Analysis of intracellular second messenger systems in ciliary epithelium.

睫状上皮细胞内第二信使系统的分析。

• 批准号: 09470377
• 负责人: ARAIE Makoto
• 金额: $ 8.06万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09470377/
• 关键词: ciliary nonpigmented epithelium; receptor; calcium ion; alpha1-adrenergic receptor; gap junction; alpha2-adrenergic; interaction; muscarinic receptor; アドレナリン受容体; 細胞内カルシウムイオン; 細胞内情報伝達; 家兎; アドレナリン作動薬; コリン作動薬

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.

睫状体上皮是产生房水的部位。在本研究中，我们研究了睫状神经非色素上皮细胞中的钙第二信使系统。以荷兰兔为实验对象。从眼内取出睫状体，在低钙介质中短期孵育，分离出睫状体无色素上皮层。细胞在37℃的含5μM Fura-2/AM的溶液中孵育50分钟。用Argus-20/CA系统(滨松光电子)和倒置显微镜(TE300、尼康)观察细胞内游离钙离子浓度([CaィイD12+ィエD1]ィイD2iィエD2)。10μM可乐定、10 0μM乙酰胆碱、10μM苯肾上腺素和1μM肾上腺素使[CaィイD12+ィエD1]ィイD2iィエD2升高。100μM异丙肾上腺素对[CaィイD12+ィエD1]ィイD2iィエD2无明显影响。在应用1μM乙酰胆碱后加入10 0μM可乐定可引起[CaィイD12+ィエD1]ィイD2iィエD2的显著升高。在10μM苯肾上腺素后加入100μM异丙肾上腺素，不引起细胞内钙离子反应。在10μM苯丙氨酸刺激前加1 mM辛醇不改变细胞的反应。结果表明，M受体激动剂与α2-肾上腺素能激动剂之间存在相互作用，而α1-肾上腺素能激动剂与β-肾上腺素能激动剂之间没有相互作用，提示缝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。