Molecular piolcgical analysises of muscarinic receptore in the rat iris dilator

外源性乙酰胆碱（ACh）激活毒蕈碱受体在大鼠虹膜扩张平滑肌中引起独特的反应；低剂量松弛，高剂量收缩(1)。毒蕈碱拮抗剂对扩张肌舒张的pA_2值与m_3型毒蕈碱受体的pA_2值最为相似，可能是由m_3样受体介导的。研究了百日咳毒素（PTX）对激动剂诱导的大鼠虹膜扩张器收缩和/或舒张的影响。前房注射PTX只影响疼痛引起的松弛。注射PTX后松弛完全消失。然而，PTX预处理对去甲肾上腺素或5-羟色胺诱导的扩张肌收缩和异丙肾上腺素诱导的舒张没有显著影响(2)。因此，百日咳毒素敏感GTP结合蛋白仅介导乙酰胆碱双相反应的松弛。为分离大鼠虹膜中表达的毒毒碱受体编码基因，采用RT-PCR方法，以大鼠眼总RNA为模板，根据大鼠心脏m2受体和大鼠脑m3受体cDNA序列设计特异性引物。因此，m2受体特异性引物(3)的扩增产物为1.4k bps， m3受体特异性引物(4)的扩增产物为1.8k bps。每个PCR产物亚克隆到质粒载体上，分析每个核苷酸序列。从大鼠眼睛中获得的m2受体cDNA蛋白序列与报道的大鼠心脏m2受体cDNA蛋白序列存在9个氨基酸残基的差异。从大鼠眼中获得的m3受体cDAN蛋白序列与报道的大鼠脑m3受体cDNA有5个氨基酸残基的差异。为了确认m2和m3受体分子亚型在大鼠虹膜组织周围的表达，我们使用特异性的PCR引物靶向第三细胞内环短片段（350-450 bps）进行了rt - pcr_s4。RT-PCR结果证实，m2和m3受体mrna在大鼠虹膜组织周围表达。此外，m1和m5受体mRNA未大量表达。少

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