Potassium softens vascular endothelium and increases nitric oxide release

Abstract

In the presence of aldosterone, plasma sodium in the high physiological range stiffens endothelial cells and reduces the release of nitric oxide. We now demonstrate effects of extracellular potassium on stiffness of individual cultured bovine aortic endothelial cells by using the tip of an atomic force microscope as a mechanical nanosensor. An acute increase of potassium in the physiological range swells and softens the endothelial cell and increases the release of nitric oxide. A high physiological sodium concentration, in the presence of aldosterone, prevents these changes. We propose that the potassium effects are caused by submembranous cortical fluidization because cortical actin depolymerization induced by cytochalasin D mimics the effect of high potassium. In contrast, a low dose of trypsin, known to activate sodium influx through epithelial sodium channels, stiffens the submembranous cell cortex. Obviously, the cortical actin cytoskeleton switches from gelation to solation depending on the ambient sodium and potassium concentrations, whereas the center of the cell is not involved. Such a mechanism would control endothelial deformability and nitric oxide release, and thus influence systemic blood pressure.

Fig. 1.

Indentation technique using AFM. (A) Cantilever with particle tip. (B) Principle of the stiffness measurement using an AFM. (C) Indentation curve with two different slopes. After engagement of the cell surface with the particle tip, the cantilever will be bent as the cell is indented. A laser beam (as displayed in B; not shown in C) reflected from the cantilever quantifies this signal. From the two linear portions of the indentation curve, the cell stiffness can be calculated over the first few hundred nanometers of indentation (cortical cell stiffness) or beyond 800 nm of indentation (cell center).

Fig. 2.

AFM imaging of living vascular endothelial cells, exposed to increasing concentrations of extracellular potassium. Paired experiments showing the same cells at different conditions. Numbers on cells indicate the respective cell heights (in μm). Numbers (Left, Lower) refer to the total volume of the cells displayed. The graph shows the mean values of cell heights at the 3 different potassium concentrations in absence and presence of 3 mM barium (potassium channel block). Cells swell in response to increasing potassium. *Significant increase in cell height compared with the initial values measured at 4 mM potassium; n = 7, P < 0.01, paired Student t test. Barium prevents this response.

Fig. 3.

Relationship between extracellular potassium concentration and cortical cell stiffness. Acute experiments were performed at four different conditions. In 2 series of experiments, aldosterone (aldo) was present in the perfusion solution (acute application) and in the culture medium 48 h before the experiment. Each series of experiments comprised 5 to 8 paired cell indentation measurements. The P values were calculated by comparing the individual mean values with the initial stiffness values at 2 mM potassium (paired Student t test).

Fig. 4.

Indentation curves obtained in 2 experiments on vascular endothelial cells. For better demonstration, 2 individual cells with a small (Left) and large (Right) initial cortical soft zone were chosen. Left, Before the application of CD, the first linear slope is short. It indicates that the cortical zone is shallow. Ten min after CD application, the cortical zone has broadened to several hundred nanometers. In addition, the first slope flattened, indicating that the cortical zone softened at the same time. Right, Before the application of trypsin, a broad and soft cortical zone is detected in this cell. Twenty-five min after addition of trypsin, this zone has shrunk. In addition, the rather steep slope indicates that the cortical zone not only shrunk but stiffened at the same time.

Fig. 5.

Time course of the mean stiffness of 5 cells measured at 2 locations. After CD, the cortical cell stiffness decreases gradually while the stiffness in the cell center remains constant. Increasing extracellular potassium (from 4 mM to 6 mM and 8 mM) after the destabilization of the cortical actin does not further affect cortical stiffness. *Significant difference versus the respective initial value (paired Student t test).

Fig. 6.

Time course of cortical cell stiffness measurements (means of 4 cells) in response to trypsin. The increase of cortical cell stiffness can be prevented by 1 μM amiloride added at the same time together with trypsin. *Significant difference versus the respective initial value (paired Student t test).

Fig. 7.

Nitrite concentrations analyzed in the supernatant culture media after exposure of the vascular endothelial cells for 24 h to different potassium concentrations. Three series of experiments were performed (low sodium, low sodium plus aldosterone, high sodium plus aldosterone). The numbers on top of the individual columns indicate the number of nitrite measurements. Asterisk on top of a column indicates a significant difference compared with the mean values of the same group of experiments. ^§Significant difference versus all other mean values of the figure except those with the same symbol. ^#Significant difference versus respective mean values in the different groups of experiments. Significantly different is P < 0.05 (unpaired Student t test).