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The Na,K-ATPase-dependent src kinase signaling changes with mesenteric artery diameter

International Journal of Molecular Sciences (2018) 19(9)
DOI: 10.3390/ijms19092489
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Abstract

Inhibition of the Na,K-ATPase by ouabain potentiates vascular tone and agonist-induced contraction. These effects of ouabain varies between different reports. In this study, we assessed whether the pro-contractile effect of ouabain changes with arterial diameter and the molecular mechanism behind it. Rat mesenteric small arteries of different diameters (150–350 µm) were studied for noradrenaline-induced changes of isometric force and intracellular Ca2+ in smooth muscle cells. These functional changes were correlated to total Src kinase and Src phosphorylation assessed immunohistochemically. High-affinity ouabain-binding sites were semi-quantified with fluorescent ouabain. We found that potentiation of noradrenaline-sensitivity by ouabain correlates positively with an increase in arterial diameter. This was not due to differences in intracellular Ca2+ responses but due to sensitization of smooth muscle cell contractile machinery to Ca2+. This was associated with ouabain-induced Src activation, which increases with increasing arterial diameter. Total Src expression was similar in arteries of different diameters but the density of high-affinity ouabain binding sites increased with increasing arterial diameters. We suggested that ouabain binding induces more Src kinase activity in mesenteric small arteries with larger diameter leading to enhanced sensitization of the contractile machinery to Ca2+.

Figures

  • Figure 1. Effect of ouabain on agonist-induced contraction varies with arterial diameter. (A,B) show representative concentration–response curves for noradrenaline-induced contractions of mesenteric arteries before (blue) and after 15 min incubation with 10 µM ouabain (red). Inner diameter of the artery in A was 167 µm, and the artery in B–314 µm. Noradrenaline was applied in concentrations of 0.1, 0.3, 1, 3, 10, and 30 µM as indicated by arrows. Sensitivity of arteries to noradrenaline was expressed as logEC50 (in (C) and was measured for each arterial segment under control conditions and in the presence of ouabain. Ouabain-induced changes in the sensitivities are shown in (D) as the difference between sensitivities in the presence of ouabain and under control conditions. (E) shows maximal tension responses to 30 µM noradrenaline under control conditions and in the presence of ouabain. Values for goodness-of-fit of linear regression (R2) are indicated by the lines. p < 0.05 (*) and p < 0.01 (**), n = 20.
  • Figure 2. The potentiating effect of ouabain on noradrenaline-induced contraction depends on arterial diameter. Wall tension changes of mesenteric arteries of different diameters was assessed in response to stimulation with either 1 µM (i), 3 µM (ii) or 10 µM (iii) noradrenaline. Note, these wall tensions were measured simultaneously with [Ca2+]i, as shown in Figure 3. The stimulations were then repeated after 15 min incubation with 10 µM ouabain. Representative curves for wall tension responses are shown in (A). (B) shows averaged noradrenaline-induced changes in wall tension correlated with arterial diameter. Linear regression quantifies goodness of fit with R2 as indicated by the lines. p < 0.01 (**) and p <0.001 (***), n = 12.
  • Figure 3. Noradrenaline-induced changes in intracellular calcium ([Ca2+]i) do not correlate with arterial di m ter. Mesenteric arteries of different diameters were loaded with the Ca2+-sensitive dye (Fura-2/AM) to access wall tension (showed in Figure 2) and [Ca2+]i changes in response to stimulation with either 1 µM (i), 3 µM (ii) or 10 µM (iii) noradrenaline. Representative curves for [Ca2+]i are shown in (A). (B) shows that averaged noradrenaline-induced changes in [Ca2+]i did not correlate with arterial diameter. Linear regression quantifies goodness of fit with R2 as indicated by the lines. No significant correlation was found. n = 12.
  • Figure 4. The potentiating effect of ouabain increases with arterial diameter for wall tension responses to 1 µM noradrenaline (A) but not for the changes in [Ca2+]i (B). (A) shows the differences in wall tension increase to 1 µM (red), 3 µM (blue) and 10 µM (green) noradrenaline stimulations after incubation with 10 µM ouabain, the data are shown in Figure 2B. (B) demonstrates the corresponding differences in [Ca2+]i changes after incubation with 10 µM ouabain, the data are shown in Figure 3B. Linear regression quantifies goodness of fit with R2, as indicated by the lines. * indicates significance of the correlation, where p < 0.05, n = 12.
  • Figure 4. The potentiating effect of ouabain increases with arterial diameter for wall tension responses to 1 µM noradrenaline (A) but not for the changes in [Ca2+]i (B). (A) shows the differences in wall tension increase to 1 µM (red), 3 µM (blue) and 10 µM (green) noradrenaline stimulations after incubation with 10 µM ouabain, the data are shown in Figure 2B. (B) demonstrates the corresponding differences in [Ca2+]i changes after incubation with 10 µM ouabain, the data are shown in Figure 3B. Linear regression quantifies goodness of fit with R2, as indicated by the lines. * indicates significance of the correlation, where p < 0.05, n = 12.
  • Figure 6. Ouabain-induced phosphorylation of Src kinase increases with arterial diameter. Arteries were fixed at resting conditions, after stimulation with 1 µM noradrenaline or incubation with 10 µM ouabain with the following 1 µM noradrenaline stimulation, as indicated in representative images ((A); bars indicates 20 µm). A phosphorylated Src (pSrc) related fluorescence intensity was measured in smooth muscle layer in the vascular wall, as indicated by SYTO-16 based nuclei staining (inserts in images in (A)). No difference in fluorescence intensity in arteries with different inner diameter was seen under resting conditions (B). In 15 experiments, the fluorescence intensity of the artery under resting conditions was compared to the intensity in the artery after noradrenaline stimulation with and without pre-incubation with 10 µM ouabain. There was no correlation between phosphorylated Src and inner diameter for noradrenaline stimulated arteries but in the presence of ouabain phosphorylated Src was significantly p < 0.01 (**) increased with arterial diameter. Linear regression quantifies goodness of fit with R2, as indicated by the graphs.
  • Figure 7. No difference in total Src expression between mesenteric arteries with different diameters. (A) shows representative images of arteries with different diameters as indicated (bars indicates 20 µm). Total Src related fluorescence intensity was measured in smooth muscle layer in the vascular wall, as indicated by SYTO-16 based nuclei staining (inserts in images). No difference in fluorescence intensity in arteries with different inner diameter was seen (B). R2 on the graph indicates linear regression quantifies goodness of fit. n = 32.
  • Figure 8. Increase in arterial diameter is associated with an increased density of the high-affinity ouabain binding sites. Arterial segments of different diameter were incubated with 10 µM BODIPY FL ouabain. (A) shows representative images of arterial segment without pharmacological intervention (i.e., baseline), two arterial segments of different diameters as indicated treated with 10 µM BODIPY FL ouabain, and artery first pre-treated with 1 mM ouabain and then exposed to 10 µM BODIPY FL ouabain (bars indicates 20 µm). Fluorescence intensity was measured in smooth muscle layer in the vascular wall, as indicated by SYTO-82 based nuclei staining (inserts in images). BODIPY FL ouabain related fluorescence intensity increases with an increase in arterial inner diameter (B). R2 on the graph indicates linear regression quantifies goodness of fit. n = 20, p < 0.05 (*).

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Zhang, L., Aalkjaer, C., & Matchkov, V. V. (2018). The Na,K-ATPase-dependent src kinase signaling changes with mesenteric artery diameter. International Journal of Molecular Sciences, 19(9). https://doi.org/10.3390/ijms19092489

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