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 [3H]-NKA in a receptor-binding assay. [3H]NKA bound to both NK-2Rs with high affinity (KD = 2-7 nM) and saturable (Bmax = 633-9000 fmol/mg protein) manner which was inhibited by GTP analogs. Competition experiments with agonists demonstrated identical order of potency in both NK-2Rs; NKA > [Nle10]NKA(4-10) > [beta-Ala8]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 rank 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.
Aharony
D
D
Department of Pharmacology, ZENECA Pharmaceuticals Group, ZENECA Inc. Wilmington, DE 19897.
Little
J
J
Thomas
C
C
Powell
S
S
Downey-Jones
M
M
Graham
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001549:Benzamides / Q000494:pharmacology; D001667:Binding, Competitive; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D015870:Gene Expression; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism / Q000494:pharmacology; D006168:Guinea Pigs; D008168:Lung / Q000737:chemistry*; D008969:Molecular Sequence Data; D015288:Neurokinin A / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D010446:Peptide Fragments / Q000494:pharmacology; D010880:Piperidines / Q000494:pharmacology; D011651:Pulmonary Artery / Q000737:chemistry*; D011817:Rabbits; D018041:Receptors, Neurokinin-2 / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017385:Sequence Homology; D015320:Tachykinins / Q000494:pharmacology; D014162:Transfection
D001549:Benzamides; D018076:DNA, Complementary; D010446:Peptide Fragments; D010880:Piperidines; D018041:Receptors, Neurokinin-2; D015320:Tachykinins; C069192:neurokinin A(4-10), Tyr(5)-Trp(6,8,9)-Lys(10)-; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C073839:SR 48968; D015288:Neurokinin A