Abstract
Abstract: :
Purpose: Previous studies have shown that pirenzepine, an M1 selective muscarinic antagonist, is effective in the inhibition of myopia development in both chicks and mammalian species. Despite this, there is currently no report of the M1 receptor protein or mRNA in the chick although the molecular properties of mammalian M1 receptor and all other known muscarinic receptor subtypes in chicks have been well characterised. The purpose of this study was to investigate the presence of the M1 mRNA and/or gene in chick tissues. Methods: Messenger RNA and genomic DNA were extracted from the following chick tissues: blood, retina-choroid complex, iris-ciliary body complex and brain. Appropriate rat tissues were used as controls. Two sets of primers were designed to target regions of M1 sequence where there is high inter-species homology. Regions of likely cross muscarinic receptor subtype homology in chicks were avoided. RT-PCR and Northern analysis were used to look for M1 mRNA expression. Southern analysis was used to validate PCR results with radiolabelled rat M1 sequences used as probes. PCR, Southern analysis and sequencing were used to investigate the presence of the M1 gene in chick. Results: No specific PCR fragments were amplified from the chick tissues, and Northern hybridisation did not detect M1 mRNA expression. Although PCR fragments were amplified from chick genomic DNA, none were found to be related to the M1 receptor gene. No signal was detected in Southern blotting and hybridisation, supporting the PCR findings. In all cases the M1 sequence was present in rat controls. Conclusions: While it is possible that the chick M1 sequence is markedly degenerate, we suggest that these results show there is no expression of the M1 receptor in the chick, and that this is due to the fact that the chick does not carry an M1 gene. These data show that pirenzepine cannot mediate its inhibitory effect on myopia in chick through an M1 mechanism.
Keywords: myopia • gene/expression • retina