Endothelial Epithelial Sodium Channel Inhibition Activates Endothelial Nitric Oxide Synthase via Phosphoinositide 3-Kinase/Akt in Small-Diameter Mesenteric Arteries
Recent studies have shown that the epithelial sodium channel (ENaC) is expressed in vascular tissue. However, the role that ENaC may play in the responses to vasoconstrictors and NO production has yet to be addressed. In this study, the contractile responses of perfused pressurized small-diameter rat mesenteric arteries to phenylephrine and serotonin were reduced by ENaC blockade with amiloride (75.1±3.2% and 16.9±2.3% of control values, respectively; P<0.01) that was dose dependent (EC50=88.9±1.6 nmol/L). Incubation with benzamil, another ENaC blocker, had similar effects. α, β, and γ ENaC were identified in small-diameter rat mesenteric arteries using RT-PCR and Western blot with specific antibodies. In situ hybridization and immunohistochemistry localized ENaC expression to the tunica media and endothelium of small-diameter rat mesenteric arteries. Patch-clamp experiments demonstrated that primary cultures of mesenteric artery endothelial cells expressed amiloride-sensitive sodium currents. Mechanical ablation of the endothelium or inhibition of eNOS with Nω-nitro-l-arginine inhibited the reduction in contractility caused by ENaC blockers. ENaC inhibitors increased eNOS phosphorylation (Ser 1177) and Akt phosphorylation (Ser 473). The presence of the phosphoinositide 3-kinase inhibitor LY294002 blunted Akt phosphorylation and eNOS phosphorylation and the decrease in the response to phenylephrine caused by blockers of ENaC, indicating that the phosphoinositide 3-kinase/Akt pathway was activated after ENaC inhibition. Finally, we observed that the effects of blockers of ENaC were flow dependent and that the vasodilatory response to shear stress was enhanced by ENaC blockade. Our results identify a previously unappreciated role for ENaC as a negative modulator of eNOS and NO production in resistance arteries.
The epithelial sodium channel (ENaC) is present in distal nephron segments and the colonic epithelium, where it mediates electrogenic Na+ influx across the apical membrane.1–3 ENaC belongs to the degenerin/ENaC family of proteins and can be reversibly inhibited by the pyrazine diuretics, eg, amiloride and benzamil.1–3 In rat epithelial tissue, ENaC is composed of 3 homologous subunits α, β, and γ.4 Recent studies have shown that ENaC subunits are also present in arteries5–7; however, specific functions that may be attributed to vascular ENaC have only recently begun to be investigated.
Vascular smooth muscle cells from rat cerebral arteries and mouse kidney arteries express β and γ ENaC,5,8 and the activation of ENaC present in vascular smooth muscle cells has been implicated in the vasoconstrictor response to elevated pressure.9 On the other hand, several studies have indicated that ENaC may be present in vascular endothelium. ENaC α mRNA transcripts and protein were identified in cultured human umbilical vein endothelial cells,6 and the activity of ENaC in human umbilical vein endothelial cell has been shown to mediate cell swelling7,10 and plasma membrane stiffening.11 Studies in oocytes have demonstrated that ENaC activity is modulated by laminar shear stress,12 and it has been suggested that endothelial ENaC activity is inhibitory on NO production stimulated by shear stress.11 However, the role of endothelial ENaC in arteries has yet to be investigated.
Here we analyzed the expression of ENaC in rat small-diameter rat mesenteric arteries (SDMAs) and rat endothelial cell primary culture. The role of ENaC in the contractile response to serotonin and phenylephrine was analyzed in isolated SDMAs in the presence of pharmacological ENaC inhibitors amiloride and benzamil. To address the relevance of the endothelium in the response to ENaC blockade, intact arteries and endothelium-denuded arteries were studied. Finally, we analyzed endothelial NO synthase (eNOS), the signaling pathways modulated by ENaC activity, and the role of ENaC in shear stress–induced vasodilation in SDMAs.
All of the methods are described in detail in the online data supplement at http://hyper.ahajournals.org.
