Müller-Berger S, Samarzija I, Kunimi M, Yamada H, Frömter E, Seki G
Zentrum der Physiologie, Johann Wolfgang Goethe-Universität, Frankfurt/Main, Germany.
Pflugers Arch. 1999 Dec;439(1-2):208-15. doi: 10.1007/s004249900171.
In the present experiments on microdissected tubules of rabbit kidney we present a refined stop-flow method for determining the rate of HCO3- absorption (J(HCO3)) or H+ secretion (JH) that can be applied to isolated microperfused tubules. Using the pH-sensitive indicator dye BCECF (2',7'-bis [2-carboxyethyl]-5[6]-carboxyfluorescein) the luminal perfusate pH is continuously measured with a microspectrofluorometric set-up, and the pH change following a sudden stop of perfusion is analysed. Because the tubules partially collapse after stop-flow the calculation of fluxes requires a correction for volume loss. This is achieved by referring all fluxes to the remaining luminal volume, which can be estimated from the decay of the 440 nm reference fluorescence. During perfusion of the lumen with pure HCO3- Ringer solution, and of the bath with the same solution but containing 5.5 mmol/l D-glucose as metabolic substrate, J(HCO3) averaged 4.4+/-0.2 pmol cm(-1) x s(-1) (n=40) and 13.4+/-0.8 pmol x cm(-1) x s(-1) (n=5) in proximal straight tubules (PST) and in proximal convoluted tubules respectively. These values agree very well with data obtained in other laboratories with the picapnotherm technique. The present method has the advantage of requiring fewer micromanipulations and a shorter measuring time, thus allowing regulatory changes in J(HCO3) to be analysed. Moreover it does not involve measurements of radioactivity, and it also allows J(H) to be measured in HCO3(-) free solutions which in PST averaged 0.9 pmol x cm(-1) x s(-1) (n=8) in the present experiments.
在目前对兔肾显微解剖小管进行的实验中，我们提出了一种改进的停流方法，用于测定HCO₃⁻重吸收速率（J(HCO₃)）或H⁺分泌速率（JH），该方法可应用于分离的微灌流小管。使用对pH敏感的指示染料BCECF（2'，7'-双[2-羧乙基]-5[6]-羧基荧光素），通过显微分光荧光测定装置连续测量管腔灌流液的pH，并分析灌注突然停止后的pH变化。由于停流后小管会部分塌陷，通量的计算需要对体积损失进行校正。这通过将所有通量参考剩余的管腔体积来实现，剩余管腔体积可根据440nm参考荧光的衰减来估计。在用纯HCO₃⁻林格溶液灌流管腔，并用相同溶液但含有5.5mmol/L D-葡萄糖作为代谢底物灌流浴槽时，近端直小管（PST）和近端曲小管中J(HCO₃)的平均值分别为4.4±0.2pmol·cm⁻¹·s⁻¹（n = 40）和13.4±0.8pmol·cm⁻¹·s⁻¹（n = 5）。这些值与其他实验室使用皮卡诺热技术获得的数据非常吻合。本方法的优点是需要的显微操作较少，测量时间较短，从而能够分析J(HCO₃)的调节变化。此外，它不涉及放射性测量，还能在无HCO₃⁻的溶液中测量JH，在本实验中，PST中JH的平均值为0.9pmol·cm⁻¹·s⁻¹（n = 8）。
