Induction of Tyrosine Phosphorylation and Na+/H+Exchanger Activation during Shrinkage of Human Neutrophils

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.