Phorbol esters downregulate expression of the sodium/calcium exchanger in renal epithelial cells

L. Smith, H. Porzig, H. W. Lee and J. B. Smith Department of Pharmacology, School of Medicine, University of Alabama at Birmingham 35294, USA. The Na+/Ca2+ exchanger (NCE) contributes to Ca2+ reabsorption by connecting tubules of the nephron. A line of renal epithelial cells from monkey kidney (LLC-MK2) was used to investigate the regulation of NCE expression. After the activation of protein kinase C (PKC) by phorbol myristate acetate (PMA), NCE activity decreased exponentially by 75% in 48 h (half time approximately 19 h). PMA decreased NCE mRNA by 85% in 24 h. The decrease in NCE transcript preceded the downregulation of NCE activity. NCE protein was quantified with a monoclonal antibody to cardiac NCE. PMA decreased the binding of 3H-labeled antibody to cell sonicates by 40% in 24 h. Immunoblots show that PMA produced a marked and extended increase in membrane-associated PKC-alpha, although PMA depleted total PKC-alpha by 65% in 24 h. In vivo 32P labeling of myristolated alanine-rich C kinase substrate, a specific PKC substrate, confirmed that PMA produced a rapid and extended activation of PKC. 4 alpha-PMA, a stereoisomer of PMA that neither binds nor activates PKC, had no effect on NCE activity or transcript. These findings indicate that activation of PKC with phorbol esters downregulates NCE mRNA, protein, and activity in renal epithelial cells.