Pressure-induced constriction is inhibited in a mouse model of reduced βENaC

Abstract

Recent studies suggest certain epithelial Na⁺ channel (ENaC) proteins may be components of mechanosensitive ion channel complexes in vascular smooth muscle cells that contribute to pressure-induced constriction in middle cerebral arteries (MCA). However, the role of a specific ENaC protein, βENaC, in pressure-induced constriction of MCAs has not been determined. The goal of this study was to determine whether pressure-induced constriction in the MCA is altered in a mouse model with reduced levels of βENaC. Using quantitative immunofluorescence, we found whole cell βENaC labeling in cerebral vascular smooth muscle cells (VSMCs) was suppressed 46% in βENaC homozygous mutant (m/m) mice compared with wild-type littermates (+/+). MCAs from βENaC +/+ and m/m mice were isolated and placed in a vessel chamber for myographic analysis. Arteries from βENaC+/+ mice constricted to stepwise increases in perfusion pressure and developed maximal tone of 10 ± 2% at 90 mmHg (n = 5). In contrast, MCAs from βENaC m/m mice developed significantly less tone (4 ± 1% at 90 mmHg, n = 5). Vasoconstrictor responses to KCl (4–80 mM) were identical between genotypes and responses to phenylephrine (10⁻⁷-10⁻⁴ M) were marginally altered, suggesting that reduced levels of VSMC βENaC specifically inhibit pressure-induced constriction. Our findings indicate βENaC is required for normal pressure-induced constriction in the MCA and provide further support for the hypothesis that βENaC proteins are components of a mechanosensor in VSMCs.

vascular smooth muscle cells (VSMCs) from most small-resistance arteries have the ability to constrict in response to increases in intraluminal pressure, a response referred to as pressure-induced, or myogenic constriction (3, 8, 9). Pressure-induced constriction contributes to the autoregulation of cerebral blood flow and may prevent transmission of systemic pressure to microvessels (3, 8, 9). The response involves transduction of pressure-induced stretch into a cellular event (vasoconstriction). Mechanosensitive ion channels are thought to participate in the initiation of the response. However, the molecules involved in initiating the response have not been fully characterized (3, 8, 9).

Recent studies suggest members of the degenerin (DEG) protein family, which includes epithelial Na⁺ channel (ENaC) and acid-sensing ion channel (ASIC) proteins, are candidates for mechanosensitive ion channels because DEG proteins are linked to mechanotransduction (5, 6, 14, 15). DEG proteins are expressed in mechanosensitive tissues, such as smooth muscle and neuronal tissue in a diverse range of species (nematode, Drosophila, and mammals). Disruption of channel expression alters normal mechanosensory responses (5, 6, 14, 15). Several lines of evidence suggest VSMC ENaC/ASIC proteins transduce mechanical stimuli in the middle cerebral artery (MCA). First, several DEG proteins are expressed in cerebral VSMCs, including βENaC, γENaC, and ASIC2 (one of several ASIC proteins) (4, 7). Second, DEG inhibition with the diuretic amiloride, or its analog benzamil, blocks pressure-induced constriction in the MCA (4). Third, evidence in genetically modified mice suggests pressure-induced constriction is impaired in ASIC2 knockout mice (7). These lines of evidence suggest vascular DEG proteins participate in pressure-induced constriction. Although genetic evidence suggests a role for ASIC2, the importance of other DEG proteins, such as βENaC, in pressure-induced constriction of MCA has never been addressed. Therefore, the goal of the current study was to determine the importance of βENaC in pressure-induced constriction in the MCA.

The importance of βENaC was evaluated in a mouse model of reduced βENaC developed by gene-targeting methods (13). The animal model was originally generated with the intention of creating a Liddle's mouse model. The mutant allele corresponds to a Liddle's mutation with the insertion of a premature stop codon at R566, however, the presence of the neomycin selection marker leads to enhanced degradation of βENaC mRNA (13). Mice homozygous for the mutation express low levels of βENaC transcripts and protein in the lung and kidney, and show delayed lung-liquid clearance and reduced colonic ENaC-mediated transport (13). These findings demonstrate βENaC expression and function are suppressed in the βENaC m/m mouse model.

The goal of the current study was to determine whether pressure-induced constriction is dependent on normal expression of VSMC βENaC. To address this aim, we evaluated 1) VSMC βENaC expression, 2) depolarization and α-agonist induced constriction, and 3) pressure-induced constriction in cerebral arteries from βENaC+/+ and m/m mice. Our results indicate pressure-induced constriction is dependent on VSMC βENaC expression.

METHODS

All protocols and procedures used in this study were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Mississippi Medical Center.

Animals.

Heterozygote βENaC+/m mating pairs were generously provided by E. Hummler and B. Rossier, University of Lausanne, Switzerland. Animals were provided standard rodent chow and water ad libitum. All mice were of similar age (∼7 wk) and body weight (∼21 g) (Table 1). Animals were exposed to 12:12-h light (0600–1800) -dark (1800–0600) cycles.

