Department of Internal Medicine, Yale University, New Haven, Connecticut, USA.
PLoS One. 2013 May 14;8(5):e62864. doi: 10.1371/journal.pone.0062864. Print 2013.
Na-HCO3 cotransport (NBC) regulates intracellular pH (pHi) and HCO3 secretion in rat colon. NBC has been characterized as a 5,5'-diisothiocyanato-2-2'-stilbene (DIDS)-sensitive transporter in several tissues, while the colonic NBC is sensitive to both amiloride and DIDS. In addition, the colonic NBC has been identified as critical for pHi regulation as it is activated by intravesicular acid pH. Molecular studies have identified several characteristically distinct NBC isoforms [i.e. electrogenic (NBCe) and electroneutral (NBCn)] that exhibit tissue specific expression. This study was initiated to establish the molecular identity and specific function of NBC isoforms in rat colon. Northern blot and reverse transcriptase PCR (RT-PCR) analyses revealed that electrogenic NBCe1B or NBCe1C (NBCe1B/C) isoform is predominantly expressed in proximal colon, while electroneutral NBCn1C or NBCn1D (NBCn1C/D) is expressed in both proximal and distal colon. Functional analyses revealed that amiloride-insensitive, electrogenic, pH gradient-dependent NBC activity is present only in basolateral membranes of proximal colon. In contrast, amiloride-sensitive, electroneutral, [H(+)]-dependent NBC activity is present in both proximal and distal colon. Both electrogenic and electroneutral NBC activities are saturable processes with an apparent Km for Na of 7.3 and 4.3 mM, respectively; and are DIDS-sensitive with apparent Ki of 8.9 and 263.8 µM, respectively. In addition to Na-H exchanger isoform-1 (NHE1), pHi acidification is regulated by a HCO3-dependent mechanism that is HOE694-insensitive in colonic crypt glands. We conclude from these data that electroneutral, amiloride-sensitive NBC is encoded by NBCn1C/D and is present in both proximal and distal colon, while NBCe1B/C encodes electrogenic, amiloride-insensitive Na-HCO3 cotransport in proximal colon. We also conclude that NBCn1C/D regulates HCO3-dependent HOE694-insensitive Na-HCO3 cotransport and plays a critical role in pHi regulation in colonic epithelial cells.
碳酸氢盐共转运蛋白（NBC）调节大鼠结肠的细胞内 pH 值（pHi）和 HCO3-分泌。在几种组织中，NBC 已被鉴定为 5,5'-二异硫氰酸-2,2'-联苯（DIDS）敏感转运体，而结肠 NBC 对阿米洛利和 DIDS 均敏感。此外，结肠 NBC 已被确定为调节 pHi 的关键，因为它被囊泡内酸 pH 值激活。分子研究已经确定了几种特征明显不同的 NBC 同工型[即电中性（NBCn）和电中性（NBCe）]，它们表现出组织特异性表达。本研究旨在确定大鼠结肠 NBC 同工型的分子特征和特定功能。Northern blot 和逆转录 PCR（RT-PCR）分析显示，电中性 NBCn1C 或 NBCn1D（NBCn1C/D）同工型在近端和远端结肠中表达，而电中性 NBCn1C 或 NBCn1D（NBCn1C/D）同工型在近端结肠中表达。功能分析表明，仅在近端结肠的基底外侧膜中存在阿米洛利不敏感、电中性、pH 梯度依赖性 NBC 活性。相比之下，阿米洛利敏感、电中性、[H+]依赖性 NBC 活性存在于近端和远端结肠中。电中性和电中性 NBC 活性都是饱和过程，Na 的表观 Km 分别为 7.3 和 4.3 mM；并且对 DIDS 敏感，表观 Ki 分别为 8.9 和 263.8 µM。除了 Na-H 交换体同工型-1（NHE1）外，pHi 酸化还受 HCO3-依赖性机制调节，该机制在结肠隐窝腺中对 HOE694 不敏感。从这些数据中，我们得出结论，电中性、阿米洛利敏感的 NBC 由 NBCn1C/D 编码，存在于近端和远端结肠中，而 NBCe1B/C 编码在近端结肠中对阿米洛利不敏感的电中性、Na-HCO3 共转运蛋白。我们还得出结论，NBCn1C/D 调节 HCO3-依赖的 HOE694 不敏感的 Na-HCO3 共转运，并在结肠上皮细胞的 pHi 调节中发挥关键作用。
