Molecular piolcgical analysises of muscarinic receptore in the rat iris dilator

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The activation of muscarinic receptors by exogenously applied acetylcholine (ACh) elicits unique responses in rat iris dilator smooth muscle ; relaxation at low doses and contraction at high doses (1). The pA_2 values of muscarinic antagonists for antagonism to relaxation in dilator muscles were most similar to those for M_3-type muscarinic receptors and contraction might be mediated by M_3-like receptor. The effects of Pertussis toxin (PTX) on contraction and/or relaxation induced by agonists were examined in the rat iris dilator. Only the ACh-induced relaxation was affected by injection of PTX into the anterior eye chamber. Relaxation had completely disappeared after injection of PTX.However, Preteatment with PTX did not significantly affect contraction induced by norepinephrine or 5-hydroxytryptamine or the relaxation induced by isoprenaline in dilator muscles (2). Therefore, only relaxation of the biphasic response to ACh was mediated by pertussis toxin sensitive GTP binding protei … More n in rat iris dilator. To isolate the gene coding muscarinic receptor expressed in rat iris, RT-PCR was carried out using total RNA of the rat eye as the template and specific primers which were designed based on the sequence of cDNA for the rat cardiac m2 receptor and cDNA for the rat brain m3 receptor.Consequently, PCR products amplified were 1.4k bps for m2 receptor specific primers (3) and 1.8k bps m3 receptor specific primers (4). Each PCR product was subcloned into plasmid vector, and each nucleotide sequence was abalyzed. The protein sequence of m2 receptor cDNA obtained from rat eye differed from that reported for rat cardiac m2 receptor cDNA by 9 amino acid residues. The protein sequence of m3 receptor cDAN obtained from rat eye differed from that reported for the rat brain m3 receptor cDNA by 5 amino acid residues. To confirm the expression of the m2 and m3 receptor molecular subtype around the tissue of rat iris, RT-PCR_Swere performed using specific PCR primers targeted short fragment (350-450 bps) of third intracellular loop. From the results of RT-PCR,it was comfirmed that m2 and m3 receptor mRNAs expressed around the tissue of rat iris. Furthermore, m1 and , m5 receptor mRNA were not expressed substantialy. Less