Epithelial sodium channel stiffens the vascular endothelium in vitro and in Liddle mice.

Liddle syndrome, an inherited form of hypertension, is caused by gain-of-function mutations in the epithelial Na(+) channel (ENaC), the principal mediator of Na(+) reabsorption in the kidney. Accordingly, the disease pathology was ascribed to a primary renal mechanism. Whether this is the sole responsible mechanism, however, remains uncertain as dysregulation of ENaC in other tissues may also be involved. Previous work indicates that ENaC in the vascular endothelium is crucial for the regulation of cellular mechanics and thus vascular function. The hormone aldosterone has been shown to concomitantly increase ENaC surface expression and stiffness of the cell cortex in vascular endothelial cells. The latter entails a reduced release of the vasodilator nitric oxide, which eventually leads to an increase in vascular tone and blood pressure. Using atomic force microscopy, we have found a direct correlation between ENaC surface expression and the formation of cortical stiffness in endothelial cells. Stable knockdown of αENaC in endothelial cells evoked a reduced channel surface density and a lower cortical stiffness compared with the mock control. In turn, an increased αENaC expression induced an elevated cortical stiffness. More importantly, using ex vivo preparations from a mouse model for Liddle syndrome, we show that this disorder evokes enhanced ENaC expression and increased cortical stiffness in vascular endothelial cells in situ. We conclude that ENaC in the vascular endothelium determines cellular mechanics and hence might participate in the control of vascular function.

Membrane potential depolarization decreases the stiffness of vascular endothelial cells.

The stiffness of vascular endothelial cells is crucial to mechanically withstand blood flow and, at the same time, to control deformation-dependent nitric oxide release. However, the regulation of mechanical stiffness is not yet understood. There is evidence that a possible regulator is the electrical plasma membrane potential difference. Using a novel technique that combines fluorescence-based membrane potential recordings with atomic force microscopy (AFM)-based stiffness measurements, the present study shows that membrane depolarization is associated with a decrease in the stiffness of endothelial cells. Three different depolarization protocols were applied, all of which led to a similar and significant decrease in cell stiffness, independently of changes in cell volume. Moreover, experiments using the actin-destabilizing agent cytochalasin D indicated that depolarization acts by affecting the cortical actin cytoskeleton. A model is proposed whereby a change of the electrical field across the plasma membrane is directly sensed by the submembranous actin network, regulating the actin polymerization:depolymerization ratio and thus cell stiffness. This depolarization-induced decrease in the stiffness of endothelial cells could play a role in flow-mediated nitric-oxide-dependent vasodilation.

Nitric oxide release follows endothelial nanomechanics and not vice versa.

In the vascular endothelium, mechanical cell stiffness (К) and nitric oxide (NO) release are tightly coupled. "Soft" cells release more NO compared to "stiff" cells. Currently, however, it is not known whether NO itself is the primary factor that softens the cells or whether NO release is the result of cell softening. To address this question, a hybrid fluorescence/atomic force microscope was used in order to measure changes in К and NO release simultaneously in living vascular endothelial cells. Aldosterone was applied to soften the cells transiently and to trigger NO release. NO synthesis was then either blocked or stimulated and, simultaneously, К was measured. Cell indentation experiments were performed to evaluate К, while NO release was measured either by an intracellular NO-dependent fluorescence indicator (DAF-FM/DA) or by NO-selective electrodes located close to the cell surface. After the application of aldosterone, К decreases, within 10 min, to 80.5 ± 1.7% of control (100%). DAF-FM fluorescence intensity increases simultaneously to 132.9 ± 2.2%, which indicates a significant increase in the activity of endothelial NO synthase (eNOS). Inhibition of eNOS (by N (ω)-nitro-L: -arginine methyl ester) blocks the NO release, but does not affect the aldosterone-induced changes in К. Application of an eNOS-independent NO donor (NONOate/AM) raises intracellular NO concentration, but, again, does not affect К. Data analysis indicates that a decrease of К by about 10% is sufficient to induce a significant increase of eNOS activity. In conclusion, these nanomechanic properties of endothelial cells in vascular endothelium determine NO release, and not vice versa.

The epithelial sodium channel (ENaC): Mediator of the aldosterone response in the vascular endothelium?

In the kidney the epithelial sodium channel (ENaC) is regulated by the mineralocorticoid hormone aldosterone, which is essential for long-term blood pressure control. Evidence has accumulated showing that ENaC is expressed in endothelial cells. Moreover, its activity modifies the biomechanical properties of the endothelium. Therefore, the vascular system is also an important target for aldosterone and responds to the hormone with an increase in cell volume, surface area, and mechanical stiffness. These changes occur in a concerted fashion from minutes to hours and can be prevented by the specific sodium channel blocker amiloride and the mineralocorticoid receptor (MR) blocker spironolactone. Aldosterone acts on cells of the vascular system via genomic and non-genomic pathways. There is evidence that the classical cytosolic MR could mediate both types of response. Using a nanosensor covalently linked to aldosterone, binding sites at the plasma membrane were identified by atomic force microscopy. The interaction of aldosterone and this newly identified surface receptor could precede the slow classic genomic aldosterone response resulting in fast activation of endothelial ENaC. Recent data suggest that aldosterone-induced ENaC activation initiates a sequence of cellular events leading to a reduced release of vasodilating nitric oxide. We propose a model in which ENaC is the key mediator of aldosterone-dependent blood pressure control in the vascular endothelium.