Arteries were obtained from adult male (180 to 240 g) Sprague-Dawley rats. Protocol was approved by the ethics committee of the faculty of medicine at the Universidad de Chile, according to National Institutes of Health Guide for the Care and Use of the Laboratory Animals.
Second-order mesenteric arteries were isolated and used for biochemical or functional studies.13
Arteries were cannulated, perfused, and pressurized, as described previously.13,14 External artery diameter was determined by videomicroscopy. Vasodilatory responses to shear stress were measured according to Matrougui et al.15 Isometric tension studies were performed according to Rahman et al.16 Vasoconstrictors were tested after 10 minutes of incubation in the presence of inhibitors (ENaC or phosphoinositide 3-kinase [PI3K]) or vehicle (control). eNOS inhibitor was added 5 minutes before ENaC inhibitors and maintained throughout the experiment.
Fluorometric Determination of NO
NO production in perfused SDMA was estimated by 4-amino-5-metilamino-2′,7′-difluorofluorescein diacetate-AM fluorescence (Molecular Probes).
Kidney and mesenteric arteries were processed for total RNA extraction,14 and PCRs were carried out with specifics primers for rat ENaC subunits.
Western Blot Analysis for ENaC, eNOS, and Akt
Total protein fractions from SDMAs were prepared and used for Western blotting with specific antibodies.13,17
In Situ Hybridization
Tissue sections were hybridized with 400 pmol/mL of digoxigenin-labeled probes and developed with antibody anti–digoxigenin-alkaline phosphatase F′ab fragments (Roche).
SDMA 4-μm sections were incubated with anti-rat ENaC subunits, and the immunoreaction was visualized with 3,3-diamino-benzidine.
Primary Culture of Mesenteric Artery Endothelial Cells
Endothelial cells were isolated by collagenase digestion from rat mesenteric arteries as described previously.18
Standard whole-cell patch-clamp recordings in primary cultures of mesenteric artery endothelial cells were performed as described.19
Values are expressed as mean±SE. Statistical comparisons were performed using 1-way ANOVA, ANOVA for repeated measurements (followed by Bonferroni-Dunn posthoc test), or unpaired Student t test when appropriate. The EC50 of the vasoconstrictor was determined for each curve using a nonlinear least-square fitting procedure for data from individual experiments (GraphPad Prism, GraphPad Software). Differences were considered statistically significant for P<0.05.
The role of ENaC in the response of SDMAs to phenylephrine was studied in isolated perfused (15 μL/min) and pressurized arteries. Vasocontraction was detectable with 0.1 μmol/L of phenylephrine (Figure 1A) and reached maximum values at 100 μmol/L (Emax: 83.6±1.1 μmol/L; Figure 1B; please see video file in the online data supplement). The presence of 500 nmol/L of amiloride starting 10 minutes before phenylephrine decreased the Emax to 24.9% of the control value (Figure 1A and 1B; Emax: 22.6±0.7 μmol/L; n=6; P<0.01). Amiloride also increased the EC50 for phenylephrine (1.9±0.8 μmol/L in vehicle-treated arteries versus 6.3±0.4 μmol/L in amiloride-treated arteries; n=6; P<0.01).
In a separate set of experiments, we analyzed the dose-response curve of SDMA to amiloride, which was added 10 minutes before the stimulus with a maximal dose of phenylephrine. The maximal effect of amiloride was attained at 1 μmol/L with an EC50 of 88.9±1.6 nmol/L (please see http://hyper.ahajournals.org), consistent with ENaC inhibition.1,2 In addition, an amiloride analog specific to the Na+/H+ antiporter (500 nmol/L of ethyl isopropyl amiloride) had no effect in the response of SDMAs to phenylephrine (please see the data supplement).
To test whether ENaC activity could also modulate the response to other vasoactive substances, the effect of amiloride on serotonin-induced constriction was tested (Figure 1C and 1D). Under control conditions, Emax was reached at a concentration of 100 μmol/L of serotonin (72.4±3.4 μm), and the EC50 of serotonin was 0.49±0.1 μmol/L. Similar to the response to phenylephrine, amiloride significantly reduced the contractile response to serotonin (Emax: 60.1±2.3 μm; n=6; P<0.01) and increased the EC50 to 2.9±0.3 μmol/L (n=6; P<0.01).
