The effect of fluoride on the structure, function, and proteome of intestinal epithelia.

Fluoride exposure is widespread, with drinking water commonly containing natural and artificially added sources of the ion. Ingested fluoride undergoes absorption across the gastric and intestinal epithelia. Previous studies have reported adverse gastrointestinal effects with high levels of fluoride exposure. Here, we examined the effects of fluoride on the transepithelial ion transport and resistance of three intestinal epithelia. We used the Caco-2 cell line as a model of human intestinal epithelium, and rat and mouse colonic epithelia for purposes of comparison. Fluoride caused a concentration-dependent decline in forskolin-induced Cl- secretion and transepithelial resistance of Caco-2 cell monolayers, with an IC50 for fluoride of about 3 mM for both parameters. In the presence of 5 mM fluoride, transepithelial resistance fell exponentially with time, with a t1/2 of about 7 hours. Subsequent imaging by immunofluorescence and scanning electron microscopy showed structural abnormalities in Caco-2 cell monolayers exposed to fluoride. The Young's modulus of the epithelium was not affected by fluoride, although proteomic analysis revealed changes in expression of a number of proteins, particularly those involved in cell-cell adhesion. In line with its effects on Caco-2 cell monolayers, fluoride, at 5 mM, also had profound effects on Cl- secretion and transepithelial resistance of both rat and mouse colonic epithelia. Our results show that treatment with fluoride has major effects on the structure, function, and proteome of intestinal epithelia, but only at concentrations considerably higher than those likely to be encountered in vivo, when much lower fluoride doses are normally ingested on a chronic basis.

Aldosterone synthase knockout mouse as a model for sodium-induced endothelial sodium channel up-regulation in vascular endothelium.

Recently, a novel feedforward activation of the endothelial epithelial sodium channel (ENaC) [endothelial sodium channel (EnNaC)] by sodium was reported that counteracts ENaC function in kidney. In the absence of aldosterone, a rise in extracellular sodium (>145 mM) increases EnNaC surface abundance, thereby stiffening the cortex of vascular endothelial cells (ECs) in vitro. The latter reduces the release of NO-the hallmark of endothelial dysfunction. Here, we test whether high extracellular sodium per se increases EnNaC expression and cortical stiffness in an aldosterone synthase (Cyp11b2)-deficient (AS(-/-)) mouse model. Therefore, we employed in situ ECs of ex vivo aorta preparations from wild-type (WT) and AS(-/-). EnNaC surface expression (-16%) and cortical stiffness (-22%) were reduced in AS(-/-), compared with WT, whereas NO secretion was exclusively detectable in AS(-/-). EnNaC inhibition with benzamil decreased stiffness in both, while mineralocorticoid receptor antagonism diminished stiffness only in the WT. In the absence of aldosterone, high sodium (150 mM) increased EnNaC surface expression ex vivo (plus 19%) and cortical stiffness ex vivo (plus 41%) and in vivo (plus 44%). Application of aldosterone adjusted the stiffness of AS(-/-) to the WT level. We conclude that high sodium per se determines EnNaC expression and consequently endothelial cortical nanomechanics, thus likely contributing to endothelial dysfunction.

The effect of fluoride on the structure, function, and proteome of a renal epithelial cell monolayer.

High concentrations of fluoride in the body may cause toxic effects. Here, we investigated the effects of fluoride on the structure, function, and proteome of a cortical collecting duct epithelium in vitro. Kidney tubule cells (M-1) were chosen because the concentration of fluoride in the kidney is 4-5-fold higher than that in plasma. Mouse M-1 cell monolayers were incubated in fluoride-containing media, and the amiloride-sensitive short-circuit current and transepithelial resistance were measured. The Young's modulus of the epithelium was determined using atomic force microscopy, and the effect of fluoride on epithelial structure was assessed using scanning and transmission electron microscopy, and immunofluorescence. Differences in the expression of membrane proteins were evaluated using proteomics and bioinformatics. Fluoride exposure reduced both transepithelial Na+ transport and resistance. The IC50 for fluoride was ∼300 µM for both effects, and the half-times for the decays of ion transport and resistance were 8.4 h and 3.6 days, respectively. Fluoride treatment did not affect the sensitivity of Na+ transport to amiloride. The Young's modulus of the epithelium was also unaffected by fluoride; however, the functional effects of fluoride were accompanied by marked structural effects. Proteomic analysis revealed changes in expression of a number of proteins, and particularly mitochondrial proteins. Treatment with fluoride had profound effects on the structure, function and proteome of a model cortical collecting duct epithelium. Significantly, however, these effects were produced only at concentrations considerably higher than those likely to be encountered in vivo. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1455-1467, 2017.

