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Am J Physiol Cell Physiol 281: C1551-C1558, 2001;
0363-6143/01 $5.00
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Vol. 281, Issue 5, C1551-C1558, November 2001

Differential regulation of the expression of Na+/H+ exchanger isoform NHE3 by PKC-alpha in Caco-2 cells

W. A. Alrefai1, B. Scaglione-Sewell2, S. Tyagi1, L. Wartman1, T. A. Brasitus2, K. Ramaswamy1, and P. K. Dudeja1

1 Section of Digestive and Liver Diseases, Department of Medicine, University of Illinois at Chicago, and Westside Veterans Affairs Medical Center, Chicago 60612; and 2 Department of Medicine, University of Chicago, Chicago, Illinois 60637

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 µM 12-O-tetradecanoylphorbol-13-acetate (TPA) for 24 h. PKC downregulation resulted in an ~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 22Na+ uptake was increased after incubation of Caco-2 cells with 1 µM 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 .

intracellular pH; phorbol esters; sodium absorption; mRNA; protein kinase C; human intestine


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