Figure 1.
Effect of ouabain and K+-free medium on intracellular Na+ (a–c) and K+ (d–f) content in HeLa (a,d), HUVEC (b,e) and RVSMC (c,f).
Cells were incubated in control or K+-free medium during 3 hr and ouabain was added at a final concentration of 3 µM (HeLa and HUVEC) or 3 mM (RVSMC). Mean ± S.E. values of experiments performed in quadruplicate are shown.
Table 1.
Lactate dehydrogenase release (LDH, %), caspase-3 activity (CAS, nmol per mg of protein per hr) and chromatin cleavage (CHR, %) in cells treated with ouabain, K+-free and Ca2+-free medium during 4 hr.
Figure 2.
Comparative analysis of the actions of Na+,K+-ATPase inhibition by ouabain and K+-free medium on HeLa, HUVEC and RVSMC transcriptomes.
A. Principal component analysis of the transcriptomes of HeLa, HUVEC and RVSMC. Cells were incubated for 3 hr in control Ca2+-containing media and processed for oligonucleotide microarray analysis as indicated in the Methods section. Ouabain was added at a final concentration of 3 µM (HeLa and HUVEC) or 3 mM (RVSMC). All experiments are repeated 4 times. Ellipsoids highlight portioning of samples based on the type of treatment. The principal components in 3-dimensional graphs (PC#1, PC#2 and PC#3) represent the variability of gene expression level within datasets. The total percentage of PCA mapping variability is shown on top. B. The total number of genes whose expression is altered by ouabain and K+-free medium by more than 1.2-fold with p<0.05 is indicated; numbers of genes affected by both stimuli appear in bold.
Figure 3.
Kinetics of elevation of intracellular Na+ in HeLa, HUVEC and RVSMC triggered by ouabain (A) or K+-free medium (B).
Ouabain was added at a final concentration of 3 µM (HeLa and HUVEC) or 3 mM (RVSMC). Intracellular Na+ content in the absence of Na+,K+-ATPase inhibitors was taken as 100%. Means obtained in experiments performed in triplicate are shown.
Table 2.
Total numbers of differentially expressed transcripts in HeLa, HUVEC and RVSMC in 3-hr of Na+,K+-ATPase inhibition in control (Ca2+ containing) medium.
Figure 4.
Correlation analysis of transcripts whose expression is altered by ouabain and K+-free medium in HeLa, HUVEC and RVSMC by more than by 1.2-fold with p<0.05.
Cells were incubated during 3 hr; ouabain was added at a final concentration of 3 µM (HeLa and HUVEC) or 3 mM (RVSMC). Incubation medium contains 1.8 mM CaCl2. The total number of transcripts subjected to analysis is shown in Figure 2B. Transcript expression in control cells was taken as 1.00.
Figure 5.
Verification of microarray results by quantitative RT-PCR.
Gene expression was quantified for 3 hr incubation of control and Ca2+-depleted HUVEC in the presence of 3 µM ouabain or in K+-free medium. Mean values obtained in 4 independent experiments are shown. Egr1 - early growth response protein 1, Ptgs2 - prostaglandin-endoperoxide synthase 2, Ppp1r15a - protein phosphatase 1, regulatory (inhibitor) subunit 15A.
Figure 6.
Na+i,K+i-sensitive transcriptomes identified in control Ca2+-containing media.
A. Pie-chart showing the numbers of Na+i,K+i-sensitive genes detected in HeLa, HUVEC and RVSMC and ubiquitous Na+i,K+i-sensitive genes found in all 3 cell types. B. Distribution of ubiquitous Na+i,K+i-sensitive genes among major functional groups. Digitals shown in italics correspond to gene numbers for each functional group.
Table 3.
Ubiquitous Na+i/K+i-sensitive genes whose expression was up- and down-regulated by more than 1.2-fold (p≤0.05) in control (Ca2+-containing) medium.
Table 4.