Simultaneous mechanical stiffness and electrical potential measurements of living vascular endothelial cells using combined atomic force and epifluorescence microscopy.

The degree of mechanical stiffness of vascular endothelial cells determines the endogenous production of the vasodilating gas nitric oxide (NO). However, the underlying mechanisms are not yet understood. Experiments on vascular endothelial cells suggest that the electrical plasma membrane potential is involved in this regulatory process. To test this hypothesis we developed a technique that simultaneously measures the electrical membrane potential and stiffness of vascular endothelial cells (GM7373 cell line derived from bovine aortic endothelium) under continuous perfusion with physiological electrolyte solution. The cellular stiffness was determined by nano-indentation using an atomic force microscope (AFM) while the electrical membrane potential was measured with bis-oxonol, a voltage-reporting fluorescent dye. These two methods were combined using an AFM attached to an epifluorescence microscope. The electrical membrane potential and mechanical stiffness of the same cell were continuously recorded for a time span of 5 min. Fast fluctuations (in the range of seconds) of both the electrical membrane potential and mechanical stiffness could be observed that were not related to each other. In contrast, slow cell depolarizations (in the range of minutes) were paralleled by significant increases in mechanical stiffness. In conclusion, using the combined AFM-fluorescence technique we monitored for the first time simultaneously the electrical plasma membrane potential and mechanical stiffness in a living cell. Vascular endothelial cells exhibit oscillatory non-synchronized waves of electrical potential and mechanical stiffness. The sustained membrane depolarization, however, is paralleled by a concomitant increase of cell stiffness. The described method is applicable for any fluorophore, which opens new perspectives in biomedical research.

Aquaporin isoforms involved in physiological volume regulation of murine spermatozoa.

Murine epididymal spermatozoa were dispersed in a medium of native osmolality and then transferred to a hypo-osmotic medium to mimic the physiological osmotic challenge, as encountered upon ejaculation into the female tract. The addition of quinine to block sperm K(+)-channels for volume regulation resulted in a size increase of viable cells. Preincubation in 0.1 mM HgCl(2), a standard aquaporin inhibitor, prevented such cell swelling. Addition of the K(+)-ionophore valinomycin to quinine-swollen sperm reversed the swelling, but not after pretreatment of the swollen sperm by HgCl(2). Aqp7, Aqp8, and Aqp9 mRNAs were identified in spermatozoa by RT-PCR, and the entire open reading frames were sequenced and compared with the GenBank database. Western blotting demonstrated specific protein signals for sperm AQP7 and AQP8 expression but probably not AQP9. The role of Hg(2+)-insensitive AQP7, if any, in sperm volume regulation was studied in transgenic mice. Spermatozoa from Aqp7(-/-) mice were the same size as wild-type sperm in basal conditions. Quinine-swollen volume, swelling reversal by valinomycin, and inhibition by Hg(2+) were also similar, indicating efficient water transport in the absence of AQP7. However, both water influx and efflux occurred faster in Aqp7(-/-) sperm than wild-type. This faster water movement in the knockout mouse spermatozoa was explainable by an upregulation of Aqp8 expression as revealed by quantitative PCR. Therefore, the Hg(2+)-sensitive AQP8, which was localized in elongated spermatids and spermatozoa, is a likely candidate for a water channel responsible for physiological sperm volume regulation crucial to in vivo fertilization.

How steroid hormones act on the endothelium--insights by atomic force microscopy.

Vascular actions of steroid hormones have gained increasing importance. Indeed, some steroid hormones favorably influence vascular structure and function, whereas others are detrimental. This review will focus on the endothelial effects of steroid hormones. In the first part, we summarize data from in vivo studies elucidating the regulation of endothelial function by steroid hormones. Accumulating data argue for an improvement of endothelium-derived relaxation and impaired vascular contraction by estradiol, whereas testosterone, progesterone, and aldosterone have contrary effects. In the second part, we present data from novel atomic force microscopy studies performed in living endothelial cells under the influence of steroid hormones. These studies provide insight into structural and functional alterations of endothelial cells characterized by changes in volume, apical surface, and stiffness. We summarize the available evidence that changes in shape of endothelial cells translate into changes of endothelial cell stiffness. Under the influence of estradiol, endothelial cells become spherical with consecutive improvement of elasticity, whereas aldosterone flattens endothelial cell-shape leading to increased stiffness. Both, endothelial cell shape and stiffness are major determinants of endothelial nitric oxide production. These studies emphasize the great potential of atomic force microscopy to investigate the function of living endothelial cells.