Differential regulation of the expression of Na+/H+ exchanger isoform NHE3by PKC-alpha in Caco-2 cells

Na+/H+ exchange (NHE) activity has been shown to be regulated by various ex ternal signals and protein kinases in many tissues and cell types. A family of six NHE isoforms has been identified. Three isoforms, NHE1, NHE2, and N HE3, have been shown to be expressed in the human intestine. The present st udies 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 sem iquantitative RT-PCR technique in response to PKC downregulation by long-te rm exposure to 1 muM 12-O-tetradecanoylphorbol-13-acetate (TPA) for 24 h. P KC downregulation resulted in an similar to 60% increase in the mRNA level for NHE3, but not for NHE1 or NHE2. Utilizing dichlorobenzimidazole ribosid e, an agent to block the synthesis of new mRNA, we demonstrated that the in crease in the NHE3 mRNA in response to downregulation of PKC was predominan tly 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 sho wed that amiloride-sensitive Na-22(+) uptake was increased after incubation of Caco-2 cells with 1 muM TPA for 24 h. To elucidate the role of PKC-alph a, an isoform downregulated by TPA, the relative abundance of NHE isoform m RNA 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-a and that apical NHE activity was higher in cells underexpressing PKC-alpha and lower in cells overexpres sing 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 PK C-alpha.