Abstract
Abstract: :
Purpose:Several small proteins called muscarinic toxins (MTs) have been isolated from venom of the green mamba. MTs recognize muscarinic receptors and MT7 is the most selective ligand for m1 muscarinic receptor. We have investigated the effect of MTs, compared them with conventional antagonists and confirmed the presence of the m1 receptor on mouse and human scleral fibroblasts (SFs). Methods:Experimental myopia was induced in 30 mice by 8 weeks of lid–suture on right eyes (OD) and left eyes (OS) were used controls. Scleras of mice and scleral rims from donor human corneas were cultured and SFs used at passage 2–4. Proliferation studies using BrdU were carried out with atropine and pirenzepine at 0.0, 0.1, 1, 10, 100 µ M, and MT7 at 0.0, 0.5, 1, 2, 4 µ M. Agonists MT1 and carbachol were used at 0.0, 0.5, 1, 2, 4 µ M, and 0.0, 0.1, 1, 10, 100 µ M respectively. Presence of the m1 receptor was confirmed by immunostaining, western blots, (m1 antibody from Biogenesis) and RT–PCR for the m1 receptor. Results:The m1 muscarinic receptor was detected in mouse and human SFs by immunostaining and western blots. RT–PCR confirmed the presence of the message for the m1 receptor in mouse and human SFs. Pirenzepine and MT7, selective m1 muscarinic receptor antagonists inhibited proliferation of SFs in a dose dependent manner (p<0.05, n=4, ANOVA). Atropine, a general muscarinic receptor antagonist, also inhibited proliferation of SFs in a dose dependent manner (p<0.05, n=4, ANOVA). At a concentration of 1µ M, MT7 was more inhibitory than either atropine or pirenzepine (0.73, 0.38, 0.24). In contrast, MT1, a reversible m1 muscarinic receptor agonist, and carbachol increased cell proliferation in a dose dependent manner (p<0.05, n=4, ANOVA); however, MT1 was more stimulatory than carbachol at 1µ M (0.24 to 0.13). Conclusions:Our results show that the mAChRs m1 subtype exists in mouse and human SFs, and that m1 receptor participates in the control of cell proliferation in mouse and human SFs.
Keywords: sclera • myopia