Pharmacological profile of non-canonical mGluR regulating mechanosensory nerve terminal firing.

Watson, S.; Zanato, C.; Dall'Angello, S.; Banks, R.W.; Greig, I.; Zanda, M.; Bewick, G.S.

Abstract

The system involved in 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). To further investigate the receptor's pharmacology, ligands selective for classical mGluRs were screened for their ability to alter stretch-evoked spindle firing. In addition, due to kainate's potency and its ease of chemical modification, novel kainate-derived compounds were tested to find more potent analogues suitable for "click chemistry" (Kolb et al, 2001). 4th lumbrical nerve-muscle preparations were excised from humanely killed (Schedule 1, ASPA, 1986) adult male Sprague-Dawley rats (305-488 g). Spindle discharges were recorded en passant from the muscle nerve during 1 mm stretch-and-hold cycles. The stretch-evoked firing in compounds applied for 60 min was compared to that in saline alone, with differences in mean firing frequencies (impulses/sec) evaluated by ANOVA with Bonferroni post test (significance threshold P = 0.05). Glutamate (1 mM) increased afferent firing by 52.3 ± 13.0% (mean ± S.E.M; n = 10; P<0.01). PCCG-13 (10 µM), a selective PLD-coupled mGluR antagonist, decreased firing by 34.6 ± 4.4% (n = 6; P < 0.01). These confirmed previous results and served as a comparison with new compounds. Quisqualate and kainate were more potent agonists, with 10 µM increasing firing by 56.3 ± 11.9% (n = 7; P < 0.001) and 29.4 ± 7.2% (n = 7; P < 0.05) respectively. Novel kainate analogues ZCZ49 and ZCZ50 produced no significant change in afferent firing. However, ZCZ90 was more potent than kainate, increasing firing by 34.3 ± 6.5% (n = 8; P<0.001) at 1 µM and by 42.0 ± 7.1% (n = 8; P<0.001) at 10 µM. Many compounds with activities at cloned mGluRs had no significant effect on afferent firing (all P > 0.05 at 10 µM); ibotenate (n = 5), DCG-IV (n = 6), ACPD (n = 8), L-AP4 (n = 8) and, importantly, 100 µM LY341495 (n = 13), a concentration that blocks all cloned mGluRs (Kingston et al, 1998). Furthermore, while 200 µM S-DHPG (n = 5) had no effect on firing, 1 µM R-DHPG decreased firing by 20.9 ± 3.7% (n = 7; P < 0.05). These data are further evidence mechanosensory terminals contain an atypical mGluR, with a ligand-activity profile distinct from cloned mGluRs but potently antagonised by PCCG-13, the specific antagonist of PLD-coupled mGluR (Albani-Torregrossa et al, 1999). Most interestingly, quisqualate is the most potent agonist, LY341495 (antagonist of all cloned mGluRs) has no effect and R-DHPG (c.f. the group I agonist, S-DHPG) is an antagonist. ZCZ90, our novel kainate analogue, is amenable to the addition of fluorescent and crosslinker tags for use in receptor visualisation and "pull down assays" in future studies.

Citation

Watson, S., Zanato, C., Dall'Angello, S., Banks, R., Greig, I., Zanda, M., & Bewick, G. (2012). Pharmacological profile of non-canonical mGluR regulating mechanosensory nerve terminal firing.

• Presentation Conference Type: Conference Paper (Published)
• Conference Name: Physiology 2012
• Publication Date: 2012
• Deposit Date: Jan 5, 2015
• Volume: 27
• Series Title: Proceedings of the Pjysiological Society
• Public URL: https://durham-repository.worktribe.com/output/1153695
• Publisher URL: http://www.physoc.org/proceedings/abstract/Proc%20Physiol%20Soc%2027PC46