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

在睫状肌肉中，补品合同需要通过细胞膜持续影响Ca^<2+>。当知道Ca^<2+>缺乏电压门控Ca^<2+>通道的Ca^<2+>的途径时，几乎没有隐藏。为了将离子通道识别为Ca^<2+>进入途径，我们研究了卡巴乔（CCH）通过全细胞电压夹对新鲜分离的牛睫状肌细胞的影响。我们还通过RT-PCR使用了人和鼠TRP的几种含义和反义构建体，研究了TRP通道基因在这种平滑肌组织中的表达，这些构建是100-130个氨基酸段，几乎完全覆盖了每个TRP的假定孔形成区域。获得的主要结果总结如下：使用装有K^+ - 免费溶液的移液器在30℃进行实验，其中包含100 mm CS天冬氨酸，5 mm-bapta（[Ca^<2+>] _ I = 70 nm）和200μm-GTP（pH 7.0）。 CCH引起了向内的电流，显示出在保持电势方面反转的极性……更接近0 mV。对当前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），其统一电导量很大（35 ps和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。 NSCC_L和NSCC_S均被剂量依赖性地抑制了1-100μm的La^<3+>，Gd^<3+>和SKF96365，这也抑制了CCH中肌肉包中肌肉包的滋补成分而不会显着影响Phasic Compontent.C。使用装有PSS的移液器进行原位膜贴片的实验确定了一个非常相似的γ（31±1 ps）和τ（10±1 ms; n = 8）的卡尔巴乔激活通道.d。在没有CCH的情况下，用GTPγs在移液溶液中替换GTP逐渐引起了该通道的发音。通过浸泡1μm-thapsigargin.e，对CCH的响应不可逆地抑制（而不是增强）。 RT-PCR的结果表明，牛睫状体的平滑肌表达了与人类TRP3和TRP6非常相似的TRP水平相对较高的水平，这被认为构建了由G蛋白连接途径控制的非选择性阳离子通道。较少的

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。

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）。

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。

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。