Janecki A J, Montrose M H, Tse C M, de Medina F S, Zweibaum A, Donowitz M
Departments of Medicine and Physiology, Division of Gastroenterology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Am J Physiol. 1999 Aug;277(2):G292-305. doi: 10.1152/ajpgi.1999.277.2.G292.
Expression of endogenous Na(+)/H(+) exchangers (NHEs) NHE3 and NHE1 at the apical (AP) and basolateral (BL) membrane domains was investigated in three clones (ATCC, PF-11, and TC-7) derived from the human adenocarcinoma cell line Caco-2. In all three clones, NHE1 was the only isoform detected at the BL domain during 3 to 22 postconfluent days (PCD). In clone PF-11, the BL NHE1 activity increased up to 7 PCD and remained stable thereafter. Both NHE1 and NHE3 were found at the AP domain at 3 PCD and contributed 67 and 33% to the total AP Na(+)/H(+) exchange, respectively. The AP NHE3 activity increased significantly from 3 to 22 PCD, from 93 to 450 microM H(+)/s, whereas AP NHE1 activity decreased from 192 to 18 microM H(+)/s during that time. Similar results were obtained with the ATCC clone, whereas very little AP NHE3 activity was observed in clone TC-7. Surface biotinylation and indirect immunofluorescence confirmed these results and also suggested an increase in the number of cells expressing NHE3 being the major mechanism of the observed overall increase in NHE3 activity in PF-11 and ATCC clones. Phorbol 12-myristate 13-acetate (PMA, 1 microM) acutely inhibited NHE3 activity by 28% of control, whereas epidermal growth factor (EGF, 200 ng/ml) stimulated the activity by 18%. The effect of PMA was abolished by the protein kinase C (PKC) inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, suggesting involvement of PKC in the PMA-induced inhibition of NHE3. Similar magnitude of inhibition by PMA and stimulation by EGF was observed at 7 and 17 PCD, suggesting the development of regulatory mechanisms in the early postconfluent period. Taken together, these data suggest a close similarity of membrane targeting and regulation of endogenous NHE3 between Caco-2 cells and native small intestinal epithelial cells and support the usefulness of some Caco-2 cell clones as an in vitro model for studies on physiology of NHE3 in the intestinal epithelium.
研究了源自人腺癌细胞系Caco-2的三个克隆（ATCC、PF-11和TC-7）中内源性钠氢交换体（NHEs）NHE3和NHE1在顶端（AP）和基底外侧（BL）膜结构域的表达。在所有三个克隆中，NHE1是在汇合后3至22天（PCD）期间在BL结构域检测到的唯一亚型。在克隆PF-11中，BL NHE1活性在7 PCD时增加，此后保持稳定。在3 PCD时，NHE1和NHE3均在AP结构域被发现，分别占总AP钠氢交换的67%和33%。AP NHE3活性在3至22 PCD期间显著增加，从93微摩尔氢离子/秒增加到450微摩尔氢离子/秒，而在此期间AP NHE1活性从192微摩尔氢离子/秒降至18微摩尔氢离子/秒。在ATCC克隆中获得了类似的结果，而在克隆TC-7中观察到的AP NHE3活性非常低。表面生物素化和间接免疫荧光证实了这些结果，并且还表明表达NHE3的细胞数量增加是PF-11和ATCC克隆中观察到的NHE3活性总体增加的主要机制。佛波醇12 -肉豆蔻酸酯13 -乙酸酯（PMA，1微摩尔）急性抑制NHE3活性，使其比对照降低28%，而表皮生长因子（EGF，200纳克/毫升）刺激其活性增加18%。蛋白激酶C（PKC）抑制剂1 -（5 -异喹啉磺酰基）-2 -甲基哌嗪消除了PMA的作用，表明PKC参与了PMA诱导的NHE3抑制。在7和17 PCD时观察到PMA的抑制和EGF的刺激幅度相似，表明在汇合后早期阶段调节机制的发展。综上所述，这些数据表明Caco-2细胞和天然小肠上皮细胞之间内源性NHE3的膜靶向和调节具有密切相似性，并支持一些Caco-2细胞克隆作为研究肠上皮中NHE3生理学的体外模型的有用性。
