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Brittany Carr, Koichiro Mihara, Rithwik Ramachandran, Mahmoud Saifeddine, Neil M Nathanson, William K Stell, Morley D Hollenberg; Myopia-Inhibiting Muscarinic Antagonists Also Block α2A-Adrenoceptor Signaling. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5465.
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Atropine is used to prevent myopia, but its target receptor is unknown. It is a potent muscarinic acetylcholine receptor (mAChR) antagonist, but there is significant evidence that it may inhibit myopia through non-mAChR means (McBrien et al. 2012. OPO). Atropine may act at α-adrenoceptors (Chang et al. 1995. Eur J Pharmacol), and the most potent myopia-inhibiting ligand found to date (MT3) has equally high affinity at mAChR M4, α1A- and α2A-adrenoceptors (Näreoja et al. 2011. BJP). We hypothesized that atropine and other myopia-inhibiting mAChRs work through α2A-adrenoceptors.
Human M4 (M4), chicken M4 (cM4), or human α2A-adrenoceptor (hADRA2A) clones were transiently co-transfected with cAMP response element luciferase vector (CRE-Luc) and constitutively active Renilla luciferase vector (RLuc) into HEK293T cells. The ability of increasing concentrations of antagonist (AT: atropine, MT3, HM: himbacine, PZ: pirenzepine, TP: tropicamide, OX: oxyphenonium, QNB; DC: dicyclomine & MP: mepenzolate) to inhibit agonist-induced CRE-Luc expression was measured using the Dual-Glo® Luciferase Assay System (Promega). Normalized data were graphed as curve-fitted log dose-responses and pIC50 values were obtained from nonlinear regression analysis.
There were no significant functional differences for antagonists at M4 compared to cM4with theexception of MT3 (pIC50 = 8.08/6.35, p<0.0001, Fig. 1); relative potencies were: QNB (9.94/9.51) > AT (9.41/9.15) ≤ OX (9.40/9.25) > MP (8.85/8.45) > HM (7.98/8.25) > DC (7.82/7.39) > PZ (7.63/7.31) > TP (6.81/6.61). At hADRA2A, relative potencies were: MT3 (7.81) > HM (4.78) > AT (4.34) > QNB (3.58) > OX (3.33) > MP (3.10) > PZ (3.062) > TP (2.83) > DC (no effect).
Here, we confirm reports that MT3 is a high affinity antagonist at hADRA2A (Fig. 2) and mAChR M4, and show that other mAChR antagonists have functional effects at hADRA2A. pIC50 data for these ligands at hADRA2A, but not M4/cM4, correlate well with their reported abilities to inhibit myopia in the chick (Luft et al. 2003. IOVS). Doses ≥ 10 mM are required for myopia inhibition by mAChR antagonists; thus, low potency of most mAChR antagonists at hADRA2A corroborates the hypothesis that hADRA2A may contribute to regulation of eye growth. While there are no species-differences in the affinities of orthosteric ligands, there is a significant difference in the affinity of MT3 at M4 compared to cM4.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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