Ndoye A, Buchli R, Greenberg B, Nguyen V T, Zia S, Rodriguez J G, Webber R J, Lawry M A, Grando S A
Department of Dermatology, University of California, Davis, USA.
J Invest Dermatol. 1998 Sep;111(3):410-6. doi: 10.1046/j.1523-1747.1998.00299.x.
Acetylcholine mediates cell-to-cell communications in the skin. Human epidermal keratinocytes respond to acetylcholine via two classes of cell-surface receptors, the nicotinic and the muscarinic cholinergic receptors. High affinity muscarinic acetylcholine receptors (mAChR) have been found on keratinocyte cell surfaces at high density. These receptors mediate effects of muscarinic drugs on keratinocyte viability, proliferation, adhesion, lateral migration, and differentiation. In this study, we investigated the molecular structure of keratinocyte mAChR and their location in human epidermis. Polymerase chain reaction amplification of cDNA sequences uniquely present within the third cytoplasmic loop of each subtype demonstrated the expression of the m1, m3, m4, and m5 mAChR subtypes. To visualize these mAChR, we raised rabbit anti-sera to synthetic peptide analogs of the carboxyl terminal regions of each subtype. The antibodies selectively bound to keratinocyte mAChR subtypes in immunoblotting membranes and epidermis, both of which could be abolished by preincubating the anti-serum with the peptide used for immunization. The immunofluorescent staining patterns produced by each antibody in the epidermis suggested that the profile of keratinocyte mAChR changes during epidermal turnover. The semiquantitative analysis of fluorescence revealed that basal cells predominantly expressed m3, prickle cells had equally high levels of m4 and m5, and granular cells mostly possessed m1. Thus, the results of this study demonstrate for the first time the presence of m1, m3, m4, and m5 mAChR in epidermal keratinocytes. Because keratinocytes express a unique combination of mAChR subtypes at each stage of their development in the epidermis, each receptor may regulate a specific cell function. Hence, a single cytotransmitter, acetylcholine, and muscarinic drugs may exert different biologic effects on keratinocytes at different stages of their maturation.
乙酰胆碱介导皮肤中的细胞间通讯。人类表皮角质形成细胞通过两类细胞表面受体，即烟碱型和毒蕈碱型胆碱能受体，对乙酰胆碱作出反应。已发现高亲和力的毒蕈碱型乙酰胆碱受体（mAChR）以高密度存在于角质形成细胞表面。这些受体介导毒蕈碱类药物对角质形成细胞活力、增殖、黏附、侧向迁移和分化的作用。在本研究中，我们调查了角质形成细胞mAChR的分子结构及其在人表皮中的定位。对每个亚型第三个细胞质环中独特存在的cDNA序列进行聚合酶链反应扩增，证实了m1、m3、m4和m5 mAChR亚型的表达。为了可视化这些mAChR，我们制备了针对每个亚型羧基末端区域合成肽类似物的兔抗血清。这些抗体在免疫印迹膜和表皮中选择性地结合角质形成细胞mAChR亚型，通过将抗血清与用于免疫的肽预孵育，两者的结合均可被消除。每种抗体在表皮中产生的免疫荧光染色模式表明，角质形成细胞mAChR的分布在表皮更替过程中发生变化。荧光的半定量分析显示，基底细胞主要表达m3，棘细胞中m4和m5水平同样较高，颗粒细胞大多含有m1。因此，本研究结果首次证明表皮角质形成细胞中存在m1、m3、m4和m5 mAChR。由于角质形成细胞在表皮发育的每个阶段都表达独特的mAChR亚型组合，每个受体可能调节特定的细胞功能。因此，单一的细胞递质乙酰胆碱和毒蕈碱类药物可能在角质形成细胞成熟的不同阶段发挥不同的生物学作用。