Atomic force microscopy imaging reveals the formation of ASIC/ENaC cross-clade ion channels.

ASIC and ENaC are co-expressed in various cell types, and there is evidence for a close association between them. Here, we used atomic force microscopy (AFM) to determine whether ASIC1a and ENaC subunits are able to form cross-clade hybrid ion channels. ASIC1a and ENaC could be co-isolated from detergent extracts of tsA 201 cells co-expressing the two subunits. Isolated proteins were incubated with antibodies against ENaC and Fab fragments against ASIC1a. AFM imaging revealed proteins that were decorated by both an antibody and a Fab fragment with an angle of ∼120° between them, indicating the formation of ASIC1a/ENaC heterotrimers.

Feedforward activation of endothelial ENaC by high sodium.

Kidney epithelial sodium channels (ENaCs) are known to be inactivated by high sodium concentrations (feedback inhibition). Recently, the endothelial sodium channel (EnNaC) was identified to control the nanomechanical properties of the endothelium. EnNaC-dependent endothelial stiffening reduces the release of nitric oxide, the hallmark of endothelial dysfunction. To study the regulatory impact of sodium on EnNaC, endothelial cells (EA.hy926 and ex vivo mouse endothelium) were incubated in aldosterone-free solutions containing either low (130 mM) or high (150 mM) sodium concentrations. By applying atomic force microscopy-based nanoindentation, an unexpected positive correlation between increasing sodium concentrations and cortical endothelial stiffness was observed, which can be attributed to functional EnNaC. In particular, an acute rise in sodium concentration (+20 mM) was sufficient to increase EnNaC membrane abundance by 90% and stiffening of the endothelial cortex by 18%. Despite the absence of exogenous aldosterone, these effects were prevented by the aldosterone synthase inhibitor FAD286 (100 nM) or the mineralocorticoid receptor (MR)-antagonist spironolactone (100 nM), indicating endogenous aldosterone synthesis and MR-dependent signaling. Interestingly, in the presence of high-sodium concentrations, FAD286 increased the transcription of the MR by 69%. Taken together, a novel feedforward activation of EnNaC by sodium is proposed that contrasts ENaC feedback inhibition in kidney.

The role of ENaC in vascular endothelium.

Once upon a time, the expression of the epithelial sodium channel (ENaC) was mainly assigned to the kidneys, colon and sweat glands where it was considered to be the main determinant of sodium homeostasis. Recent, though indirect, evidence for the possible existence of ENaC in a non-epithelial tissue was derived from the observation that the vascular endothelium is a target for aldosterone. Inhibitory actions of the intracellular aldosterone receptors by spironolactone and, more directly, by ENaC blockers such as amiloride supported this view. Shortly after, direct data on the expression of ENaC in vascular endothelium could be demonstrated. There, endothelial ENaC (EnNaC) could be defined as a major regulator of cellular mechanics which is a critical parameter in differentiating between vascular function and dysfunction. Foremost, the mechanical stiffness of the endothelial cell cortex, a layer 50-200 nm beneath the plasma membrane, has been shown to play a crucial role as it controls the production of the endothelium-derived vasodilator nitric oxide (NO) which directly affects the tone of the vascular smooth muscle cells. In contrast to soft endothelial cells, stiff endothelial cells release reduced amounts of NO, the hallmark of endothelial dysfunction. Thus, the combination of endothelial stiffness and myogenic tone might increase the peripheral vascular resistance. An elevation of arterial blood pressure is supposed to be the consequence of such functional changes. In this review, EnNaC is discussed as an aldosterone-regulated plasma membrane protein of the vascular endothelium that could significantly contribute to maintaining of an appropriate arterial blood pressure but, if overexpressed, could participate in the pathogenesis of arterial hypertension.

Direct evidence for functional TRPV1/TRPA1 heteromers.

