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Hypertonicity activates Na+/H+ exchange through Janus kinase 2 and calmodulin
Maria N Garnovskaya1, Yurii V Mukhin, Tamara M Vlasova
1Medical and Research Services, Ralph H. Johnson Veterans Affairs Medical Center, Charleston, South Carolina 29425, USA. garnovsk@musc.edu
The Journal of Biological Chemistry
|March 11, 2003
Summary
Hypertonicity activates the sodium-hydrogen exchanger (NHE-1) via a pathway involving Janus kinase 2 (Jak2) and calmodulin (CaM). This signaling cascade, crucial for cell volume regulation, highlights novel molecular mechanisms.
Area of Science:
- Cellular Physiology
- Membrane Transport
- Signal Transduction
Background:
- The type 1 sodium-hydrogen exchanger (NHE-1) is a critical electroneutral transporter involved in maintaining intracellular pH and cell volume.
- Osmotic regulation of NHE-1 activity is vital for cellular homeostasis, yet the underlying molecular mechanisms remain incompletely understood.
- Understanding these mechanisms is essential for various physiological processes, including cell volume restoration.
Purpose of the Study:
- To identify key signaling molecules involved in the hypertonicity-induced activation of NHE-1 in Chinese Hamster Ovary K1 (CHO-K1) cells.
- To elucidate the molecular pathway mediating osmotic regulation of NHE-1.
- To investigate the roles of calcium/calmodulin (CaM) and Janus kinase 2 (Jak2) in this process.
Main Methods:
- Utilized proton microphysiometry and fluorometric imaging plate reader (FLIPR) to assess NHE-1 activity.
- Employed inhibitors for various signaling pathways, including Ca(2+)/calmodulin (CaM), Janus kinase 2 (Jak2), protein kinase C, Ras-ERK1/2, and Src kinase.
- Conducted immunoprecipitation studies to analyze protein-protein interactions and Western blotting to detect protein phosphorylation.
Main Results:
- Hypertonicity rapidly activated NHE-1 in a concentration-dependent manner in CHO-K1 cells.
- Inhibitors of Ca(2+)/calmodulin (CaM) and Janus kinase 2 (Jak2) significantly attenuated hypertonicity-induced NHE-1 activation.
- Hypertonicity led to tyrosine phosphorylation of Jak2 and CaM, and stimulated the assembly of a signaling complex involving CaM, Jak2, and NHE-1.
Conclusions:
- Hypertonicity activates NHE-1 in CHO-K1 cells primarily through a pathway involving Jak2 phosphorylation and activation.
- This activation is mediated by the tyrosine phosphorylation of CaM, its association with NHE-1, and subsequent NHE-1 activation.
- The findings reveal a novel signaling cascade for osmotic regulation of NHE-1, implicating Jak2 and CaM.