Submitted on April 29, 2004
Accepted on December 29, 2004
A role for ERK1/2 in EGF- and ATP-dependent regulation of amiloride-sensitive sodium absorption
Rebecca Falin1, I. Elias Veizis1, and Calvin U. Cotton1*
1 Pediatrics, Case Western Reserve University, Cleveland, Ohio, USA; Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio, USA
* To whom correspondence should be addressed. E-mail: cuc{at}po.cwru.edu.
Receptor-mediated inhibition of amiloride-sensitive sodium absorption was observed in primary and immortalized (mCT12) murine renal collecting duct cell monolayers. Addition of epidermal growth factor (EGF) to the basolateral bathing solution of polarized monolayers, reduced amiloride-sensitive short-circuit current (Isc) by 15-25%; whereas, addition of ATP to the apical bathing solution decreased Isc 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 Isc 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 (PD98059; 30 µM) prevented phosphorylation of p42/p44 and significantly reduced EGF, ATP, and PMA-induced inhibition of amiloride-sensitive Isc. In contrast, pretreatment of monolayers with a PKC inhibitor (Bisindolylmaleimide I; GF109203x; 1 µM) almost completely blocked the PMA-induced decrease in Isc, but did not alter the EGF- or ATP-induced inhibition of Isc. The DBHQ-mediated decrease in Isc 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 (ENaC 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 of MAP kinase signaling as a negative regulator of electrogenic sodium absorption in epithelia.
