Grueb Matthias, Mielke Joerg, Bartz-Schmidt Karl Ulrich, Rohrbach Jens Martin
Department of Ophthalmology, University of Tuebingen, Schleichstr. 12, 72076, Tuebingen, Germany.
Graefes Arch Clin Exp Ophthalmol. 2007 Apr;245(4):595-9. doi: 10.1007/s00417-006-0443-y. Epub 2006 Oct 6.
Muscarinic cholinoceptors are distributed widely in both the central and peripheral nervous system. The presence of muscarinic cholinoceptors in corneal tissue is well established. Previous reports have shown that corneal muscarinic cholinoceptors are of the m2 or m4 subtype. However, recent studies have indicated the presence of the m5 muscarinic cholinoceptor subtype in human corneal epithelium and endothelium. The aim of the study was to confirm the presence of the m5 cholinoceptor subtype in bovine corneal epithelium and endothelium and the activation of phosphatidyl inositol pathway by its stimulation.
Muscarinic m5 cholinoceptor sites, phosphatidyl inositol 4,5-biphosphate, inositol 1,4,5-triphosphate and protein kinase C, were studied using immunocytochemistry and immunofluorescence. Activation of protein kinase C after stimulation of the m5 muscarinic cholinoceptor subtype was measured using the HTS protein kinase C assay kit.
Immunocytochemistry/immunofluorescence revealed the presence of the m5 muscarinic cholinoceptor subtype, phosphatidyl inositol 4,5-biphosphate and protein kinase C in bovine corneal epithelial and endothelial cells. In bovine corneal epithelium and endothelium, protein kinase C activity was stimulated by acetylcholine in a dose-dependent manner (P<0.0001).
Our findings indicate that acetylcholine-induced stimulation of muscarinic m5 cholinoceptors activates the phosphatidyl inositol pathway in corneal epithelial and endothelial cells, resulting in increased protein kinase C activity. Further work will be needed to clear the physiologic role of this signaling pathway in corneal epithelium and endothelium.
毒蕈碱型胆碱能受体广泛分布于中枢和外周神经系统。角膜组织中存在毒蕈碱型胆碱能受体已得到充分证实。先前的报道表明，角膜毒蕈碱型胆碱能受体为m2或m4亚型。然而，最近的研究表明，人角膜上皮和内皮中存在m5毒蕈碱型胆碱能受体亚型。本研究的目的是证实牛角膜上皮和内皮中m5胆碱能受体亚型的存在及其刺激对磷脂酰肌醇途径的激活作用。
采用免疫细胞化学和免疫荧光技术研究毒蕈碱型m5胆碱能受体位点、磷脂酰肌醇4,5-二磷酸、肌醇1,4,5-三磷酸和蛋白激酶C。使用HTS蛋白激酶C检测试剂盒测定m5毒蕈碱型胆碱能受体亚型刺激后蛋白激酶C的激活情况。
免疫细胞化学/免疫荧光显示牛角膜上皮和内皮细胞中存在m5毒蕈碱型胆碱能受体亚型、磷脂酰肌醇4,5-二磷酸和蛋白激酶C。在牛角膜上皮和内皮中，乙酰胆碱以剂量依赖方式刺激蛋白激酶C活性（P<0.0001）。
我们的研究结果表明，乙酰胆碱诱导的毒蕈碱型m5胆碱能受体刺激激活了角膜上皮和内皮细胞中的磷脂酰肌醇途径，导致蛋白激酶C活性增加。需要进一步的研究来明确该信号通路在角膜上皮和内皮中的生理作用。
