Hotchkiss Brain Institute, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada.
Alberta Children's Hospital Research Institute, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada.
Invest Ophthalmol Vis Sci. 2018 Jun 1;59(7):2778-2791. doi: 10.1167/iovs.17-22562.
Myopia is a refractive disorder that degrades vision. It can be treated with atropine, a muscarinic acetylcholine receptor (mAChR) antagonist, but the mechanism is unknown. Atropine may block α-adrenoceptors at concentrations ≥0.1 mM, and another potent myopia-inhibiting ligand, mamba toxin-3 (MT3), binds equally well to human mAChR M4 and α1A- and α2A-adrenoceptors. We hypothesized that mAChR antagonists could inhibit myopia via α2A-adrenoceptors, rather than mAChR M4.
Human mAChR M4 (M4), chicken mAChR M4 (cM4), or human α2A-adrenergic receptor (hADRA2A) clones were cotransfected with CRE/promoter-luciferase (CRE-Luc; agonist-induced luminescence) and Renilla luciferase (RLuc; normalizing control) into human cells. Inhibition of normalized agonist-induced luminescence by antagonists (ATR: atropine; MT3; HIM: himbacine; PRZ: pirenzepine; TRP: tropicamide; OXY: oxyphenonium; QNB: 3-quinuclidinyl benzilate; DIC: dicyclomine; MEP: mepenzolate) was measured using the Dual-Glo Luciferase Assay System.
Relative inhibitory potencies of mAChR antagonists at mAChR M4/cM4, from most to least potent, were QNB > OXY ≥ ATR > MEP > HIM > DIC > PRZ > TRP. MT3 was 56× less potent at cM4 than at M4. Relative potencies of mAChR antagonists at hADRA2A, from most to least potent, were MT3 > HIM > ATR > OXY > PRZ > TRP > QNB > MEP; DIC did not antagonize.
Muscarinic antagonists block hADRA2A signaling at concentrations comparable to those used to inhibit chick myopia (≥0.1 mM) in vivo. Relative potencies at hADRA2A, but not M4/cM4, correlate with reported abilities to inhibit chick form-deprivation myopia. mAChR antagonists might inhibit myopia via α2-adrenoceptors, instead of through the mAChR M4/cM4 receptor subtype.
近视是一种降低视力的屈光不正。它可以用毒蕈碱乙酰胆碱受体（mAChR）拮抗剂阿托品治疗，但机制尚不清楚。阿托品在浓度≥0.1mM 时可能会阻断α-肾上腺素能受体，另一种强效的抑制近视的配体曼巴毒素-3（MT3）同样能很好地结合人 mAChR M4 和α1A-和α2A-肾上腺素能受体。我们假设 mAChR 拮抗剂可以通过α2A-肾上腺素能受体抑制近视，而不是 mAChR M4。
将人 mAChR M4（M4）、鸡 mAChR M4（cM4）或人α2A-肾上腺素能受体（hADRA2A）克隆与 CRE/启动子-荧光素酶（CRE-Luc；激动剂诱导的发光）和海肾荧光素酶（RLuc；正常化对照）共转染到人细胞中。用 Dual-Glo 荧光素酶测定系统测量拮抗剂（ATR：阿托品；MT3；HIM：海巴碱；PRZ：哌仑西平；TRP：托吡卡胺；OXY：氧苯丙胺；QNB：3-奎宁环基苯甲酸盐；DIC：二氢环庚甲胺；MEP：甲哌啶嗪）对正常化激动剂诱导的发光的抑制作用。
mAChR 拮抗剂在 M4/cM4 上的相对抑制效力，从最强到最弱，依次为 QNB>OXY≥ATR>MEP>HIM>DIC>PRZ>TRP。MT3 在 cM4 上的效力比在 M4 上低 56 倍。mAChR 拮抗剂在 hADRA2A 上的相对效力，从最强到最弱，依次为 MT3>HIM>ATR>OXY>PRZ>TRP>QNB>MEP；DIC 没有拮抗作用。
毒蕈碱拮抗剂在与体内抑制小鸡近视（≥0.1mM）相当的浓度下阻断 hADRA2A 信号传导。在 hADRA2A 上的相对效力，而不是在 M4/cM4 上的效力，与报告的抑制小鸡形觉剥夺性近视的能力相关。mAChR 拮抗剂可能通过α2-肾上腺素能受体而不是通过 mAChR M4/cM4 受体亚型抑制近视。