Transient receptor potential cation channel, subfamily V, member 1 (TRPV1) plays a key role in sensing environmental hazards and in enhanced pain sensation following inflammation. A considerable proportion of TRPV1-expressing cells also express transient receptor potential cation channel, subfamily A, member 1 (TRPA1). There is evidence for a TRPV1-TRPA1 interaction that is predominantly calcium-dependent, and it has been suggested that the two proteins might form a heteromeric channel. Here, we constructed subunit concatemers to search for direct evidence for such an interaction. We found that a TRPV1::TRPV1 concatemer and TRPV1 formed channels with similar properties. A TRPV1::TRPA1 concatemer was responsive to TRPV1 agonists capsaicin, acidic pH and ethanol, but not to TRPA1 agonists. Isolated TRPV1 and TRPV1::TRPA1 imaged by atomic force microscopy (AFM) both had molecular volumes consistent with the formation of tetrameric channels. Antibodies decorated epitope tags on TRPV1 with a four-fold symmetry, as expected for a homotetramer. In contrast, pairs of antibodies decorated tags on TRPV1::TRPA1 predominantly at 180°, indicating the formation of a channel consisting of two TRPV1::TRPA1 concatemers arranged face to face. TRPV1::TRPA1 was sensitized by PKC activation and could be inhibited by a TRPV1 antagonist. TRPV1::TRPA1 was activated by heat and displayed a threshold and temperature coefficient similar to TRPV1. However, the channel formed by TRPV1::TRPA1 has only two binding sites for capsaicin and shows less total current and a smaller capsaicin-induced shift in voltage-dependent gating than TRPV1::TRPV1 or TRPV1. We conclude that the presence of TRPA1 exerts a functional inhibition on TRPV1.

Nanomechanics of vascular endothelium.

The mechanical characteristics of endothelial cells reveal four distinct compartments, namely glycocalyx, cell cortex, cytoplasm and nucleus. There is accumulating evidence that endothelial nanomechanics of these individual compartments control vascular physiology. Depending on protein composition, filament formation and interaction with cross-linker proteins, these four compartments determine endothelial stiffness. Structural organization and mechanical properties directly influence physiological processes such as endothelial barrier function, nitric oxide release and gene expression. This review will focus on endothelial nanomechanics and its impact on 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.

Role of cellular mechanics in the function and life span of vascular endothelium.

The vascular endothelium plays a crucial role in vessel homeostasis and is implicated in the pathogenesis of cardiovascular disease. The function and life span of endothelial cells, therefore, have a large impact upon the quality and expectancy of an individual's life. Exposure to haemodynamic forces determines the phenotype of endothelial cells. Turbulent blood flow, disturbed shear stress and a rising tension of the vessel wall result in endothelial dysfunction and an enhanced endothelial cell turnover. In this scenario, the role of endothelial mechanics is yet poorly described. The streaming blood exerts shear forces transmitted to the soft cortical actin mesh immediately underneath the plasma membrane. The mechanical properties of this actin cortex seem to be an important regulator of endothelial function. Aldosterone and high plasma sodium stiffen the endothelial cell cortex which is accompanied by a decrease in NO release. If endothelial stiffening is only transient, it may be a useful mechanism to compensate for any decrease in arterial blood pressure. Long-term stiffening of the cell, however, may lead to endothelial dysfunction and may contribute to cardiovascular disorders, as observed in disturbed aldosterone/sodium homeostasis. In this case, the mineralocorticoid receptor antagonist spironolactone maintains the endothelial cell cortex soft and thereby preserves normal endothelial function and longevity. This may explain the recently observed beneficial effects of spironolactone on the cardiovascular system. Taken together, the review highlights the importance of elasticity for normal endothelial function.

The endothelial αENaC contributes to vascular endothelial function in vivo.

The Epithelial Sodium Channel (ENaC) is a key player in renal sodium homeostasis. The expression of α ß Î³ ENaC subunits has also been described in the endothelium and vascular smooth muscle, suggesting a role in vascular function. We recently demonstrated that endothelial ENaC is involved in aldosterone-modulated endothelial stiffness. Here we explore the functional role of the endothelial αENaC subunit in vascular function in vivo. Compared to littermates, mice with conditional αENaC subunit gene inactivation in the endothelium only (endo-αENaC Knock Out mice) had no difference in their physiological parameters such as systolic blood pressure or heart rate. Acute and long-term renal Na+ handlings were not affected, indicating that endothelial αENaC subunit is not involved in renal sodium balance. Pharmacological inhibition of ENaC with benzamil blunted acetylcholine-induced nitric oxide production in mesenteric arteries from wild type mice but not in endo-αENaC KO mice, suggesting a critical role of endothelial ENaC in agonist-induced nitric oxide production. In endo-αENaC KO mice, compensatory mechanisms occurred and steady state vascular function was not altered except for flow-mediated dilation. Our data suggest that endothelial αENaC contributes to vascular endothelial function in vivo.

The cytotoxic effect of TiF4 and NaF on fibroblasts is influenced by the experimental model, fluoride concentration and exposure time.

