HORMONAL-REGULATION OF MAP KINASE IN CULTURED RAT INNER MEDULLARY COLLECTING TUBULE CELLS

Mitogen-activated protein (MAP) kinase is a widely expressed protein serine/threonine kinase that serves as a convergence point for many signaling pathways including receptor tyrosine kinases, G protein-coupled receptors, and protein kinase C (PKC). The hormonal regulation of MAP kinase was studied in cultured established rat inner medullary collecting tubule (RIMCT) cells. Neither vasopressin nor beta-adrenergic agonists stimulated MAP kinase, despite clear stimulation of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase. In contrast, carbachol, ATP, and epidermal growth factor (EGF), which are known to antagonize vasopressin action in the RIMCT, stimulated the MAP kinase pathway. This stimulation was mimicked by the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, which directly activates PKC. The potency with which EGF and carbachol activated MAP kinase was similar to the potency with which they inhibited vasopressin-stimulated cAMP accumulation. To assess the role of G(i) proteins in these stimulatory events, RIMCT cells were pretreated with pertussis toxin to inhibit G(i)-mediated signaling. Pertussis toxin did not influence ATP- or EGF-stimulated MAP kinase, but completely inhibited carbachol stimulation, suggesting that G(i) proteins mediate muscarinic stimulation. Prolonged exposure of RIMCT cells to high phorbol ester concentrations to downregulate PKC ablated carbachol- and ATP-stimulated MAP kinase, but not EGF-stimulated MAP kinase, suggesting that PKC is a component of the network involved in MAP kinase activation by purinergic and muscarinic agonists. Investigation of the sidedness of the hormonal stimulations indicated that EGF-stimulated MAP kinase was highly polarized, occurring exclusively from the basolateral surface, whereas carbachol stimulated MAP kinase similarly from either cell surface. Thus EGF, ATP, and carbachol, three hormones that negatively influence vasopressin-stimulated water transport in the collecting duct, signal MAP kinase activation through distinct mechanisms. The findings highlight the MAP kinases and their upstream activators as a potential pathway, in addition to PKC, involved in hormonal regulation of vasopressin action in the collecting duct.