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

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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.