Wall S J, Yasuda R P, Li M, Wolfe B B
Department of Pharmacology, Georgetown University School of Medicine, Washington, D. C. 20007.
Mol Pharmacol. 1991 Nov;40(5):783-9.
A synthetic oligopeptide (QCDKRKRRKQQYQQRQSV) corresponding to a carboxyl-terminal sequence of the rat m3 receptor (amino acids 561-578) was coupled to carrier proteins and used to generate a polyclonal antiserum. This serum selectively immunoprecipitates at least 90% of the m3 receptors expressed by A9 cells transfected with the cDNA encoding the m3 muscarinic receptor but does not precipitate receptors from cells transfected with cDNA encoding m1, m2, m4, or m5 receptors. Using this m3 antiserum, the density of m3 receptors in various regions of rat brain was quantified. Areas expressing the highest density of m3 receptors are the cortex, hippocampus, striatum, and olfactory tubercle, with 232 fmol/mg, 197 fmol/mg, 140 fmol/mg, and 130 fmol/mg, respectively. Hindbrain regions (i.e., cerebellum, thalamus/hypothalamus, and pons/medulla) expressed fewer m3 receptors, both as a percentage of total muscarinic receptors (5-6%) and in terms of absolute receptor density (12-70 fmol/mg). A panel of subtype-selective antisera (m1, m2, and m3) was used to determine receptor distribution in several peripheral tissues of the rat (lung, ileum, and bladder). The m2 receptor subtype constitutes the majority of total receptors in the bladder (86%), lung (91%), and ileum (69%). The m3 receptor was found at lower densities in these tissues (5-11%), whereas the m1 receptor is present in highest amounts in the ileum (17%). Human clonal cell lines, in which regulation of muscarinic receptors has been commonly studied, were also examined. The SK-N-SH neuroblastoma line, which has been reported to express M3 receptors, on the basis of pharmacology and molecular size, was found to express a mixture of subtypes (m1 = 31%, m2 = 21%, m3 = 43%). Interestingly, SH-SY-5Y and SH-IN cells, both derived from SK-N-SH cells, exhibit predominantly m3 receptors (74% for SH-SY-5Y; 58% for SH-IN), with lower levels of m1 and m2 receptors (5% and 8% for SH-SY-5Y; 4% and 23% for SH-IN, respectively.) Another commonly studied cell line, 132-1-N1 astrocytoma cells, reportedly expressing M3 receptors, based upon mRNA measurements and second messenger linkage, also expresses a predominance of m3 receptors (91% of total). This m3-selective antiserum should prove useful not only for localizing and quantifying m3 muscarinic receptors but also for examining mechanisms involved in the regulation of receptor expression in human tissues or animal models of disease, as well as in cell culture.
一种与大鼠m3受体羧基末端序列（氨基酸561 - 578）相对应的合成寡肽（QCDKRKRRKQQYQQRQSV）与载体蛋白偶联，并用于产生多克隆抗血清。该血清能选择性免疫沉淀至少90%由转染了编码m3毒蕈碱受体cDNA的A9细胞所表达的m3受体，但不会沉淀转染了编码m1、m2、m4或m5受体cDNA的细胞中的受体。使用这种m3抗血清，对大鼠脑不同区域的m3受体密度进行了定量。表达m3受体密度最高的区域是皮质、海马体、纹状体和嗅结节，分别为232 fmol/mg、197 fmol/mg、140 fmol/mg和130 fmol/mg。后脑区域（即小脑、丘脑/下丘脑和脑桥/延髓）表达的m3受体较少，无论是占总毒蕈碱受体的百分比（5 - 6%）还是绝对受体密度（12 - 70 fmol/mg）。一组亚型选择性抗血清（m1、m2和m3）用于确定大鼠几种外周组织（肺、回肠和膀胱）中的受体分布。m2受体亚型占膀胱（86%）、肺（91%）和回肠（69%）中总受体的大部分。在这些组织中发现m3受体密度较低（5 - 11%），而m1受体在回肠中含量最高（17%）。人们通常研究毒蕈碱受体调节的人克隆细胞系也进行了检测。据报道，基于药理学和分子大小，SK - N - SH神经母细胞瘤系表达M3受体，结果发现它表达多种亚型的混合物（m1 = 31%，m2 = 21%，m3 = 43%）。有趣的是，均源自SK - N - SH细胞的SH - SY - 5Y和SH - IN细胞主要表达m3受体（SH - SY - 5Y为74%；SH - IN为58%），m1和m2受体水平较低（SH - SY - 5Y分别为5%和8%；SH - IN分别为4%和23%）。另一种通常研究的细胞系132 - 1 - N1星形细胞瘤细胞，据报道基于mRNA测量和第二信使联系表达M3受体，也主要表达m3受体（占总数的91%）。这种m3选择性抗血清不仅应被证明对定位和定量m3毒蕈碱受体有用，而且对研究人类组织或疾病动物模型以及细胞培养中受体表达调节所涉及的机制也有用。
