Molecular analysis of muscarinic receptors and regulation mechanism of dilation in the rat iris.

用RT-PCR方法半定量测定大鼠虹膜中毒蕈碱型乙酰胆碱受体亚型m1、m2、m3、m4和m5的mRNA含量。与其他平滑肌组织一样，m2和m3在大鼠虹膜中的mRNA表达水平高于m1和m5。m4 mRNA高表达是大鼠虹膜的特征。此外，利用RT-PCR方法克隆了大鼠虹膜中编码m2、m3和m4亚型的cDNA。大鼠虹膜m2亚型与心脏m2亚型的氨基酸序列有9处差异，与基因组m2亚型的氨基酸序列100%相同。为了研究副交感神经切除诱导的大鼠虹膜平滑肌效应的机制，测定了睫状神经节切除大鼠虹膜中毒蕈碱受体亚型的mRNA表达水平。结果表明，副交感神经切除对mRNA水平无明显影响。实验还表明，在静息状态下，大鼠虹膜扩张肌的胞内Ca^<2 +>浓度（[Ca^<2+]_i）高于虹膜括约肌和许多其他没有固有张力的平滑肌。由于毒蕈碱受体的兴奋可导致[Ca^<2+>] i的减少，因此，毒蕈碱舒张可能是由于[Ca^<2+>] i的减少。在去神经的虹膜扩张肌中，刺激M受体既不引起[Ca^<2+>] i的降低，也不引起舒张。

The amount of mRNAs of muscarinic acetylcholine receptor subtypes, m1, m2, m3, m4 and m5, in the rat iris was determined quasi-quantitatively using RT-PCR methods. The mRNA expression levels of m2 and m3 were higher than those of m1 and m5 in the rat iris as in other smooth muscle tissues. It was characteristic in the rat iris that the m4 mRNA expression level was high. In addition, the cloning of cDNAs encoding m2, m3 and m4 subtypes in the rat iris was performed using RT-PCR methods. Amino acids sequence of m2 subtype in the rat iris included nine differences in comparison with that in the heart, whereas it was 100% identical to genomic one. To examine the mechanisms underlying the parasympathectomy-induced effects in the rat iris smooth muscles, the mRNA expression levels of muscarinic receptor subtypes were determined in iris from ciliaryganglionectomized rats. The results suggested that the parasympathectomy does not markedly affect the mRNA levels. It was also clarified that the intracellular Ca^<2+> concentration ([Ca^<2+>]_i) in the rat iris dilator under the resting conditions is higher than those in the iris sphincter and many other smooth muscles which do not have an inherent tone. Since the muscarinic receptor stimulation resulted in the decrease in [Ca^<2+>]_i in the rat iris dilator, the muscarinic relaxation appeared to be due to the decrease in [Ca^<2+>]_i. In denervated iris dilators, muscarinic receptor stimulation induced neither the decrease in [Ca^<2+>]_i nor the relaxation.