Abstract
The type 1 sodium-hydrogen exchanger (NHE-1) is a ubiquitous electroneutral membrane transporter that is activated by hypertonicity in many cells. NHE-1 may be an important pathway for Na(+) entry during volume restoration, yet the molecular mechanisms underlying the osmotic regulation of NHE-1 are poorly understood. In the present study we conducted a screen for important signaling molecules that could be involved in hypertonicity-induced activation of NHE-1 in CHO-K1 cells. Hypertonicity rapidly activated NHE-1 in a concentration-dependent manner as assessed by proton microphysiometry and by measurements of intracellular pH on a FLIPR (fluorometric imaging plate reader). Inhibitors of Ca(2+)/calmodulin (CaM) and Janus kinase 2 (Jak2) attenuated this activation, whereas neither calcium chelation nor inhibitors of protein kinase C, the Ras-ERK1/2 pathway, Src kinase, and Ca(2+)/calmodulin-dependent enzymes had significant effects. Hypertonicity also resulted in the rapid tyrosine phosphorylation of Jak2 and STAT3 (the major substrate of Jak2) and CaM. Phosphorylation of Jak2 and CaM were blocked by AG490, an inhibitor of Jak2. Immunoprecipitation studies showed that hypertonicity stimulates the assembly of a signaling complex that includes CaM, Jak2, and NHE-1. Formation of the complex could be blocked by AG490. Thus, we propose that hypertonicity induces activation of NHE-1 in CHO-K1 cells in large part through the following pathway: hypertonicity --> Jak2 phosphorylation and activation --> tyrosine phosphorylation of CaM --> association of CaM with NHE-1 --> NHE-1 activation.
Highlights
The type 1 sodium-hydrogen exchanger (NHE-1) is a ubiquitous electroneutral membrane transporter that is activated by hypertonicity in many cells
The increased extracellular acidification rate (ECAR) induced by exposure to hypertonic media was both sodium-dependent and inhibitable by an NHE inhibitor, suggesting the involvement of NHE-1 (CHO-K1 cells only express NHE-1)
In the current report we studied the regulation of NHE-1 activity by hypertonicity in CHO-K1 cells
Summary
Hypertonicity-induced shrinkage of mammalian cells is a powerful stimulant for many protein kinases that could play important direct or indirect roles in activating NHE-1 These include mitogen-activated protein kinases such as extracellular signal-regulated protein kinase (ERK), stress-activated protein kinases (c-Jun N-terminal kinases) [7,8,9,10], Src family tyrosine kinases p59fgr and p56/59hck [11], protein kinase C [12,13,14], Janus kinase [15], and phosphatidylinositol 3-kinase [13], there is not a consensus that any of those kinases mediate hypertonicity-induced activation of NHE-1. Our results demonstrate that this pathway may be a fundamental mechanism for the rapid regulation of NHE-1 in multiple cell types
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