Integrated Analysis in Peristaltic Pacemaker Mechanisms of Ureter using Two-Photon Excitation

双光子激发输尿管蠕动起搏机制的综合分析

• 批准号: 14570047
• 负责人: YAMASHITA Toshikazu
• 金额: $ 1.86万
• 依托单位: Nihon University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14570047/
• 关键词: ureter; smooth muscle; peristalsis; pacemaker; two-photon excitation

The purpose of this study was to identify pacemaker cells and to perform integrated analysis on mechanisms of the ureteral peristalsis. Two photon excitation (TPE) technique with mode-lock pulse laser (710 nm, 40 fs, 80 MHz) enabled Ca^<2+> imaging in deeper muscle layer in situ. In rat ureter isolated along with renal pelvis, we identified two types of spontaneous intracellular Ca^<2+> movements in longitudinal smooth muscle cells: 1) Ca^<2+> transients rapidly propagating through the longitudinal muscle bundle (velocity, 6-7 mm ・ s^<-1>); 2) Ca^<2+> waves propagating slowly in the individual smooth muscle cells (velocity, 〜25 μm ・ s^<-1>). Pharmacological analyses of the Ca^<2+> waves revealed involvement of IP_3-induced Ca^<2+> release in their production. TPE combined with conventional confocal microscopy also enabled photolysis of caged compounds and simultaneous measurement of fluorescence in micro-space. Spatial resolution of TPE photolysis measured with caged F1TC in our system was 1.6 and 1.7 μm in horizontal and vertical axes, respectively. With this technique we successfully applied neurotransmitter such as caged ATP, caged ACh, and caged NO to single cell and observed its Ca^<2+> movements simultaneously. Modification of Ca^<2+> movements by these neurotransmitters in the ureteral smooth muscle layer is now being investigated. Development of this study will clearly indicate pacemaking and propagation mechanisms of the ureteral peristalsis.

本研究的目的是鉴定起搏细胞，并对输尿管狭窄的发生机制进行综合分析。采用锁模脉冲激光（710 nm，40 fs，80 MHz）的双光子激发（TPE）技术，可在更深的肌肉层原位进行Ca^2+成像。在沿着肾盂分离的大鼠输尿管中，我们发现纵向平滑肌细胞内自发的细胞内Ca^&lt;2+&gt;运动有两种类型：1）Ca^&lt;2+&gt;瞬变通过纵向肌束快速传播（速度，6-7 mm · s^<-1>）; 2）Ca^&lt;2+&gt;波在单个平滑肌细胞中缓慢传播（速度，~ 25 μm · s^<-1>）。对Ca^&lt;2+&gt;波的药理学分析表明，IP_3诱导的Ca^&lt;2+&gt;释放参与了Ca ^&lt;2+&gt;波的产生。TPE与传统的共聚焦显微镜相结合，也使笼状化合物的光解和同时测量荧光在微空间。在我们的系统中，用笼状F1 TC测量TPE光解的空间分辨率在水平和垂直轴上分别为1.6和1.7 μm。利用这一技术，我们成功地将笼状ATP、笼状ACh和笼状NO等神经递质应用于单个细胞，同时观察了其Ca^&lt;2+&gt;运动。目前正在研究这些神经递质对输尿管平滑肌层中Ca^2+运动的影响。这项研究的发展将清楚地表明输尿管扩张的起搏和传播机制。