The incubation of SDMA in the presence of benzamil, another ENaC blocker, also reduced the contractile response to phenylephrine. As shown in Figure 1E, the Emax to phenylephrine decreased (25.3% of control value; n=6; P<0.01), and the EC50 shifted to the right (4.0±0.1 μmol/L; P<0.01). Contraction elicited by serotonin (Figure 1F) was also affected by benzamil, as shown by a shift to the right of the dose-response curve to serotonin (EC50: 1.8±0. 2 μmol/L; n=6; P<0.01). As in the case of amiloride, the dose-response curve to benzamil suggested ENaC blockade, with an EC50 of 8.2±0.2 nmol/L (please see the data supplement).
To assess expression of ENaC subunits in SDMAs, total RNA samples obtained from isolated arteries were analyzed by RT-PCR. Using primers specific for α, β, and γ ENaCs, all 3 of the ENaC subunit transcripts were detected (Figure 2A). Consistent with the RT-PCR results, α, β, and γ ENaC transcripts were detected in endothelial cell mesenteric arteries by in situ hybridization (Figure 2B). Labeling for all 3 of the subunits was also observed in smooth muscle cells, although with lower intensity for β ENaC. The presence of α, β, and γ ENaC proteins in small diameter arteries was confirmed by Western blot analysis with anti-ENaC–specific antibodies (Figure 2C). Finally, immunohistochemical analysis of SDMA sections identified expression of α, β, and γ ENaC proteins in mesenteric endothelium and vascular smooth muscle cell (please see the data supplement).
The decrease in the contractile response observed on ENaC inhibition could have been attributed to the inhibition of the channel in either endothelial and/or vascular smooth muscle cells. To distinguish between these possibilities, phenylephrine dose-response curves were measured for small diameter arteries after mechanical ablation of the endothelium. This procedure neither altered the dose-response curve to phenylephrine nor vasodilation in response to acetylcholine (please see the data supplement). However, the inhibitory action of benzamil on the phenylephrine concentration-response curve was almost undetectable in endothelium-denuded arteries (Figure 3A). Benzamil had no effect on Emax, and only vasoconstriction in response to low concentration of phenylephrine was reduced by the ENaC blocker. Similarly, amiloride had no effect on phenylephrine-evoked vasoconstriction after endothelium ablation (data not shown). These data suggest that reduced contractile response observed after blocking ENaC could be because of an endothelium-derived vasodilator.
To evaluate the possibility that NO might be involved in vasoconstriction, the NO-fluorescent indicator 4-amino-5-metilamino-2′,7′-difluorofluorescein diacetate-AM fluorescence was used in perfused arteries. After loading with the fluorophore, arteries were incubated either in the presence of benzamil and phenylephrine or the eNOS inhibitor Nω-nitro-l-arginine (l-NNA) and phenylephrine. A significant increase in 4-amino-5-methylamino-2′,7′-difluorescein fluorescence was observed in benzamil-treated arteries (Figure 3B), consistent with the interpretation that NO was being produced after ENaC inhibition. In addition, the perfusion of endothelium-intact arteries with l-NNA (100 μmol/L) for 5 minutes before assessing the effect of amiloride or benzamil on the phenylephrine dose-response curves (Figure 4A and 4B) greatly diminished the effect of ENaC blockers. Similarly, l-NNA prevented the inhibitory effect of amiloride or benzamil on serotonin-induced contraction (Figure 4C and 4D).
The presence of amiloride-sensitive Na+ currents in the endothelium was studied by whole-cell patch clamp in primary cultures of mesenteric artery endothelial cells. Endothelial cell primary cultures expressed α, β, and γ ENaC transcripts, as demonstrated by RT-PCR (please see http://hyper.ahajournals.org). Currents elicited by voltage pulses between −80 and +80 mV are shown in the online Data Supplement. Using KCl-rich intracellular solution and an NaCl-rich extracellular solution, under this condition were recording inward and outward currents with a reversal potential of −16.7±3.2 mV (n=27). Replacing extracellular Na+ with N-methyl-d-glucamine chloride markedly decreased inward currents (please see the data supplement) and shifted the reversal potential to approximately −22.8±2.6 mV (n=3). The addition of 500 nmol/L of amiloride to the medium led to an inhibition by ≈60% to 70% of the total current at −80 mV (please see http://hyper.ahajournals.org). The amiloride concentration-dependence studies showed an EC50 of 5.4±0.04 nmol/L of amiloride. These results support ENaC as being active in endothelial cells.