HeLa cells: the list of genes whose expression was changed by more than 4-fold in 3 hr of Na+,K+-ATPase inhibition in control (Ca2+ containing) medium.
Table 5.
HUVEC: the list of genes whose expression was changed in by more than 4-fold in 3 hr of Na+,K+-ATPase inhibition in control (Ca2+ containing) medium.
Table 6.
RVSMC: the list of genes whose expression was changed by more than 4-fold in 3 hr of Na+,K+-ATPase inhibition in control (Ca2+ containing) medium.
Figure 7.
Effect of EGTA and BAPTA on Ca2+ signalling triggered by activation of purinergic receptors.
A. Representative records showing kinetics of elevation of intracellular Ca2+-concentration (F340/F360 ratio) in HeLa cells triggered by addition of 100 µM ATP. 1 – Control (Ca2+-containing medium); 2 – cells were preincubated for 10 min in Ca2+-free medium containing 50 µM EGTA and 10 µM BAPTA-AM. B. Baseline and maximal values of [Ca2+]i in ATP-treated HeLa, HUVEC and RVSMC in control and Ca2+-free medium containing 50 µM EGTA and 10 µM BAPTA-AM. Mean ± S.E. values obtained in 4 experiments are shown.
Figure 8.
Comparative analysis of the actions of Na+,K+-ATPase inhibition by ouabain and K+-free medium on the transcriptome of Ca2+-depleted HeLa, HUVEC and RVSMC.
A. Principal component analysis of HeLa, HUVEC and RVSMC transcriptomes. Cells were incubated for 3 hr in Ca2+-free medium containing 50 µM EGTA and 10 µM BAPTA-AM and processed for oligonucleotide microarray analysis as indicated in the Methods section. Ouabain was added at a final concentration of 3 µM (HeLa and HUVEC) or 3 mM (RVSMC). Ellipsoids highlight portioning of samples based on the type of cell treatment. The principal components in 3-dimensional graphs (PC#1, PC#2 and PC#3) represent the variability in gene expression level within datasets. The total percentage of PCA mapping variability is shown on top. B. Total numbers of genes whose expression is altered by ouabain and K+-free medium by more than 1.2-fold with p<0.05 are indicated; numbers of genes affected by both stimuli appears in bold.
Figure 9.
Correlation analysis of transcripts whose expression is altered by ouabain and K+-free medium in Ca2+-depleted HeLa, HUVEC and RVSMC more than by 1.2-fold with p<0.05.
Cells were incubated during 3 hr in Ca2+-free medium containing 50 µM EGTA and 10 µM BAPTA-AM. Ouabain was added at a final concentration of 3 µM (HeLa and HUVEC) or 3 mM (RVSMC). The total number of transcripts subjected to analysis is shown in Figure 8B. Transcript expression in control cells was taken as 1.00.
Figure 10.
Na+i,K+i-sensitive transcriptomes identified in Ca2+-depleted cells.
A. Pie-chart showing the numbers of and cell type-specific, Na+i,K+i-sensitive genes detected in HeLa, HUVEC and RVSMC and ubiquitous Na+i,K+i-sensitive genes found in all 3 types of cells. Experiments were performed in Ca2+-free medium containing 50 µM EGTA and 10 µM BAPTA-AM. B. The distribution of ubiquitous and cell type-specific Na+i,K+i-sensitive among major functional groups. Digitals shown in italics correspond to gene numbers in each functional group.
Table 7.
Total numbers of differentially expressed transcripts in HeLa, HUVEC and RVSMC in 3-hr of Na+,K+-ATPase inhibition in Ca2+-free medium containing extra- and intracellular Ca2+ chelators.
Figure 11.
Pie-chart showing the action of Ca2+ depletion on Na+i,K+i-sensitive transcriptomes.
Ca2+-depletion was triggered by omission of extracellular Ca2+ and addition of 50 µM EGTA and 10 µM BAPTA-AM. The numbers of ubiquitous Na+i,K+i-sensitive genes and Na+i,K+i-sensitive genes detected in HUVEC, HeLa and RVCSM are shown in italics.
