Development of fluorescent and biotinylated agonists for a novel glutamate receptor in mechanosensory terminals.

Mechanotransduction by proprioceptive sensory organs, such as muscle spindles, is poorly understood. We recently reported that stretch releases glutamate from synaptic-like vesicles within spindle terminals, and activates a non-canonical mGluR (Bewick et al, 2005) coupled to phospholipase D (PLD), modulating afferent firing. In further investigations into this receptor's pharmacology, ligands selective for classical mGluRs were screened for their ability to alter stretch-evoked spindle firing. We now report on the development of a fluorescent and a biotinylated agonist. These agonists will be in the key in the next stages of the investigation, isolating the receptor using a pull-down/precipitation assays. Nerve-muscle preparations were excised from humanely killed (Schedule 1, ASPA, 1986) adult male Sprague-Dawley rats. Spindle discharges during 1mm stretch-and-hold cycles were compared with and without drug. Differences in mean firing frequencies were evaluated by two-way ANOVA with Bonferroni post test (sig. thresh- old P=0.05). Four compounds were found to increase afferent firing; kainate (1 µM, n = 7, P = 0.01) quisqualate (1 µM, n = 8, P = 0.05), t-ADA (1 µM, n = 9, P = 0.05) and L-CSA (0.1 µM, n = 7, P = 0.05). NBQX, a kainate and AMPA receptor antagonist, had no effect on afferent firing when applied alone (n=4) or in combination with kainate (n = 7), adding further evidence that classical kainate or AMPA ionotropic receptors are not responsible for the increase in stretch-induced afferent firing with application of kainate. In addition PCCG-13 was able to attenuate the effect of kainate (n = 4) suggesting kainate is acting on the PLD-coupled mGluR. Kainate was used as the parent compound in the synthesis of click-chemistry amenable (Kolb et al, 2001) analogues; ZCZ49, ZCZ50 and ZCZ90. Only ZCZ90 retained activity in the system, increasing afferent firing at 1 µM (n = 7, P = 0.01). From this fluorescent (ZCZ172) and biotinylated (ZCZ180) versions were synthesised. Both retained activity, increasing afferent firing at 10 µM (n = 8, P = 0.001) and 1 µM (n = 8, P = 0.01) respectively. These data show kainate is a useful base ligand for the development of tools to isolate and further characterise this intriguing receptor. [ABSTRACT FROM AUTHOR]