Kunimi M, Müller-Berger S, Hara C, Samarzija I, Seki G, Frömter E
Zentrum der Physiologie, Johann Wolfgang Goethe-Universität, Frankfurt/Main, Germany.
Pflugers Arch. 2000 Oct;440(6):908-17. doi: 10.1007/s004240000361.
Using a new stop-flow perfusion technique with microspectrofluorometric determination of luminal fluid pH, we have studied which substrates or incubation conditions allow isolated rabbit proximal tubules to attain in-vivo-like rates of HCO3- absorption (J(HCO3)) and maximal responses of J(HCO3) to norepinephrine (NE). Essentially three incubation media were tested: plasma-like HCO(3-)-Ringer solution containing 5 mmol/l D-glucose (G-Ringer sol.), the same solution also containing 10 mmol/l lactate and 5 mmol/l L-alanine, (LAG-Ringer sol.), and two tissue culture media (DMEM and RPMI 1640). Compared to G-Ringer sol., application of LAG-Ringer sol. in the bath and/or lumen, or application of DMEM or RPMI 1640 in the bath either slightly increased or decreased J(HCO3) with borderline significance. However, RPMI 1640 plus 1 mmol/l pyruvate stimulated J(HCO3) by 55%. While NE (10(-5) mol/l), if applied in G-Ringer sol., had no effect, in the presence of LAG-Ringer sol. it increased J(HCO3) by approximately =40%, and in the presence of DMEM or RPMI 1640 it increased J(HCO3) by approximately =100%. This stimulation by NE followed Michaelis-Menten kinetics with an EC50 value of 0.25 micromol/l and was probably mediated by alpha1-adrenergic receptors. Additional cell pH measurements suggest that NE stimulates the basolateral Na+-HCO3- cotransporter which then becomes susceptible to inhibition by cAMP. We conclude that incubation in tissue culture media allows isolated proximal tubules to maintain a better functional state than the commonly used solutions with unphysiologically high substrate concentrations.
我们采用一种新的停流灌注技术，通过微量光谱荧光法测定管腔液pH值，研究了哪些底物或孵育条件能使分离的兔近端小管达到类似体内的HCO₃⁻重吸收率（J(HCO₃)）以及J(HCO₃)对去甲肾上腺素（NE）的最大反应。基本上测试了三种孵育介质：含5 mmol/L D -葡萄糖的血浆样HCO₃⁻ -林格溶液（G -林格溶液）、同样含10 mmol/L乳酸盐和5 mmol/L L -丙氨酸的该溶液（LAG -林格溶液），以及两种组织培养基（DMEM和RPMI 1640）。与G -林格溶液相比，在浴槽和/或管腔中应用LAG -林格溶液，或在浴槽中应用DMEM或RPMI 1640，J(HCO₃)略有增加或减少，差异接近显著水平。然而，RPMI 1640加1 mmol/L丙酮酸盐可使J(HCO₃)增加55%。当在G -林格溶液中应用NE（10⁻⁵ mol/L）时无作用，但在LAG -林格溶液存在时，它使J(HCO₃)增加约40%，在DMEM或RPMI 1640存在时，它使J(HCO₃)增加约100%。NE的这种刺激遵循米氏动力学，EC50值为0.25 μmol/L，可能由α1 -肾上腺素能受体介导。额外的细胞pH测量表明，NE刺激基底外侧Na⁺ - HCO₃⁻共转运体，然后该转运体变得易受cAMP抑制。我们得出结论，在组织培养基中孵育能使分离的近端小管比常用的具有非生理性高底物浓度的溶液维持更好的功能状态。
