The Presence of m1 to m5 Receptors in Human Sclera: Evidence of the Sclera as a Potential Site of Action for Muscarinic Receptor Antagonists

Abstract

PURPOSE: The aim of this study was to identify the presence of muscarinic acetylcholine receptors (mAChRs) in human sclera in order to determine whether the sclera is a potential site of action for mAChR antagonists. METHODS: Cell lines of human scleral fibroblasts were cultured in Dulbecco modified Ealge's medium. Reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot analysis were used to detect mRNA expression of muscarinic acetylcholine receptors in the cell lines of the fibroblasts. Western blot analysis and immunocytochemistry were used to detect proteins of mAChRs in the cell lines. Immunohistochemical study was used to further detect the presence of mAChR proteins in the frozen scleral sections. RESULTS: The cultured fibroblasts demonstrated mRNA expression of five mAChRs (m1 to m5) in RT-PCR and Northern blot analysis. The molecular size of mRNA expression was largest for the m3 receptor, followed by the m2, m4, m5, and m1 in both RT-PCR and Northern blot analysis. Proteins of the m1 to m5 receptors were present in cell line fibroblasts under Western blot analysis and immunocytochemistry with a range of molecular weight from 80 kDa (m5) to 60 kDa (m1) in Western blot analysis. The presence of these five receptors was also detected in scleral tissues with immunohistochemistry. CONCLUSIONS: This study demonstrated the presence of mAChR subtypes (m1 to m5) in human scleral fibroblasts at both mRNA and protein levels. This finding indicates that the sclera is a potential site of action for the currently used mAChR antagonists in prevention of human myopia.