12-o-retinoylphorbol-13-acetate and 2-5-di-tert-butylhydroquinone

Receptor-mediated inhibition of amiloride-sensitive sodium absorption was observed in primary and immortalized murine renal collecting duct cell (mCT12) monolayers. The addition of epidermal growth factor (EGF) to the basolateral bathing solution of polarized monolayers reduced amiloride-sensitive short-circuit current (I(sc)) by 15-25%, whereas the addition of ATP to the apical bathing solution decreased I(sc) by 40-60%. Direct activation of PKC with phorbol 12-myristate 13-acetate (PMA) and mobilization of intracellular calcium with 2,5-di-tert-butyl-hydroquinone (DBHQ) reduced amiloride-sensitive I(sc) in mCT12 monolayers by 46 +/- 4% (n = 8) and 22 +/- 2% (n = 8), respectively. Exposure of mCT12 cells to EGF, ATP, PMA, and DBHQ caused an increase in phosphorylation of p42/p44 (extracellular signal-regulated kinase; ERK1/2). Pretreatment of mCT12 monolayers with an ERK kinase inhibitor (PD-98059; 30 microM) prevented phosphorylation of p42/p44 and significantly reduced EGF, ATP, and PMA-induced inhibition of amiloride-sensitive I(sc). In contrast, pretreatment of monolayers with a PKC inhibitor (bisindolylmaleimide I; GF109203x; 1 microM) almost completely blocked the PMA-induced decrease in I(sc), but did not alter the EGF- or ATP-induced inhibition of I(sc). The DBHQ-mediated decrease in I(sc) was due to inhibition of basolateral Na(+)-K(+)-ATPase, but EGF-, ATP-, and PMA-induced inhibition was most likely due to reduced apical sodium entry (epithelial Na(+) channel activity). The results of these studies demonstrate that acute inhibition of amiloride-sensitive sodium transport by extracelluar ATP and EGF involves ERK1/2 activation and suggests a role for MAP kinase signaling as a negative regulator of electrogenic sodium absorption in epithelia.

Topics: Adenosine Triphosphate; Amiloride; Animals; Biological Transport; Cells, Cultured; Enzyme Inhibitors; Epidermal Growth Factor; Epithelial Cells; Extracellular Signal-Regulated MAP Kinases; Flavonoids; Hydroquinones; Indoles; Kidney Tubules, Collecting; Maleimides; Mice; Phorbol Esters; Phosphorylation; Protein Kinase C; Sodium