THE AMILORIDE-SENSITIVE EPITHELIAL SODIUM-CHANNEL ALPHA-SUBUNIT IS TRANSCRIPTIONALLY DOWN-REGULATED IN RAT PAROTID CELLS BY THE EXTRACELLULAR SIGNAL-REGULATED PROTEIN-KINASE PATHWAY

Previous studies have shown that an inducible Raf-1 kinase protein, De lta Raf-1:ER, activates the mitogen-activated protein kinase/extracell ular signal-regulated protein kinase (ERK)-signaling pathway, which is required for the transformation of the rat salivary epithelial cell l ine, Pa-4. Differential display polymerase chain reaction was employed to search for mRNAs repressed by Delta Raf-1:ER activation. Through t his approach, the gene encoding the cu-subunit of the amiloride-sensit ive epithelial sodium channel (alpha-ENac) was identified as a target of activated Raf-1 kinases. alpha-ENaC down-regulation could also be s een in cells treated with 12-O-tetradecanoyl-1-phorbol-13-acetate (TPA ), indicating that the repression of steady-state alpha-ENaC mRNA leve l was dependent upon the activity of protein kinase C, the target of T PA, as well. Pretreatment of cells with a specific inhibitor of the ER K kinase pathway, PD 98059, markedly abolished the down-regulation of alpha-ENaC expression, consistent with the hypothesis that the ERR kin ase-signaling pathway is involved in TPA-mediated repression. Moreover , through the use of transient transfection assays with alpha-ENaC-rep orter and activated Raf expression construct(s), we provide the first evidence that activation of the ERR pathway down-regulates alpha-ENaC expression at the transcriptional level. Elucidating the molecular pro gramming that modulates the expression of the alpha-subunit may provid e new insights into the modulation of sodium reabsorption across epith elia.