Analysis of intracellular second messenger systems in ciliary epithelium.

睫状上皮是产生房水的部位。在这项研究中，我们研究了睫状非色素上皮细胞中的钙第二信使系统。荷兰兔用于实验。从眼睛中提取睫状体，并通过将组织在低钙培养基中孵育短时间来分离睫状体非色素上皮层。将细胞在含有 5 μM fura-2/AM 的溶液中于 37℃ 孵育 50 分钟。使用ARGUS-20/CA系统（Hamamatsu photonics）和倒置显微镜（TE300，Nikon）观察细胞内游离钙离子（[CaiD12 D1]D2iD2）的浓度。 10 μM 可乐定、100 μM 乙酰胆碱、10 μM 去氧肾上腺素和 1 μM 肾上腺素诱导 [CaiD12 D1]D2iD2 增加。 100 μM 异丙肾上腺素不会导致 [CaiD12 D1]D2iD2 增加。应用 1 μM 乙酰胆碱后添加 100 μM 可乐定诱导 [CaiD12 D1]D2iD2 强烈增加。添加 10 μM 去氧肾上腺素后添加 100 μM 异丙肾上腺素不会引起细胞内钙离子反应。在用 10 μM 去氧肾上腺素刺激之前应用 1 mM 辛醇不会改变细胞的反应。由此证明，毒蕈碱激动剂与α2-肾上腺素激动剂对细胞内钙离子的动员之间存在相互作用，而α1-肾上腺素激动剂与β-肾上腺素激动剂之间未观察到相互作用，表明间隙连接在钙离子第二信使系统的信号转导中可能不起主要作用。

Ciliary epithelium is the site producing aqueous humor. In this study, we investigated the calcium second messenger system in the ciliary nonpigment epithelial cells. Dutch rabbit were used for the experiments. Ciliary body was extracted from the eye and ciliary nonpigmented epithelial layer was isolated by incubating the tissue in low calcium medium for a short period. Cells were incubated at 37℃ for 50 minutes in solution containing 5 μM fura-2/AM. The concentration of the intracellular free calcium ion ([CaィイD12+ィエD1]ィイD2iィエD2) was observed with an ARGUS-20/CA system (Hamamatsu photonics) and an inverted microscope (TE300, Nikon). Ten μM clonidine, 100 μM acetylcholine, 10 μM phenylephrine, and 1 μM epinephrine induced the increase of [CaィイD12+ィエD1]ィイD2iィエD2. One hundred μM isoproterenol caused no increase of [CaィイD12+ィエD1]ィイD2iィエD2. Addition of 100 μM clonidine after the application of 1 μM acetylcholine induced strong increase of [CaィイD12+ィエD1]ィイD2iィエD2. Addition of 100 μM isoproterenol after the 10 μM phenylephrine caused no intracellular calcium ion response. The application of 1 mM octanol before the stimulation by 10 μM phenylephirine did not change the response of the cells. Thus, it was proved that there was an interaction between the intracelluar calcium ion mobilization by muscarinic agonists and alpha2-adrenergic agonists while the interaction between alpha1-adrenergic agonists and beta-adrenergic agonists could not be observed and it is suggested that the gap junction may not play a major role in signal transduction of calcium ion second messenger system.