Osmotic stimulation of the Na+/H+ exchanger NHE1: Relationship to the activation of three MAPK pathways

The Na+/H+ exchanger (NHE) becomes activated by hyperosmolar stress, thereb y contributing to cell volume regulation. The signaling pathway(s) responsi ble for the shrinkage-induced activation of NHE, however, remain unknown. A family of mitogen-activated protein kinases (MAPK), encompassing p42/p44 E rk, p38 MAPK and SAPK, has been implicated in a variety of cellular respons es to changes in osmolarity. We therefore investigated whether these kinase s similarly signal the hyperosmotic activation of NHE. The time course and osmolyte concentration dependence of hypertonic activation of NHE and of th e three sub-families of MAPK were compared in U937 cells. The temporal cour se and dependence on osmolarity of Eric and p38 MAPK activation were found to be similar to that of NHE stimulation. However, while pretreatment of U9 37 cells with the kinase inhibitors PD98059 and SB203580 abrogated the osmo tic activation of Erk and p38 MAPK, respectively, it did not prevent the as sociated stimulation of NHE. Thus, Erk1/2 and/or p38 MAPK are unlikely to m ediate the osmotic regulation of NHE. The kinetics of NHE activation by hyp erosmolarity appeared to precede SAPK activation. In addition, hyperosmotic activation of NHE persisted in mouse embryonic fibroblasts lacking SEK1/MK K4, an upstream activator of SAPK. Moreover, shrinkage-induced activation o f NHE still occurred in COS-7 cells that were transiently transfected with a dominant-negative form of SEK1/MKK4 (SEK1/MKK4-A/L) that is expected to i nhibit other isoforms of SEK as well. Together, these results demonstrate t hat the stimulation of NHE and the activation of Erk, p38 MAPK and SAPK are parallel but independent events.