Hypertonicity Activates Na+/H+ Exchange through Janus Kinase 2 and Calmodulin

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 TM (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؉ /calmodulindependent 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 3 Jak2 phosphorylation and activation 3 tyrosine phosphorylation of CaM 3 association of CaM with NHE-1 3 NHE-1 activation.The ubiquitous isoform of the Na ϩ /H ϩ exchanger (NHE-1) 1 is essential for the regulation of cellular volume and intracellular pH. NHE-1 is nearly quiescent in resting cells but is activated by a variety of hormones and growth factors (1, 2). NHE-1 is also rapidly activated by hypertonic stress in many cells (3), and this may be an important pathway for Na ϩ entry during volume restoration. Despite the potential importance of this process, the molecular mechanisms underlying the regulation of NHE-1 by hypertonicity have not been fully elucidated. The rapid activation of NHE-1 is often associated with an increase in its phosphorylation (3). Kinases that have been shown to directly phosphorylate NHE-1 include p90 S6 kinase (4) and the Nck-interacting kinase (5). However, deletion of the major phosphorylation sites contained within residues 636 -815 of NHE-1 only reduces its response to growth factors by about 50% (6), suggesting that mechanisms of regulation other than direct phosphorylation of NHE-1 are also important.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) (7-10), Src family tyrosine kinases p59 fgr and p56/59 hck (11), protein kinase C (12-14), Jan...

Dynamic Effects of Hg2+-induced Changes in Cell Volume

“…A schematic diagram for the ionic pathways identified from the CHO cells are shown in Fig. 1 [25][26][27][28][29][30]. The sensor chip allows us to repeatedly expose cultured cells to mercurials and other reagents and to follow the response in real time.…”

Signalling pathways involved in hypertonicity- and acidification-induced activation of Na+/H+ exchange in trout hepatocytes

“…In parallel with its reduction in the intracellular alkalinization, calmidazolium reduced the hypertonicityinduced proton secretion rate by 15% in the trout cells (Fig.·7B). This is considerably less than the 55 to 94% inhibition of hypertonicity-induced proton secretion seen in CHO-K1 cells exposed to various CaM inhibitors (Garnovskaya et al, 2003b). Compared with the severe reduction in alkalinization, indicating that a significant amount of protons remained in the cytoplasm during hypertonic stress in the presence of calmidazolium, the reduction in the rate of proton secretion was smaller than might be expected.…”

“…Ca 2+ -calmodulin (CaM) has been shown to be involved in the activation process of NHE-1 in response to a variety of stimuli including mitogenic factors, ionomycin and serotonin (Wakabayashi et al, 1994;Bertrand et al, 1994;Garnovskaya et al, 2003a), and, importantly, osmotic shrinkage (Dascalu et al, 1992;Shrode et al, 1995;Shrode et al, 1997;Garnovskaya et al, 2003b). Consistent with the latter studies, our data confirmed an important role of a CaMdependent pathway in the hypertonicity-induced NHE-1 activation in trout hepatocytes, as the inhibition of CaM by calmidazolium reduced the hypertonicity-induced pHi increase by 54% (Fig.·7A).…”

Cell volume regulation in mammalian oocytes and preimplantation embryos

“…One tyrosine kinase activated by decreased cell volume is Janus kinase 2 (Jak2) (Gatsios et al, 1998). Jak2 has been found to be required for full activation of NHE1 by decreased cell volume in Chinese Hamster Ovary (CHO) cells, where Jak2 activates NHE1 through phosphorylating and activating Calmodulin (CaM), which then binds to and activates NHE1 (Garnovskaya et al, 2003). This mechanism also appears to operate in mouse preimplantation embryos, where activation and tyrosine phosphorylation of Jak2 is markedly increased upon a cell volume decrease, and expression of a dominant negative Jak2 or pharmacological Jak2 or CaM inhibitors block NHE1 activation (Zhou and Baltz, 2012).…”