Bidet M, Tauc M, Gastineau M, Poujeol P
Département de Biologie Cellulaire et Moléculaire, Centre d'Etudes de Saclay, Gif sur Yvette, France.
Pflugers Arch. 1992 Sep;421(6):523-9. doi: 10.1007/BF00375047.
To examine the intracellular pH (pHi) regulation in primary cultures of rabbit distal convoluted tubules (DCTb) we used the pH-sensitive dye 2,7-bis-carboxyethyl-5(6)-carboxyfluorescein (BCECF/AM) and a video-microscopy technique. DCTb segments were microdissected from rabbit kidney cortex and cultured in a hormonally defined medium. The culture epithelia were grown on semi-transparent permeable supports. Before pHi measurement, DCTb primary cultures were maintained for 48-96 h in growth-factor-free medium to obtain quiescent cells. We had previously shown that two mechanisms are involved in the regulation of intracellular pH: a basolateral Na+/H+ exchanger and an apical Cl-/HCO3- exchanger. The pHi of DCTb cells was significantly decreased by the addition of 60 nM human calcitonin (from 7.30 +/- 0.04 to 7.08 +/- 0.04). This response to calcitonin was dose-dependent and mimicked by both forskolin and permeant cyclic AMP derivatives. An initial acidification (of 0.25 pH unit in 7-8 min) was observed after the addition of basolateral amiloride (1 mM). The persistence of the effect induced by human calcitonin in these conditions, suggests that the Na+/H+ exchanger is not involved in the response. However, the acidification response was blocked in both the absence of chloride at the apical side and by the apical addition of 0.1 mM 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS). These experiments suggest that the target for the human calcitonin effect on pHi is the Cl-/HCO3- exchanger. This study confirms the importance of this transporter in pHi regulation within the physiological pHi range and the influence of calcitonin in the regulation of DCTb cell function.
为了研究兔远曲小管（DCTb）原代培养物中的细胞内pH（pHi）调节，我们使用了pH敏感染料2,7 - 双羧乙基 - 5(6) - 羧基荧光素（BCECF/AM）和视频显微镜技术。从兔肾皮质显微解剖出DCTb节段，并在激素限定培养基中培养。培养的上皮细胞生长在半透明的可渗透支持物上。在测量pHi之前，将DCTb原代培养物在无生长因子的培养基中维持48 - 96小时以获得静止细胞。我们之前已经表明，细胞内pH调节涉及两种机制：一种是基底外侧Na⁺/H⁺交换体，另一种是顶端Cl⁻/HCO₃⁻交换体。添加60 nM人降钙素后，DCTb细胞的pHi显著降低（从7.30±0.04降至7.08±0.04）。这种对降钙素的反应是剂量依赖性的，并且被福斯可林和渗透性环磷酸腺苷衍生物模拟。添加基底外侧氨氯吡咪（1 mM）后，观察到初始酸化（在7 - 8分钟内为0.25个pH单位）。在这些条件下，人降钙素诱导的效应持续存在，这表明Na⁺/H⁺交换体不参与该反应。然而，在顶端侧无氯离子的情况下以及顶端添加0.1 mM 4,4'-二异硫氰酸芪 - 2,2'-二磺酸（DIDS）时，酸化反应均被阻断。这些实验表明，人降钙素对pHi的作用靶点是Cl⁻/HCO₃⁻交换体。这项研究证实了该转运体在生理pHi范围内pHi调节中的重要性以及降钙素对DCTb细胞功能调节的影响。
