Plasma sodium stiffens vascular endothelium and reduces nitric oxide release

Abstract

Dietary salt plays a major role in the regulation of blood pressure, and the mineralocorticoid hormone aldosterone controls salt homeostasis and extracellular volume. Recent observations suggest that a small increase in plasma sodium concentration may contribute to the pressor response of dietary salt. Because endothelial cells are (i) sensitive to aldosterone, (ii) in physical contact with plasma sodium, and (iii) crucial regulators of vascular tone, we tested whether acute changes in plasma sodium concentration, within the physiological range, can alter the physical properties of endothelial cells. The tip of an atomic force microscope was used as a nanosensor to measure stiffness of living endothelial cells incubated for 3 days in a culture medium containing aldosterone at a physiological concentration (0.45 nM). Endothelial cell stiffness was unaffected by acute changes in sodium concentration <135 mM but rose steeply between 135 and 145 mM. The increase in stiffness occurred within minutes. Lack of aldosterone in the culture medium or treatment with the epithelial sodium channel inhibitor amiloride prevented this response. Nitric oxide formation was found down-regulated in cells cultured in aldosterone-containing high sodium medium. The results suggest that changes in plasma sodium concentration per se may affect endothelial function and thus control vascular tone.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Force–distance curves obtained in a single endothelial cell exposed subsequently to increasing sodium concentrations (exposure time to one individual sodium concentration was ≈3 min). Each curve was obtained in ≈1 s and repeated 5 to 10 times. The slopes of the curves were analyzed from the cantilever deflections related to the indentation depth.

Fig. 2.

Changes in endothelial cell stiffness in response to increasing sodium concentration measured in buffered electrolyte solution (constant osmolality). Sodium concentration was varied between 120 and 160 mM. Osmolality was kept constant by adequate additions of mannitol. Measurements were started with 120 mM sodium in the solution (reference solution). Single cells in the monolayer were chosen and force curves were obtained. Then, the reference solution was exchanged stepwise for solutions containing increasing sodium concentrations (exposure time to one individual salt concentration was ≈3 min). The stiffness measurement obtained in cells bathed with 120 mM sodium served as the reference value and deviations from this value (in %) are reported here. Mean values of 10 independent measurements per series of experiments are given ± SEM. In aldosterone-treated endothelium, mean values of 140 mM sodium and higher are significantly different compared with mean values measured at 120 mM sodium (P < 0.01). Also, comparisons of mean values between eplerenone-treated endothelium and aldosterone-treated endothelium, obtained at the same sodium concentrations, reveal statistical significances (P < 0.01) at 140 mM sodium and higher.

Fig. 3.

Changes in endothelial cell stiffness in response to increasing sodium concentration in human plasma. Blood was drawn from volunteers in heparin-coated syringes and centrifuged, and then plasma sodium was analyzed. Plasma with a sodium concentration of 137 mM was used as a reference (mean data given as deviation from this value in %). NaCl was added to achieve concentrations of 142 and 147 mM sodium. Monolayers were exposed subsequently to the three different plasma samples and stiffness was measured in individual cells under all three conditions. Mean values of 10 independent measurements are given ± SEM. In eplerenone-treated endothelium cell stiffness did not change significantly in high sodium (147 mM) compared with a sodium concentration of 142 mM but increased in aldosterone-treated cells.

Fig. 4.

Surface line scans versus time in endothelial cells using AFM. (A) Schematic showing living endothelial cells and the AFM tip engaged to the apical membrane surface. The AFM tip scans at 1 Hz (temporal resolution 1 s) along one line (lateral resolution ≈100 nm) forth and back in horizontal direction (x axis). (B) Line scan of a single endothelial cell. The profile shows the transient increase in cell height (h) when the sodium concentration in the buffer is increased from 135 to 150 mM sodium. (C) As described in B except that amiloride (1 μM) was present in the high sodium solution. (D) Mean data (± SEM; n = 15) of the transient increases of cell heights.

Fig. 5.

AFM images of living (aldosteroen-treated) endothelial cells and their respective profiles. An endothelial cell monolayer was scanned with constant force (5 nN) and constant frequency (1 Hz) at two different sodium concentrations in the bath, 135 mM sodium (Left) and 150 mM sodium (Right). Lines and arrows indicate the location of the profiles shown below the images. At low sodium, cells are flattened by the force of the AFM stylus applied to the endothelial cells. At high sodium, cells resist the applied forces and thus appear prominent.

Fig. 6.

Nitrite concentrations of different culture media after exposure to GM7373 bovine aortic endothelial cells. Culture flasks were mounted on a shaker in the incubator for mimicking shear forces. Aldosterone concentration = 0.45 nM. Three independent sets of experiments were performed (three nitrite measurements per set; number of single measurements per column = 9; ± SEM). The asterisk (150 mM sodium + aldosterone) indicates a significant difference of mean nitrite concentration in comparison with each of the other mean values (P < 0.01).

Fig. 7.

Scheme defining the role of extracellular sodium in the regulation of vascular tone. Sodium enters the endothelial cell through ENaC. After a transient increase in cell volume, the endothelium stiffens. Thus vascular pulse pressure exerted by the working heart has a reduced impact on endothelial cell shape. The reduced rhythmic deformation of the endothelial cells decreases nitric oxide synthesis and release. As a result, vascular smooth muscle tone increases.