Elevation of Intracellular Na+ Contributes to Expression of Early Response Genes Triggered by Endothelial Cell Shrinkage

Abstract

Background/aims: Prolonged hyperosmotic shrinkage evokes expression of osmoprotective genes via nuclear factor NFAT5-mediated pathway and activates Na⁺ influx via hypertonicity-induced cation channels (HICC). In human umbilical vein endothelial cells (HUVEC) elevation of intracellular sodium concentration ([Na⁺]_i) triggers transcription of dozens of early response genes (ERG). This study examined the role of monovalent cations in the expression of Na⁺_i-sensitive ERGs in iso- and hyperosmotically shrunken HUVEC.

Methods: Cell volume was measured by 3D reconstruction of cell shape and as ¹⁴C-urea available space. Intracellular Na⁺ and K⁺ content was measured by flame atomic absorption spectrometry. ERG transcription was estimated by RT-PCR.

Results: Elevation of medium osmolality by 150 mM mannitol or cell transfer from hypo- to isosmotic medium decreased cell volume by 40-50%. Hyperosmotic medium increased [Na⁺]_i by 2-fold whereas isosmotic shrinkage had no impact on this parameter. Hyperosmotic but not isosmotic shrinkage increased up-to 5-fold the content of EGR1, FOS, ATF3, ZFP36 and JUN mRNAs. Expression of these ERGs triggered by hyperosmotic shrinkage and Na⁺,K⁺-ATPase inhibition by 0.1 µM ouabain exhibited positive correlation (R²=0.9383, p=0.0005). Isosmotic substitution of NaCl by N-methyl-D-glucamine abolished an increment of [Na⁺]_i and ERG expression triggered by mannitol addition.

Conclusion: Augmented expression of ERGs in hyperosmotically shrunken HUVEC is mediated by elevation of [Na⁺]_i.

Keywords: Cell shrinkage; Early response genes; Endothelium; Intracellular Na+; Ouabain; Transcription.