Alrefai W A, Scaglione-Sewell B, Tyagi S, Wartman L, Brasitus T A, Ramaswamy K, Dudeja P K
Section of Digestive and Liver Diseases, Department of Medicine, University of Illinois at Chicago, and Westside Veterans Affairs Medical Center, Chicago 60612, USA.
Am J Physiol Cell Physiol. 2001 Nov;281(5):C1551-8. doi: 10.1152/ajpcell.2001.281.5.C1551.
Na(+)/H(+) exchange (NHE) activity has been shown to be regulated by various external signals and protein kinases in many tissues and cell types. A family of six NHE isoforms has been identified. Three isoforms, NHE1, NHE2, and NHE3, have been shown to be expressed in the human intestine. The present studies were designed to study regulation of these human NHE isoforms by the alpha-isoform of protein kinase C (PKC) in the Caco-2 cell line. The mRNA levels of the NHE isoforms in Caco-2 cells were initially measured by a semiquantitative RT-PCR technique in response to PKC downregulation by long-term exposure to 1 microM 12-O-tetradecanoylphorbol-13-acetate (TPA) for 24 h. PKC downregulation resulted in an approximately 60% increase in the mRNA level for NHE3, but not for NHE1 or NHE2. Utilizing dichlorobenzimidazole riboside, an agent to block the synthesis of new mRNA, we demonstrated that the increase in the NHE3 mRNA in response to downregulation of PKC was predominantly due to an increase in the rate of transcription, rather than a decrease in the NHE3 mRNA stability. Consistent with the mRNA results, our data showed that amiloride-sensitive (22)Na(+) uptake was increased after incubation of Caco-2 cells with 1 microM TPA for 24 h. To elucidate the role of PKC-alpha, an isoform downregulated by TPA, the relative abundance of NHE isoform mRNA levels and the apical NHE activity were assessed in Caco-2 cells over- and underexpressing PKC-alpha. Our results demonstrated that NHE3, but not NHE1 or NHE2, mRNA was downregulated by PKC-alpha and that apical NHE activity was higher in cells underexpressing PKC-alpha and lower in cells overexpressing PKC-alpha than in control cells. In conclusion, these data demonstrate a differential regulation of NHE3, but not NHE2 or NHE1, expression by PKC in Caco-2 cells, and this regulation appears to be predominantly due to PKC-alpha.
钠/氢交换（NHE）活性已被证明在许多组织和细胞类型中受多种外部信号和蛋白激酶调节。已鉴定出一个由六种NHE亚型组成的家族。三种亚型，即NHE1、NHE2和NHE3，已被证明在人类肠道中表达。本研究旨在研究蛋白激酶C（PKC）的α亚型在Caco-2细胞系中对这些人类NHE亚型的调节作用。通过半定量RT-PCR技术，最初检测了长期暴露于1μM 12-O-十四酰佛波醇-13-乙酸酯（TPA）24小时导致PKC下调后，Caco-2细胞中NHE亚型的mRNA水平。PKC下调导致NHE3的mRNA水平增加约60%，但NHE1或NHE2的mRNA水平未增加。利用二氯苯并咪唑核糖苷（一种阻断新mRNA合成的试剂），我们证明，PKC下调后NHE3 mRNA的增加主要是由于转录速率的增加，而不是NHE3 mRNA稳定性的降低。与mRNA结果一致，我们的数据表明，Caco-2细胞与1μM TPA孵育24小时后，amiloride敏感的（22）Na+摄取增加。为了阐明TPA下调的PKC-α亚型的作用，在过表达和低表达PKC-α的Caco-2细胞中评估了NHE亚型mRNA水平的相对丰度和顶端NHE活性。我们的结果表明，PKC-α下调了NHE3的mRNA，但未下调NHE1或NHE2的mRNA，并且低表达PKC-α的细胞中顶端NHE活性高于对照细胞，而过表达PKC-α的细胞中顶端NHE活性低于对照细胞。总之，这些数据表明，PKC在Caco-2细胞中对NHE3（而非NHE2或NHE1）的表达有差异调节作用，并且这种调节似乎主要归因于PKC-α。