Table 8.
Intracellular concentration of BAPTA, Ca2+ and expression of EGR1, PTGS2 and PPP1R15A in HeLa cells.
Table 9.
Genes whose expression was changed in HeLa, HUVEC and RVSMC by more than 1.2-fold (p≤0.05) in 3 hr of Na+,K+-ATPase inhibition in Ca2+-free medium containing extra- and intracellular Ca2+ chelators.
Figure 12.
Effect of Ca2+-depletion on expression of ubiquitous Na+i,K+i-sensitive genes in HeLa, HUVEC and RVSMC.
Cells were incubated during 3 hrs in control medium containing 1.8 mM CaCl2 or in Ca2+-containing K+-free medium. To trigger Ca2+-depletion, CaCl2 was omitted and 50 µM EGTA and 10 µM BAPTA-AM were added. Increments of gene expression triggered by Na+,K+-ATPase inhibition in Ca2+-containing medium were taken as 100%. For absolute values of gene expression, see Tables 3 and 8.
Figure 13.
The signaling network possessing the highest score of 48 for association with differential expression of ubiquitous Na+i,K+i-sensitive genes.
The p-value and geometric fold change for each gene listed in Table 3 were overlaid onto a global molecular network developed from information within the IPA Knowledge Base. Genes are represented as nodes of various shapes to represent the functional category of gene product as shown in the top corner, and the biological relationship between two nodes is represented as a line. The up-regulated Na+i,K+i-sensitive genes are shown in pink. Note that network's Score of 48 is equal to -log(Fisher's Exact test result). It means that there is a 1 in 1048 chance of getting a network from Ingenuity Knowledge Base containing at least the same number of eligible molecules by chance when randomly picking molecules derived form 80 ubiquitous Na+i,K+i-sensitive genes listed in Table 3.
Figure 14.
Top significantly altered biological functions associated with differential expression of ubiquitous Na+i,K+i-sensitive genes.
The p-value and geometric fold change for each gene listed in Table 3 were imported into Ingenuity Pathway Analysis. The significance criteria with a threshold of p = 0.05 (or 1.3 when expressed as -log(p-value) is shown by line.
Figure 15.
Mechanisms underlying transcriptomic alterations in normal and pathophysiological conditions: a working hypothesis.
Activation of Na+-permeable channels such as NMDA receptors (1), Na+/H+ exchanger (2), voltage-gated K+ channels (6) and inhibition of Na+,K+-ATPase (4) lead to elevation of the [Na+]i/[K+]i ratio. In cells abundant with Na+/Ca2+ exchanger (3), the dissipation of transmembrane gradients of monovalent cations is accompanied by elevation of [Ca2+]i. Elevation of the [Na+]i/[K+]i ratio affects the expression of X1,2,3… and Z1,2,3… genes via activation of unknown Na/K sensor(s) and Na/K response elements (Na/KRE). Expression of Z1,2,3… genes is also subjected to regulation by elevated [Ca2+]i via its interaction with calmodulin (CaM) and other Ca2+i sensors and diverse Ca2+-response elements (CaRE), whereas Y1,2,3… genes lacking Na/KRE are controlled by [Ca2+]i only. The set of Na+i,K+i-sensitive transcription regulators shown as Xn, Yn and Zn contributes to overall transcriptomic changes via activation of canonical response elements (RE) within V1,2,3… genes. Autocrine pathways triggered by the release of interleukin 6 and other [Na+]i/[K+]i-sensitive regulators of gene expression (Zm) may also contribute to overall transcriptomic changes via activation of their receptors (7).
Figure 16.
Disorders significantly associated with differential expression of ubiquitous Na+i,K+i-sensitive genes.
The p-value and geometric fold change for each genes listed in Table 3 were imported into Ingenuity Pathway Analysis. The criteria with a threshold for significance of p = 0.05 (or 1.3 when expressed as -log(p-value) is shown by line.