Kuwahara M, Verkman A S
Department of Medicine, University of California, San Francisco 94143.
Biophys J. 1988 Oct;54(4):587-93. doi: 10.1016/S0006-3495(88)82993-X.
A fluorescence method has been developed for accurate and instantaneous measurement of transepithelial diffusional water permeability (Pd) in perfused kidney tubules based on the sensitivity of the fluorophore aminonapthelane trisulfonic acid (ANTS) to solution H2O/D2O content. The fluorescence of ANTS was 3.2-fold lower in an H2O buffer than in a D2O buffer. The response of ANTS fluorescence to a change in solution H2O/D2O content occurred in less than 1 ms and was due to a collisional quenching mechanism. Isolated cortical (CCT) and outer medullary (OMCT) collecting tubules from rabbit were perfused with an isosmotic D2O buffer at specified lumen flow rates (2-100 nl/min); tubules were bathed in isosmotic H2O or D2O buffers in which vasopressin (VP) could be added rapidly. Lumen fluorescence was monitored by quantitative epifluorescence microscopy at 380 +/- 5 nm excitation and greater than 530 emission wavelengths. Pd was determined from tubule geometry, lumen flow, ANTS fluorescence, and ANTS fluorescence vs. H2O/D2O calibration relation. The instrument response time for a change in bath H2O/D2O content was less than 4 s. At 37 degrees C, Pd values (mean +/- SE in cm/s x 10(4] were 6.4 +/- 1.0 (-VP, n = 9) and 14.3 +/- 1.1 (+250 microU/ml bath VP, n = 9) in the CCT, and 5.8 +/- 1.0 (-VP, n = 6) and 15.3 +/- 2.0 (+VP, n = 6) in the OMCT; at 23 degrees C, Pd was 5.1 +/- 0.6 (-VP, n = 4) and 7.8 +/- 0.6 (+VP, n = 4) in the CCT. In response to rapid addition of 250 micro U/ml vasopressin to the bath, CCT Pd remained unchanged for 71 +/- l0s (n = 9, 37 degree C) and 170 +/- 45 s (n = 4, 23 degree C); this was followed by a slow increase in Pd(TI/2 = 91 +/- 17 s, 37 degree C; 119 +/- 31 s, 23 degree C) to the new steady-state value. These results provide a new approach for study of transepithelial water transport in kidney tubules. Compared with 3H20 methods, the fluorescence method is superior in technical simplicity, time resolution, and accuracy. The improved time resolution is important for examination of the pre-steady-state kinetics of vasopressin-induced signalling events resulting in the hydroosmotic response.
基于荧光团氨基萘三磺酸（ANTS）对溶液H₂O/D₂O含量的敏感性，已开发出一种荧光方法，用于精确、即时测量灌注肾小管中的跨上皮扩散水通透性（Pd）。ANTS在H₂O缓冲液中的荧光比在D₂O缓冲液中低3.2倍。ANTS荧光对溶液H₂O/D₂O含量变化的响应在不到1毫秒内发生，这是由于碰撞猝灭机制。用等渗D₂O缓冲液以特定的管腔流速（2 - 100 nl/min）灌注来自兔子的分离皮质（CCT）和外髓质（OMCT）集合小管；小管浸浴在等渗H₂O或D₂O缓冲液中，其中可快速添加血管加压素（VP）。通过定量落射荧光显微镜在380±5 nm激发波长和大于530 nm发射波长下监测管腔荧光。根据小管几何形状、管腔流速、ANTS荧光以及ANTS荧光与H₂O/D₂O校准关系确定Pd。浴液H₂O/D₂O含量变化时仪器的响应时间小于4秒。在37℃时，CCT中的Pd值（以cm/s×10⁴为单位的平均值±标准误）为6.4±1.0（-VP，n = 9）和14.3±1.1（浴液中添加250 μU/ml VP，n = 9），OMCT中的Pd值为5.8±1.0（-VP，n = 6）和15.3±2.0（+VP，n = 6）；在23℃时，CCT中的Pd值为5.1±0.6（-VP，n = 4）和7.8±0.6（+VP，n = 4）。响应于向浴液中快速添加250 μU/ml血管加压素，CCT的Pd在37℃下保持不变71±10秒（n = 9），在23℃下保持不变170±45秒（n = 4）；随后Pd缓慢增加（37℃时半衰期TI/2 = 91±17秒；23℃时半衰期TI/2 = 119±31秒）至新的稳态值。这些结果为研究肾小管中的跨上皮水转运提供了一种新方法。与³H₂O方法相比，荧光方法在技术简便性、时间分辨率和准确性方面更具优势。改进的时间分辨率对于检查血管加压素诱导的信号事件导致渗透反应的稳态前动力学很重要。
