Ouabain at Low Concentrations Affects Transcription without Any Impact on Intracellular Content of Sodium and Potassium in Rat Brain Neurons

Abstract—

In several types of cells, inhibition of ubiquitous α1-isoform of the Na⁺,K⁺-ATPase leads to drastic transcriptomic changes mediated by dissipation of transmembrane concentration gradients of monovalent cations. In the present study, we employed the sharp differences in the affinity of α1- and α3-Na⁺,K⁺-ATPase for ouabain to examine the role of α3 isoform in the regulation of intracellular Na⁺ and K⁺ content and gene expression in primary cultures of rat cerebellum granule cells. Addition of 100 nM ouabain decreased the Na⁺,K⁺-ATPase activity by 20% due to the inhibition of α3 isosyme. At this concentration, ouabain changed transcription of 17 genes with maximal ~2-fold activation and 1.5-fold inhibition. The full-scale inhibition of α1- and α3-Na⁺,K⁺-ATPase by 1 mM ouabain was accompanied by a ~50-fold elevation of the [Na⁺]_i/[K⁺]_i ratio and altered the content of mRNA encoding 673 genes with maximal 20-fold activation and 3-fold inhibition. Unlike 1 mM ouabain, 100 nM ouabain did not affect phosphorylation of the Ca²⁺-sensitive transcription regulator CREB. Our results show that transcriptomic changes in neurons subjected to inhibition of α3-Na⁺,K⁺-ATPase by low doses of ouabain are not mediated by elevation of the [Na⁺]_i/[K⁺]_i, ratio and activation-sensitive mechanisms of excitation–transcription coupling.