To gain further insight into the mechanisms underlying NO production after ENaC inhibition, we evaluated the possibility that eNOS was being activated by protein kinase B–dependent phosphorylation at serine 1177. Protein kinase B is activated through recruitment to the cellular membrane by PI3K lipid products and phosphorylation by 3′-phosphoinositide-dependent kinase 1. Western blotting experiments with total protein homogenates obtained from SDMAs showed that ENaC inhibition with amiloride or benzamil for 10 minutes produced a significant increase in eNOS phospho-Ser 1177 (Figure 5). The increase of eNOS phosphorylation at Ser 1177 was also observed when phenylephrine was present in the perfusion media. To evaluate Akt activation after ENaC blockade, the levels of phospho-Akt were determined. Incubation of arteries in the presence of phenylephrine plus benzamil caused a significant increase in phospho-Akt levels (Ser 473; Figure 6A) that was blocked in the presence of the LY294002, a PI3K inhibitor. Benzamil-induced eNOS phosphorylation was also blocked by LY294002 (Figure 6B).
On the basis of the results described above, we hypothesized that pharmacological inhibition of PI3K should also be effective in preventing reduced responsiveness to vasoconstrictors in the presence of ENaC blockers. As expected, the presence of LY294002 in the perfusion solution greatly reduced the ability of benzamil to inhibit phenylephrine-induced contraction (Figure 6C). In addition, LY294002 also prevented the increase in phenylephrine EC50 and the reduction in phenylephrine Emax caused by amiloride (Figure 6D).
ENaC has been implicated as part of flow sensor mechanisms,11,12 and shear stress activates eNOS. We evaluated whether the reduction of intraluminal flow modulated the action of ENaC blockers in contractility evoked by phenylephrine. The magnitude of the effect of both amiloride and benzamil decreased at lower flow (Figure 7A), and in the absence of flow (isometric tension studies) we observed no significant effect of amiloride (Figure 7B). Finally, to test whether ENaC inhibition enhanced the vasodilatory response to shear stress, we measured flow-induced dilation in the presence of amiloride. Flow pressure-diameter relationships were established by step increases in flow (4 to 40 μL/min) to SDMAs. As shown in Figure 7C, external diameter increased when flow was raised, and this effect was increased by amiloride. The pressure-diameter relationships showed that, in amiloride-treated arteries, the diameter was significantly greater than in vehicle-treated arteries for similar pressures (Figure 7D).
In the present study, we confirmed ENaC expression in SDMAs, and we found that ENaC blockers reduced the contractile response to phenylephrine and serotonin. The EC50 values obtained in dose-response experiments with pharmacological ENaC inhibitors are similar to the published inhibition constant and IC50 values for ENaC in mammalian distal nephron segments and Xenopus oocytes expressing α, β, and γ ENaC (amiloride: 10 to 100 nmol/L; benzamil: 10 nmol/L).1,2 In addition, an amiloride derivative (ethyl isopropyl amiloride) did not affect the contractile response to phenylephrine. In situ hybridization and immunohistochemistry studies showed α, β, and γ ENaC expression in the tunica media and endothelial cells of SDMAs. In addition, amiloride-sensitive sodium currents were detected in primary cultures of mesenteric artery endothelial cells. These data indicate expression of all 3 of the ENaC subunits in SDMAs that would form trimeric channels with biophysical properties similar to the channels present in kidney tubules.1–3 However, our experiments also show that the efficacy of amiloride is higher than the efficacy of benzamil, as evidenced by the higher Emax observed in arteries treated with amiloride. This could be indicative of the presence of nonclassical ENaC channels.
