R396. The lank order of antagonist affinity is consistent with that in cloned human NK-2R and the observations of NK-2 receptor pharmacology in native human, guinea pig and rabbit tissues.