Time- and dose dependent actions of cardiotonic steroids on transcriptome and intracellular content of Na+ and K+: a comparative analysis

Recent studies demonstrated that in addition to Na+,K+-ATPase inhibition cardiotonic steroids (CTSs) affect diverse intracellular signaling pathways. This study examines the relative impact of [Na+]i/[K+]i-mediated and -independent signaling in transcriptomic changes triggered by the endogenous CTSs ouabain and marinobufagenin (MBG) in human umbilical vein endothelial cells (HUVEC). We noted that prolongation of incubation increased the apparent affinity for ouabain estimated by the loss of [K+]i and gain of [Na+]i. Six hour exposure of HUVEC to 100 and 3,000 nM ouabain resulted in elevation of the [Na+]i/[K+]i ratio by ~15 and 80-fold and differential expression of 258 and 2185 transcripts, respectively. Neither [Na+]i/[K+]i ratio nor transcriptome were affected by 6-h incubation with 30 nM ouabain. The 96-h incubation with 3 nM ouabain or 30 nM MBG elevated the [Na+]i/[K+]i ratio by ~14 and 3-fold and led to differential expression of 880 and 484 transcripts, respectively. These parameters were not changed after 96-h incubation with 1 nM ouabain or 10 nM MBG. Thus, our results demonstrate that elevation of the [Na+]i/[K+]i ratio is an obligatory step for transcriptomic changes evoked by CTS in HUVEC. The molecular origin of upstream [Na+]i/[K+]i sensors involved in transcription regulation should be identified in forthcoming studies

Deoxygenation affects composition of membrane-bound proteins in human erythrocytes

Background/Aims: ATP release from erythrocyte plays a key role in hypoxia-induced elevation of blood flow in systematic circulation. We have previously shown that hemolysis contributes to erythrocyte ATP release triggered by several stimuli, including hypoxia, but the molecular mechanisms of hypoxia-increased membrane fragility remain unknown. Methods: In this study, we compared the action of hypoxia on hemolysis, ATP release and the composition of membrane-bound proteins in human erythrocytes. Results: Twenty minutes incubation of human erythrocytes in the oxygen-free environment increased the content of extracellular hemoglobin by ∼1.5 fold. Paired measurements of hemoglobin and ATP content in the same samples, showed a positive correlation between hemolysis and ATP release. Comparative analysis of SDS-PAGE electrophoresis of erythrocyte ghosts obtained under control and deoxygenated conditions revealed a ∼2-fold elevation of the content of membrane-bound protein with Mr of ∼60 kDa. Conclusion: Deoxygenation of human erythrocytes affects composition of membrane-bound proteins. Additional experiments should be performed to identify the molecular origin of 60 kDa protein and its role in the attenuation of erythrocyte integrity and ATP release in hypoxic conditions

Time- and dose dependent actions of cardiotonic steroids on transcriptome and intracellular content of Na+ and K+: a comparative analysis

Recent studies demonstrated that in addition to Na+,K+-ATPase inhibition cardiotonic steroids (CTSs) affect diverse intracellular signaling pathways. This study examines the relative impact of [Na+]i/[K+]i-mediated and -independent signaling in transcriptomic changes triggered by the endogenous CTSs ouabain and marinobufagenin (MBG) in human umbilical vein endothelial cells (HUVEC). We noted that prolongation of incubation increased the apparent affinity for ouabain estimated by the loss of [K+]i and gain of [Na+]i. Six hour exposure of HUVEC to 100 and 3,000 nM ouabain resulted in elevation of the [Na+]i/[K+]i ratio by ~15 and 80-fold and differential expression of 258 and 2185 transcripts, respectively. Neither [Na+]i/[K+]i ratio nor transcriptome were affected by 6-h incubation with 30 nM ouabain. The 96-h incubation with 3 nM ouabain or 30 nM MBG elevated the [Na+]i/[K+]i ratio by ~14 and 3-fold and led to differential expression of 880 and 484 transcripts, respectively. These parameters were not changed after 96-h incubation with 1 nM ouabain or 10 nM MBG. Thus, our results demonstrate that elevation of the [Na+]i/[K+]i ratio is an obligatory step for transcriptomic changes evoked by CTS in HUVEC. The molecular origin of upstream [Na+]i/[K+]i sensors involved in transcription regulation should be identified in forthcoming studies

Deoxygenation affects composition of membrane-bound proteins in human erythrocytes

Background/Aims: ATP release from erythrocyte plays a key role in hypoxia-induced elevation of blood flow in systematic circulation. We have previously shown that hemolysis contributes to erythrocyte ATP release triggered by several stimuli, including hypoxia, but the molecular mechanisms of hypoxia-increased membrane fragility remain unknown. Methods: In this study, we compared the action of hypoxia on hemolysis, ATP release and the composition of membrane-bound proteins in human erythrocytes. Results: Twenty minutes incubation of human erythrocytes in the oxygen-free environment increased the content of extracellular hemoglobin by ∼1.5 fold. Paired measurements of hemoglobin and ATP content in the same samples, showed a positive correlation between hemolysis and ATP release. Comparative analysis of SDS-PAGE electrophoresis of erythrocyte ghosts obtained under control and deoxygenated conditions revealed a ∼2-fold elevation of the content of membrane-bound protein with Mr of ∼60 kDa. Conclusion: Deoxygenation of human erythrocytes affects composition of membrane-bound proteins. Additional experiments should be performed to identify the molecular origin of 60 kDa protein and its role in the attenuation of erythrocyte integrity and ATP release in hypoxic conditions