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

• Published in: British journal of pharmacology Vol. 128; no. 3; pp. 627 - 636
• Main Authors: Medhurst, Andrew D, Hirst, Warren D, Jerman, Jeffery C, Meakin, Jacqueline, Roberts, Jennifer C, Testa, Tania, Smart, Darren
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01-10-1999; Nature Publishing
• Subjects: Amino Acid Sequence; Animals; Autoradiography; Base Sequence; Biological and medical sciences; CHO Cells; ciliary processes; cloned receptor; Cloning, Molecular; Cricetinae; Fundamental and applied biological sciences. Psychology; Humans; Iris - metabolism; Molecular Sequence Data; Muscle, Smooth - metabolism; rabbit iris sphincter muscle; Rabbits; Radioligand Assay; Rats; Receptors, Neurokinin-3 - drug effects; Receptors, Neurokinin-3 - genetics; Receptors, Neurokinin-3 - physiology; RNA, Messenger - genetics; Sequence Homology, Amino Acid; Tachykinin NK3 receptors; Vertebrates: skin, associated glands, phaneres, light organs, various exocrine glands (salt gland, uropygial gland...), adipose tissue, connective tissue; Rabbit; Tissue specificity; Smooth muscle; Lagomorpha; In vitro; Eye; Polymerase chain reaction; Vertebrata; Mammalia; Structure activity relation; Pupillary sphincter; Animal; Distribution; Established cell line; NK3 Tachykinin receptor; Recombinant protein
• ISSN: 0007-1188; 1476-5381
• DOI: 10.1038/sj.bjp.0702854

A functional tachykinin NK3 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 NK3 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 NK3 receptors respectively.
In CHO‐K1 cells transiently expressing the recombinant rabbit NK3 receptor, the relative order of potency of NKB>>NKASP to displace [125I]‐[MePhe7]‐NKB binding and to increase intracellular calcium, together with the high affinity of NK3 selective agonists (e.g. senktide, [MePhe7]‐NKB) and antagonists (e.g. SR 142801, SB 223412) in both assays was consistent with NK3 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 NK3 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 NK3 receptor mRNA in the iris muscle and associated ciliary processes. Discrete specific labelling of NK3 receptors with the selective NK3 receptor agonist [125I]‐[MePhe7]‐NKB was also observed in the ciliary processes using autoradiography.
Our study reveals a high molecular similarity between rabbit and human NK3 receptor mRNAs, as predicted from previous pharmacological studies, and provide the first evidence that NK3 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’ NK3 receptor subtypes previously reported.
British Journal of Pharmacology (1999) 128, 627–636; doi:10.1038/sj.bjp.0702854