Weill A E, Tisher C C, Conde M F, Weiner I D
Division of Nephrology, Hypertension and Transplantation, University of Florida College of Medicine, Gainesville 32610.
Am J Physiol. 1994 Mar;266(3 Pt 2):F466-76. doi: 10.1152/ajprenal.1994.266.3.F466.
The inner medullary collecting duct (IMCD) is the final portion of the mammalian renal tubule that is able to significantly regulate systemic acid-base balance. Although the H+ transporters of this segment are relatively well studied, little is known regarding the mechanisms of HCO3- transport. The mechanisms of HCO3- transport in primary cultures of rabbit IMCD were studied using the pH-sensitive dye, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, in CO2/HCO3(-)-containing solutions at 37 degrees C. Removal of Cl- from the extracellular solution caused reversible intracellular alkalinization, demonstrating the presence of Cl-/HCO3- exchange. Alkalinization with Cl- removal was independent of changes in membrane potential, did not require the presence of extracellular Na+, and was inhibited by the disulfonic stilbene, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS, 10(-4) M). Half-maximal intracellular pH (pHi) recovery with readdition of Cl- to the extracellular solution occurred at a Cl- concentration of 37.4 +/- 5.7 mM. When rabbit IMCD were cultured on permeable support membranes, Cl-/HCO3- exchange activity was found only on the basolateral membrane. However, there was no evidence of band 3 protein immunoreactivity. In contrast, no evidence for Na(+)-(HCO3-)n > 1 cotransport activity was found. Depolarization of IMCD cells by acute increases in extracellular K+ did not alter pHi, nor was Na(+)-dependent, 5-(N-ethyl-N-isopropyl)amiloride-insensitive pHi recovery from an acid load inhibited by DIDS (10(-4) M). Finally, recovery from intracellular alkalosis induced by incubation in 0 mM Cl-, 50 mM HCO3- extracellular solution required Cl- and was independent of Na+. These studies indicate that the major mechanism of HCO3- transport in primary cultures of the rabbit IMCD is via a band 3 protein-negative, Na(+)-independent, basolateral, Cl-/HCO3- exchanger.
髓质内集合管（IMCD）是哺乳动物肾小管的最后一部分，能够显著调节全身酸碱平衡。尽管该节段的H⁺转运体已得到较为充分的研究，但关于HCO₃⁻转运机制却知之甚少。本研究使用pH敏感染料2',7'-双（2-羧乙基）-5（6）-羧基荧光素，在37℃含CO₂/HCO₃⁻的溶液中，对兔IMCD原代培养物中HCO₃⁻的转运机制进行了研究。从细胞外溶液中去除Cl⁻会导致细胞内可逆性碱化，表明存在Cl⁻/HCO₃⁻交换。去除Cl⁻引起的碱化与膜电位变化无关，不需要细胞外Na⁺的存在，并且被二磺酸芪4,4'-二异硫氰酸芪-2,2'-二磺酸（DIDS，10⁻⁴ M）抑制。当向细胞外溶液中重新添加Cl⁻时，细胞内pH（pHi）恢复到半最大值时的Cl⁻浓度为37.4±5.7 mM。当兔IMCD在可渗透支持膜上培养时，Cl⁻/HCO₃⁻交换活性仅在基底外侧膜上被发现。然而，没有证据表明存在带3蛋白免疫反应性。相反，未发现Na⁺-（HCO₃⁻）n＞1共转运活性的证据。细胞外K⁺急性增加使IMCD细胞去极化，并未改变pHi，DIDS（10⁻⁴ M）也未抑制从酸负荷中恢复的Na⁺依赖性、5-（N-乙基-N-异丙基）氨氯地平不敏感的pHi。最后，在0 mM Cl⁻、50 mM HCO₃⁻细胞外溶液中孵育诱导的细胞内碱中毒的恢复需要Cl⁻，且与Na⁺无关。这些研究表明，兔IMCD原代培养物中HCO₃⁻转运的主要机制是通过一种不依赖Na⁺、位于基底外侧的带3蛋白阴性的Cl⁻/HCO₃⁻交换体。
