Induction of tyrosine phosphorylation and Na+/H+ exchanger activation during shrinkage of human neutrophils.

Eric Krump,Kaliopi Nikitas,Sergio Grinstein

doi:10.1074/jbc.272.28.17303

Eric Krump, Kaliopi Nikitas + Show 1 more

Open Access

https://doi.org/10.1074/jbc.272.28.17303

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Abstract

The ubiquitous isoform of the Na+/H+ exchanger (NHE1) is essential for the regulation of cellular volume. The underlying molecular mechanism, which is poorly understood, was studied in human polymorphonuclear leukocytes (PMN). Suspension of PMN in hypertonic media induced rapid cellular shrinkage and activation of NHE1, which is measurable as a cytosolic alkalinization. Concomitantly, hypertonic stress also induced extensive tyrosine phosphorylation of several proteins. Pretreatment of PMN with genistein, a tyrosine kinase inhibitor, prevented not only the tyrosine phosphorylation in response to a hypertonic shock but also the activation of NHE1. The signal elicited by hyperosmolarity that induces activation of tyrosine kinases and NHE1 was investigated. Methods were devised to change medium osmolarity without altering cell volume and vice versa. Increasing medium and intracellular osmolarity in normovolemic cells failed to activate tyrosine kinases or NHE1. However, shrinkage of cells under iso-osmotic conditions stimulated both tyrosine phosphorylation and NHE1 activity. These findings imply that cells detect alterations in cell size but not changes in osmolarity or ionic strength. The identity of the proteins that were tyrosine-phosphorylated in response to cell shrinkage was also investigated. Unexpectedly, the mitogen-activated protein kinases SAPK, p38, erk1, and erk2 were not detectably phosphorylated or activated. In contrast, the tyrosine kinases p59(fgr) and p56/59(hck) were phosphorylated and activated upon hypertonic challenge. We propose that cells respond to alterations in cell size, but not to changes in osmolarity, with increased tyrosine phosphorylation, which in turn leads to the activation of NHE1. The resulting changes in ion content and cytosolic pH contribute to the restoration of cell volume in shrunken cells.

Highlights

The Naϩ/Hϩ exchanger isoform 1 (NHE1)1 is a ubiquitously expressed cation antiporter that is involved in the regulation of
We investigated the relationship between the stimulation of protein kinases and the activation of NHE1, and we attempted to determine whether reduced cell volume or increased cytosolic osmolarity were the signals leading to the activation of these effectors
The current study investigated the mechanism that regulates the activation of a major, volume-sensitive ion transporter in human polymorphonuclear leukocytes (PMN), namely NHE1

Summary

EXPERIMENTAL PROCEDURES

Materials—Dextran T-500 and Ficoll-Paque were from Pharmacia Biotech Inc. Genistein and erbstatin analog were from Calbiochem. Isotonic NaCl buffer contained (in mM) 5 KCl, 10 glucose, 140 NaCl, 1 CaCl2, 1 MgCl2, 10 Hepes, pH 7.4. Hypertonic NaCl buffer contained 5 KCl, 10 glucose, 240 NaCl, 1 CaCl2, 1 MgCl2, and 10 Hepes, pH 7.4. Iso-osmotic sucrose buffer contained 5 KCl, 10 glucose, 280 sucrose, 1 CaCl2, 1 MgCl2, and 10 Hepes, pH 7.4. Isolation of cells was performed using dextran sedimentation and centrifugation on Ficoll-Paque cushions as described previously [16]. The cell pellet was dissolved in ice-cold Nonidet P-40 buffer containing protease and phosphatase inhibitors (1 mM phenylmethylsulfonyl fluoride, 10 ␮g/ml pepstatin, 10 ␮g/ml leupeptin, 10 ␮g/ml aprotinin, 10 ␮M sodium vanadate, and 1 mM NaF) and kept on ice for at least 10 min. A score of p Ͻ 0.05 was considered significant

RESULTS

Role of Osmolarity in the Induction of Tyrosine Phosphorylation

Role of Cell Shrinkage in the Induction of Tyrosine Phosphorylation

DISCUSSION