In experiments with endothelium-denuded arteries, we observed a strong reduction in the ability of ENaC blockers to reduce the vasoconstriction. Benzamil treatment stimulated production of the NO, and pharmacological inhibition of eNOS greatly decreased the ability of amiloride and benzamil to reduce the response to vasoconstrictors. We conclude that reduced contractile responses observed on blocking ENaC are endothelium dependent and require eNOS activation, as well as NO production.
Our studies revealed a significant increase of eNOS phosphorylation at Ser 1177 after ENaC inhibition, implying the PI3K/Akt pathway as the mechanism leading to eNOS phosphorylation. Pharmacological inhibition of PI3K with LY294002 was effective in reducing both Akt phosphorylation and eNOS phosphorylation. Moreover, PI3K inhibition was also effective in preventing the reduction in the contractile response to phenylephrine and serotonin caused by ENaC inhibitors. These results indicated that the endothelial PI3K/Akt pathway is necessary to evoke eNOS activation after ENaC inhibition. Phosphorylation of eNOS at Ser 1177 enhances electron flux through the eNOS reductase domain by reducing calmodulin dissociation from the enzyme when calcium concentrations are low.20 Therefore, ENaC blocking should decrease the dependence of eNOS activation on cytosolic calcium.
We observed that ENaC inhibition in SDMAs caused increased eNOS phosphorylation and NO production. Shear stress is the most potent physiological stimulus for NO production in endothelial cells.21,22 The phosphorylation of eNOS at Ser 1177 by a sequential activation of PI3K and Akt pathway is one of the mechanisms by which shear stress can stimulate NO production.23 In the present study, we observed that the action of ENaC blockers depended on intraluminal flow. In addition, vasodilatory response to shear stress was enhanced by amiloride. These observations support the notion that ENaC activity reduces endothelial PI3K/Akt activation by shear stress and subsequent phosphorylation of eNOS.23
Oberleithner et al,11 using atomic force microscopy in human umbilical vein endothelial cells, demonstrated that small increments of extracellular [Na+] under isotonic conditions increased cell volume and cell stiffening. These changes were amiloride sensitive, suggesting that ENaC mediated sodium influx. Furthermore, increased sodium influx resulted in decreased nitrite concentration in the culture medium of endothelial cells,11 suggesting that ENaC activity could reduce endothelial NO production in response to shear stress and/or blood flow because of a loss of the cell’s deformability. An alternative mechanism that may contribute to the effects of ENaC inhibitors in small-diameter mesenteric arteries is membrane hyperpolarization.24 Endothelial cell membrane hyperpolarization is known to increase eNOS activity.25 Under physiological conditions, ENaC in endothelial cells would have a depolarizing effect, acting as a negative modulator of eNOS activity in response to shear stress.
Our studies indicate that endothelial ENaC is a negative modulator of eNOS and vasodilation in response to shear stress. Endothelial cells express mineralocorticoid receptors,26 and aldosterone decreases endothelial NO output.27 Blocking of the mineralocorticoid receptor increases NO bioavailability and improves the impaired endothelial function in hypertension, atherosclerosis, myocardial infarction, and heart failure.28–31 Aldosterone upregulates ENaC expression and activity in kidney collecting duct cells,1–3 and increases the expression of α ENaC in human umbilical vein endothelial cells.10 Therefore, in vivo, aldosterone may act directly in the endothelium by promoting ENaC expression and activity, contributing to endothelial dysfunction. Recently, mineralocorticoid receptor–dependent downregulation of eNOS phosphorylation (Ser 1177) in the endothelium has been reported.27,31 Thus, further studies will be needed to clarify the relevance of endothelial ENaC in mediating the deleterious effects of aldosterone on eNOS.
We thank Dr Andrew Quest and Peter W. Murphy for their editorial advice, as well as Andrea Birkner her for technical assistance.
Sources of Funding
This work was supported by Fondo Nacional de Desarrollo Cientifico y Tecnológico grant 1050690, Fondo de Areas Prioritarias 15010006, and Millennium Nucleus on Immunology and Immunotherapy (P04/030-F).
- Received December 24, 2008.
- Revision received January 7, 2009.
- Accepted March 31, 2009.
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