Transcriptomic changes in Ca²⁺-depleted cells: Role of elevated intracellular [Na⁺]/[K⁺] ratio

Previously, we reported that Ca(2+) depletion increased permeability of the plasma membrane for Na(+). This study examined the relative impact of [Na(+)]i/[K(+)]i-mediated signaling on transcriptomic changes in cultured vascular smooth muscle cells from rat aorta (VSMC) subjected to Ca(2+)-depletion by extra-(EGTA) and intracellular (BAPTA-AM) Ca(2+) chelators. Na(+),K(+)-ATPase inhibition in K(+)-free medium during 3 h led to elevation of [Na(+)]i and attenuation of [K(+)]i by ∼7- and 10-fold, whereas Ca(2+)-depletion resulted in alteration of these parameters by ∼3- and 2-fold, respectively. Augmented VSMC permeability for Na(+) and elevation of the [Na(+)]i/[K(+)]i ratio was triggered by addition to Ca(2+)-free medium 50 μM EGTA and was not affected by 10 μM BAPTA-AM. Na(+),K(+)-ATPase inhibition and Ca(2+)-depletion changed expression of 3677 and 4610 mRNA transcripts, respectively. We found highly significant (p<10(-12)) positive (R(2)>0.51) correlation between levels of expression of 2071 transcripts whose expression was affected by both stimuli. Among genes whose expression in Ca(2+)-depleted cells was augmented by more than 7-fold we noted cyclic AMP-dependent transcription factor Atf3, early growth response protein Egr1 and nuclear receptor subfamily 4, group A member Nr4a1. Dissipation of transmembrane gradients of monovalent cations in high-K(+), low-Na(+)-medium abolished the increments of the [Na(+)]i/[K(+)]i ratio as well as the augmented expression of these genes triggered by incubation of VSMC in EGTA containing medium. Thus, our results demonstrate, for the first time, that robust transcriptomic changes triggered by Ca(2+)-depletion in the presence of extracellular Ca(2+)-chelators are at least partially mediated by elevation of the [Na(+)]i/[K(+)]i ratio and activation of Ca(2+)i-independent, [Na(+)]i/[K(+)]i-mediated mechanism of excitation-transcription coupling. These results shad a new light on analysis of data obtained in cells subjected to long-term exposure to Ca(2+) chelators.