OBJECTIVE: Titanium tetrafluoride (TiF4) has shown promising effect in preventing tooth lesions. Therefore, we compared the cytotoxicity of TiF4 with sodium fluoride (NaF) (already applied in Dentistry) considering different fluoride concentrations, pH values and experimental models. MATERIALS AND METHODS: Step 1) NIH/3T3 fibroblasts were exposed to mediums containing NaF or TiF4 (from 0.15 to 2.45% F), both at native and adjusted pH, for 6 h. Step 2) NIH/3T3 were exposed to NaF or TiF4 varnishes with 0.95, 1.95 or 2.45% F (native pH), for 6, 12 or 24 h. We applied MTT (1st and 2nd steps) and Hoescht/PI stain (2nd step) assays. Step 3) NIH/3T3 were exposed to NaF or TiF4 varnish (2.45% F), at native pH, for 6 or 12 h. The cell stiffness was measured by atomic force microscopy (AFM). RESULTS: Step 1) All cells exposed to NaF or TiF4 mediums died, regardless of the F concentration and pH. Step 2) Both varnishes, at 1.90 and 2.45% F, reduced cell viability by similar extents (33-86% at 6 h, 35-93% at 12 h, and 87-98% at 24 h) compared with control, regardless of the type of fluoride. Varnishes with 0.95% F did not differ from control. Step 3) TiF4 and NaF reduced cell stiffness to a similar extent, but only TiF4 differed from control at 6 h. CONCLUSIONS: Based on the results of the 3 experimental steps, we conclude that TiF4 and NaF have similar cytotoxicity. The cytotoxicity was dependent on F concentration and exposure time. This result gives support for testing the effect of TiF4 varnish in vivo.

Exocytotic fusion pores are composed of both lipids and proteins.

During exocytosis, fusion pores form the first aqueous connection that allows escape of neurotransmitters and hormones from secretory vesicles. Although it is well established that SNARE proteins catalyze fusion, the structure and composition of fusion pores remain unknown. Here, we exploited the rigid framework and defined size of nanodiscs to interrogate the properties of reconstituted fusion pores, using the neurotransmitter glutamate as a content-mixing marker. Efficient Ca(2+)-stimulated bilayer fusion, and glutamate release, occurred with approximately two molecules of mouse synaptobrevin 2 reconstituted into ∼6-nm nanodiscs. The transmembrane domains of SNARE proteins assumed distinct roles in lipid mixing versus content release and were exposed to polar solvent during fusion. Additionally, tryptophan substitutions at specific positions in these transmembrane domains decreased glutamate flux. Together, these findings indicate that the fusion pore is a hybrid structure composed of both lipids and proteins.

Functional analysis of the interface between the tandem C2 domains of synaptotagmin-1.

C2 domains are widespread motifs that often serve as Ca(2+)-binding modules; some proteins have more than one copy. An open issue is whether these domains, when duplicated within the same parent protein, interact with one another to regulate function. In the present study, we address the functional significance of interfacial residues between the tandem C2 domains of synaptotagmin (syt)-1, a Ca(2+) sensor for neuronal exocytosis. Substitution of four residues, YHRD, at the domain interface, disrupted the interaction between the tandem C2 domains, altered the intrinsic affinity of syt-1 for Ca(2+), and shifted the Ca(2+) dependency for binding to membranes and driving membrane fusion in vitro. When expressed in syt-1 knockout neurons, the YHRD mutant yielded reductions in synaptic transmission, as compared with the wild-type protein. These results indicate that physical interactions between the tandem C2 domains of syt-1 contribute to excitation-secretion coupling.

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.

Cortical actin nanodynamics determines nitric oxide release in vascular endothelium.

The release of the main vasodilator nitric oxide (NO) by the endothelial NO synthase (eNOS) is a hallmark of endothelial function. We aim at elucidating the underlying mechanism how eNOS activity depends on cortical stiffness (К(cortex)) of living endothelial cells. It is hypothesized that cortical actin dynamics determines К(cortex) and directly influences eNOS activity. By combined atomic force microscopy and fluorescence imaging we generated mechanical and optical sections of single living cells. This approach allows the discrimination between К(cortex) and bulk cell stiffness (К(bulk)) and, additionally, the simultaneous analysis of submembranous actin web dynamics. We show that К(cortex) softens when cortical F-actin depolymerizes and that this shift from a gel-like stiff cortex to a soft G-actin rich layer, triggers the stiffness-sensitive eNOS activity. The results implicate that stiffness changes in the ∼100 nm phase of the submembranous actin web, without affecting К(bulk), regulate NO release and thus determines endothelial function.