SFKs, Ras, and the classic MAPK pathway couple muscarinic receptor activation to increased Na-HCO3cotransport activity in renal epithelial cells

Cholinergic agents are known to affect the epithelial transport of H2O and electrolytes in the kidney. In proximal tubule cells, cholinergic agonists increase basolateral Na-HCO(3) cotransport activity via M(1) muscarinic receptor activation. The signaling intermediates that couple these G protein-coupled receptors to cotransporter activation, however, are not well defined. We therefore sought to identify distal effectors of muscarinic receptor activation that contribute to increased NBC activity in cultured proximal tubule cells. As demonstrated previously for acute CO2-regulated cotransport activity, we found that inhibitors of Src family kinases (SFKs) or the classic mitogen-activated protein kinase (MAPK) pathway prevented the stimulation of NBC activity by carbachol. The ability of carbachol to activate Src, as well as the proximal (Raf) and distal [extracellular signal-regulated kinases 1 and 2 (ERK1/2)] elements of the classic MAPK module, was compatible with these findings. Cholinergic stimulation of ERK1/2 activity was also completely prevented by overexpression of a dominant negative mutant of Ras (N17-Ras). Taken together, these findings suggest a requirement for the sequential activation of SFKs, Ras, and the classic MAPK pathway [Raf-->MAPK/ERK kinase (MEK)-->ERK]. These findings provide important insights into the molecular mechanisms underlying cholinergic regulation of NBC activity in renal epithelial cells. They also suggest a specific mechanism whereby cholinergic stimulation of the kidney can contribute to pH homeostasis.