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，紧张性收缩需要持续的Ca^<2+>通过细胞膜流入。对于缺乏电压门控性Ca^2+通道的组织中Ca^2+内流的途径，迄今知之甚少。为了鉴定Ca^2+进入通道，我们用全细胞电压钳技术研究了卡巴胆碱（CCh）对新鲜分离的牛睫状肌细胞的作用。我们还通过RT-PCR检测了trp通道基因在该平滑肌组织中的表达，所述RT-PCR使用几种跨越100-130个氨基酸区段的人和鼠trp的有义和反义构建体，所述区段几乎完全覆盖每个trp的推定的孔形成区域。主要研究结果如下：a.实验在30℃下进行，使用装有无K^+溶液的移液管，该溶液含有100 mM天冬氨酸铯、5 mM-BAPTA（[Ca^<2+>]_i=70 nM）和200 μM-GTP（pH 7.0）。CCh在保持电位时诱发一种极性反转的内向电流 ...更多信息 接近0 mV。对电流噪声的分析区分了两种类型的非选择性阳离子通道（NSCC_L和NSCC_S），其具有广泛不同的单位电导（35 pS和100 fS）。通过总离子置换法估算的NSCC_L对Li^+、Na^+、Cs^+、Mg^<2 +>、Ca^<2+>、Sr^<2+>和Ba^<2+>的渗透率之比分别为0.9：1.0：1.5：0.2：0.3：0.4：0.5和1.0：1.0：1.8：2.5：2.6：0.5。3.2：5.0（对于NSCC_S.b.）1-100 μM的La^<3+>、Gd^<3+>和SKF 96365对NSCC_L和NSCC_S均有剂量依赖性抑制作用，它们也抑制CCh引起的肌束收缩的紧张性成分，但不显著影响相位成分。使用填充有PSS的移液管的原位膜贴片的实验鉴定了具有非常相似的γ（31± lpS）和τ（10± lms; n=8）的卡巴胆碱激活通道。在不存在CCh的情况下，用GTPγS取代移液器溶液中的GTP逐渐导致通道自发开放。1 μ M-thapsiglavin对CCh的反应被不可逆地抑制（而不是增强）。RT-PCR的结果表明，牛睫状体的平滑肌表达相对高水平的trp的非常类似于人的trp 3和trp 6，这被认为是构建由G蛋白连接的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。