Presence of M1 to M5 Acetylcholine Receptors in Human Sclera: An Evidence of Local Mechanism in Control of Axial Myopia

Abstract

Abstract: : Purpose: This study was to demonstrate the presence of mAChR subtypes in human scleral fibroblasts in order to understand the mechanisms associated with muscarinic receptor antagonists in control of axial myopia. Methods: Cell lines of scleral fibroblasts were cultured in DMEM and mRNA expression of mAChR subtypes from the cell lines was identified with RT–PCR and Northern blot analysis. Western blot analysis and indirect immunofluorescene (IIF) were used to detect proteins of mAChRs from the cell lines and immunohistochemical study on frozen scleral sections was used to further confirm the existence of mAChRs proteins. Results: The mRNA expression of m1–m5 mAChRs from the cell lines was demonstrated by RT–PCR and Northern blot analysis. Proteins of m1–m5 were confirmed by Western blot, indirect immunofluorescene and immunohistochemical studies. In RT–PCR and Northern Blot analysis, the amount of m1 and m3 was higher than that of m2, m4 and m5. In Western blot, the molecular weight of the subtype receptors was 60kDa for m1, 65kDa for m2, 70kDa for m3, 72kDa for m4 and 80kDa for m5 and the main subtypes appeared to be m1, m3 and m4. Conclusions: This study demonstrated a profile of mAChR subtypes (m1 to m5) present in human sclera. This result provides an evidence of the local mechanisms involved in myopic development at molecular level and the possible acting site of currently used mAChR antagonists in prevention of myopia in humans.

Keywords: myopia • sclera • acetylcholine