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Molecular and pharmacological characterization of a functional tachykinin NK 3 receptor cloned from the rabbit iris sphincter muscle
Author(s) -
Medhurst Andrew D,
Hirst Warren D,
Jerman Jeffery C,
Meakin Jacqueline,
Roberts Jennifer C,
Testa Tania,
Smart Darren
Publication year - 1999
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1038/sj.bjp.0702854
Subject(s) - tachykinin receptor , receptor , agonist , biology , microbiology and biotechnology , ciliary muscle , endocrinology , medicine , biochemistry , substance p , neuropeptide , accommodation , neuroscience
A functional tachykinin NK 3 receptor was cloned from the rabbit iris sphincter muscle and its distribution investigated in ocular tissues. Standard polymerase chain reaction (PCR) techniques were used to clone a full length rabbit NK 3 receptor cDNA consisting of 1404 nucleotides. This cDNA encoded a protein of 467 amino acids with 91 and 87% homology to the human and rat NK 3 receptors respectively. In CHO‐K1 cells transiently expressing the recombinant rabbit NK 3 receptor, the relative order of potency of NKB>>NKASP to displace [ 125 I]‐[MePhe 7 ]‐NKB binding and to increase intracellular calcium, together with the high affinity of NK 3 selective agonists (e.g. senktide, [MePhe 7 ]‐NKB) and antagonists (e.g. SR 142801, SB 223412) in both assays was consistent with NK 3 receptor pharmacology. In binding and functional experiments, agonist concentration response curves were shallow (0.7–0.8), suggesting the possibility of multiple affinity states of the receptor. Quantitative real time PCR analysis revealed highest expression of rabbit NK 3 receptor mRNA in iris sphincter muscle, lower expression in retina and iris dilator muscle, and no expression in lens and cornea. In situ hybridization histochemistry revealed discrete specific localization of NK 3 receptor mRNA in the iris muscle and associated ciliary processes. Discrete specific labelling of NK 3 receptors with the selective NK 3 receptor agonist [ 125 I]‐[MePhe 7 ]‐NKB was also observed in the ciliary processes using autoradiography. Our study reveals a high molecular similarity between rabbit and human NK 3 receptor mRNAs, as predicted from previous pharmacological studies, and provide the first evidence that NK 3 receptors are precisely located on ciliary processes in the rabbit eye. In addition, there could be two affinity states of the receptor which may correspond to the typical and ‘atypical’ NK 3 receptor subtypes previously reported.British Journal of Pharmacology (1999) 128 , 627–636; doi: 10.1038/sj.bjp.0702854