Molecular-biological approach to the regulatory mechanism of ciliary muscle contraction

睫状肌收缩调节机制的分子生物学方法

• 批准号: 13470365
• 负责人: TAKAI Akira
• 金额: $ 8.51万
• 依托单位: Asahikawa Medical College (2002)Nagoya University (2001)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13470365/
• 关键词: Visual accommodation; Intraocular pressure; Ciliary muscle; Aqueous humor outflow; Whole-cell voltage clamp; trp channel; Molecular biology; Recombinant DNA

In the ciliary muscle, the tonic contraction requires a sustained influx of Ca^<2+> through the cell membrane. Very little has hitherto been known about the route(s) of Ca^<2+> influx in this tissue that lacks voltage-gated Ca^<2+> channels. To identify ion channels as the Ca^<2+> entry pathway we investigated effects of carbachol (CCh) on freshly isolated bovine ciliary muscle cells by whole-cell voltage clamp. We have also examined the expression of the trp channel gene in this smooth muscle tissue by RT-PCR using several sense and anti-sense constructs for human and murine trp's spanning 100-130 amino acid segments which almost entirely cover the putative pore forming region of each trp. The major results obtained are summarized as follows:a. Experiments were carried out at 30℃ using pipettes filled with K^+-free solution containing 100 mM Cs aspartate, 5 mM-BAPTA ([Ca^<2+>]_i=70 nM) and 200 μM-GTP (pH 7.0). CCh evoked an inward current showing polarity reversal at holding potential … More near 0 mV. Analysis of the current noise distinguished two types of non-selective cation channel (NSCC_L, and NSCC_S) with widely different unitary conductances (35 pS and 100 fS). The ratios of the permeabilities to Li^+, Na^+, Cs^+, Mg^<2+>, Ca^<2+>, Sr^<2+> and Ba^<2+>, estimated by total ion replacement procedures, were 0.9 : 1.0 : 1.5 : 0.2 : 0.3 : 0.4 : 0.5 for NSCC_L and 1.0 : 1.0 : 1.8 : 2.5 : 2.6 : 3.2 : 5.0 for NSCC_S.b. Both NSCC_L and NSCC_S were dose-dependently inhibited by 1-100 μM of La^<3+>, Gd^<3+> and SKF96365, which also inhibited the tonic component of the contraction produced in muscle bundles by CCh without markedly affecting the phasic component.c. Experiments with in-situ membrane patches using pipettes filled with PSS identified a carbachol-activated channel with very similar γ(31±1 pS) and τ(10±1 ms; n=8).d. Replacement of GTP in the pipette solution with GTPγS gradually caused a spontaneous opening of the channel in the absence of CCh. The response to CCh was irreversibly inhibited (rather than augmented) by bath application of 1 μM-thapsigargin.e. The results of RT-PCR indicated that the smooth muscle of the bovine ciliary body expresses a relatively high levels of trp's very similar to human trp3 and trp6, which are thought to construct non-selective cation channels controlled by G-protein-linked pathways. Less

在睫状肌中，强直性收缩需要Ca^<2+>通过细胞膜持续流入。迄今为止，对于缺乏电压门控Ca^<2+>通道的组织中Ca^<2+>内流的途径知之甚少。为了确定Ca^<2+>进入途径的离子通道，我们用全细胞电压钳法研究了碳二醇（CCh）对新鲜分离的牛纤毛肌细胞的影响。我们还通过RT-PCR检测了trp通道基因在平滑肌组织中的表达，使用了人类和小鼠trp跨越100-130个氨基酸片段的几种正义和反义结构，这些片段几乎完全覆盖了每个trp的假定孔隙形成区域。主要研究结果总结如下：实验在30℃下进行，移液填充无K^+溶液，其中含有100 mM Cs天冬氨酸，5 mM- bapta （[Ca^<2+>]_i=70 nM）和200 μM-GTP （pH 7.0）。在接近0 mV时，CCh诱发了一个向内电流，显示极性反转。电流噪声分析区分了两种非选择性阳离子通道（NSCC_L和NSCC_S），它们的单位电导（35 pS和100 fS）差异很大。通过总离子置换程序估计，NSCC_L的渗透率与Li^+、Na^+、Cs^+、Mg^<2+>、Ca^<2+>、Sr^<2+>和Ba^<2+>的比值分别为0.9:1.0:1.5:0.2:0.3:0.4:0.5,NSCC_S.b为1.0:1.0:1.8:2.5:2.6:3.2:5.0。1 ~ 100 μM的La^<3+>、Gd^<3+>和SKF96365对NSCC_L和NSCC_S均有剂量依赖性抑制作用，同时对CCh在肌束中产生的收缩的强力性成分也有抑制作用，但对相性成分无明显影响。利用PSS填充的移液器进行原位膜贴片实验，发现了一个γ（31±1 pS）和τ(10±1 ms; n=8) d非常相似的碳甾醇激活通道。移液液中GTP被GTP - γ s取代，在没有CCh的情况下，通道逐渐自发打开。1 μ m -thapsigargin对CCh的反应不可逆地抑制（而不是增强）。RT-PCR结果表明，牛纤毛体平滑肌中表达了相对较高水平的trp，与人的trp3和trp6非常相似，被认为是构建由g蛋白连接途径控制的非选择性阳离子通道。少

项目成果

Kita, A., Matsunaga, S., Takai, A., Kataiwa, H., Wakimoto, T., et al.: "Crystal structure of the complex between calyculin A and catalytic subunit of protein phosphatase 1"Structure. 10. 715-724 (2002)

Kita, A.、Matsunaga, S.、Takai, A.、Kataiwa, H.、Wakimoto, T.等人：“花萼蛋白 A 和蛋白磷酸酶 1 催化亚基之间复合物的晶体结构”结构。

Ito, E., Yasumoto, T., Takai, A., Imanishi, S., Harada, K.: "Investigation of the distribution and excretion of okadaic acid in mice using immunostaining method"Toxicon. 40. 159-165 (2002)

Ito, E.、Yasumoto, T.、Takai, A.、Imanishi, S.、Harada, K.：“使用免疫染色方法研究小鼠冈田酸的分布和排泄”Toxicon。

Takai, A., Takai, Y.: "Two types of non-selective cation channels activated by cacbachol in freshly isolated smooth muclce cells from the bovine ciliary body"Journal of Physiology (London). (印刷中).

Takai，A.，Takai，Y.：“从牛睫状体新鲜分离的平滑粘液细胞中由卡巴酚激活的两种非选择性阳离子通道”生理学杂志（伦敦）（出版中）。

Wakimoto, T., Matsunaga, S., Takai, A., Fusetani, N.: "Insight into binding of calyculin a to protein phosphatase 1. Isolation of hemicalyculin a and chemical transformation of calyculin A"Chemistry & Biology. 9. 309-319 (2002)

Wakimoto, T.、Matsunaga, S.、Takai, A.、Fusetani, N.：“深入了解花萼蛋白 a 与蛋白磷酸酶的结合 1. 半萼蛋白 a 的分离和花萼蛋白 A 的化学转化”化学

Ito, E, Takai, A., Kondo, F., Masui, H., Imanishi, S., Harada, K.: "Comparison of protein phosphatase inhibitory activity and apparent toxicity of microcystins and related compounds"Toxicon. 40. 1017-1025 (2002)

Ito, E、Takai, A.、Kondo, F.、Masui, H.、Imanishi, S.、Harada, K.：“微囊藻毒素及相关化合物的蛋白磷酸酶抑制活性和表观毒性的比较”Toxicon。