Molecular and pharmacological characterization of a functional tachykinin NK3 receptor cloned from the rabbit iris sphincter muscle

1 A functional tachykinin NK3 receptor was cloned from the rabbit iris sphi ncter muscle and its distribution investigated in ocular tissues. 2 Standard polymerase chain reaction (PCR) techniques were used to clone a full length rabbit NK3 receptor cDNA consisting of 1404 nucleotides. This c DNA encoded a protein of 467 amino acids with 91 and 87% homology to the hu man and rat NK3 receptors respectively. 3 In CHO-KI cells transiently expressing the recombinant rabbit NK3 recepto r, the relative order of potency of NKB >> NKA greater than or equal to SP to displace [I-125]-[MePhe(7)]-NKB binding and to increase intracellular ca lcium, together with the high affinity of NK3 selective agonists (e.g. senk tide, [MePhe(7)]-NKB) and antagonists (e.g. SR 142801, SE 223412) in both a ssays was consistent with NK3 receptor pharmacology. In binding and functio nal experiments, agonist concentration response curves were shallow (0.7-0. 8), suggesting the possibility of multiple affinity states of the receptor. 4 Quantitative real time PCR analysis revealed highest expression of rabbit NK3 receptor mRNA in iris sphincter muscle, lower expression in retina and iris dilator muscle, and no expression in lens and cornea. In situ hybridi zation histochemistry revealed discrete specific localization of NK3 recept or mRNA in the iris muscle and associated ciliary processes. Discrete speci fic labelling of NK3 receptors with the selective NK3 receptor agonist [I-1 25]-[MePhe(7)]-NKB was also observed in the ciliary processes using autorad iography. 5 Our study reveals a high molecular similarity between rabbit and human NK 3 receptor mRNAs, as predicted from previous pharmacological studies, and p rovide the first evidence that NK3 receptors are precisely located on cilia ry processes in the rabbit eye. In addition, there could be two affinity st ates of the receptor which may correspond to the typical and 'atypical' NK3 receptor subtypes previously reported.