Table 1.

Age and body weights of βENaC+/+ and m/m mice

	Genotype		P
+/+	m/m
Age, wk	7.1±0.6	6.9±0.4	0.51
Body weight, g	20.9±1.5	22.0±0.8	0.76
n	9	9

Data are expressed as means ± SE. m/m, βENaC homozygous mutant; ++, βENaC wild-type littermates.

Genotyping.

Offspring of heterozygote mating pairs were genotyped at 3 wk of age using DNA-isolated (DirectTail PCR, Viagen) from tail samples and reconfirmed following phenotypic analysis using liver samples. Expression of wild-type and mutant DNA were determined using polymerase chain reaction (AccuPrime Supermix II, Invitrogen) (13). Oligonucleotides for the wild-type allele were 5′-CTTCCAAGAGTTCAACTACCG-3′ and 5′-TCTACCAGCTCAGCCACAGTG-3′. Oligonucleotides for the mutant allele were 5′-CTTCCAAGAGTTCAACTACCG-3′ and 5′-CTGCTATTGGCCCGCTGCCCCA-3′. The oligonucleotides for the mutant allele detect the presence of the neomycin cassette. DNA samples were incubated at 92°C for 1 min, 53°C for 1 min, and 72°C for 1 min for 32 cycles and then incubated at 72°C for another 5 min. PCR products were separated by gel electrophoresis.

Cerebral VSMC dissociation and quantitative immunolabeling.

We used quantitative immunolabeling as previously described to determine whether βENaC expression was suppressed in βENaC m/m cerebral VSMCs. For these studies, cerebral vessels were isolated, and VSMCs were enzymatically dispersed and fixed, as described previously (10). Samples were labeled with rabbit anti βENaC antibodies as previously described (10, 11). All samples were examined using quantitative fluorescence confocal microscopy (TCS-SP2, Leica Microsystems). All samples were collected, labeled, and imaged side-by-side under identical conditions. Background signal from no-primary antibody controls was subtracted from all samples. We have used this approach to quantify ENaC expression in VSMCs previously (7, 10, 11). Data were averaged from two animals in each group.

Cannulation of middle cerebral arteries for dimensional analysis.

Animals were anesthetized with isofluorane, decapitated, and the brain was excised and placed in ice-cold physiological saline solution (PSS), as previously described (10, 11). Middle cerebral arteries were dissected and mounted in a vessel chamber (CH/1/SH, Living Systems) and analyzed using MetaMorph software (Universal Imaging) as described previously (7, 10, 11). Following an initial incubation period (30 min at 50 mmHg), a pressure-diameter curve was generated to determine the effect of βENaC suppression on myogenic constriction, as previously described (7). The pressure-diameter curve was repeated in the presence of Ca²⁺-free PSS (same as above PSS plus 2 mM EGTA and omit 1.8 mM CaCl₂) plus papaverine (10⁻⁴ M) to determine the passive pressure-diameter curve. Myogenic constriction was calculated as the percent difference between the active (PSS) and passive (Ca²⁺-free PSS) inner diameter at each pressure. As a control for depolarization-induced vascular reactivity, we assessed vasoconstriction to KCl (20, 40, and 80 mM) immediately following pressurization. Additionally, following the pressure-ramp, we assessed vasoconstriction to the α₁-adrenergic receptor agonist, phenylephrine (PE; 10⁻⁹-10⁻⁵ M). Phenylephrine reactivity was used as a supplemental control to ensure the vessel had not been injured during the pressure ramp, usually incurred by passage of small air bubbles. The log of the half-maximal effective concentration (EC₅₀) for PE was calculated using Prism software (GraphPad).

Calculations of wall thickness, circumferential stress, and strain.

Wall thickness was calculated as (outer diameter-inner diameter/2), where diameter is in micrometers, in vessels following pressurization at 50 mmHg. Wall-to-lumen ratio was calculated as the wall thickness/inner diameter. For these measurements, we included all vessels with robust KCl responses, regardless of responsiveness to PE. Circumferential strain was calculated as inner diameter_p − inner diameter₁₅, where 15 is the lowest pressure step (15 mmHg) under Ca²⁺-free conditions. For calculation of circumferential stress intraluminal pressure was converted from mmHg to Newtons (where 1 mmHg =1.334 × 102 N/m²). Circumferential stress was calculated as (intraluminal pressure × intraluminal diameter)/ (2 × wall thickness) under Ca²⁺-free conditions. For these measurements, only vessels with robust responses to PE were used.

Statistics.

All data are expressed as mean ± SE. An independent t-test or two-way repeated-measure ANOVA was used to make comparisons where appropriate. The Student-Newman-Keuls post hoc test was used to make comparisons among groups. Statistical significance was considered at P < 0.05.

RESULTS

βENaC expression in cerebral VSMCs.

To confirm VSMC βENaC was suppressed in the mouse model, we used quantitative immunolabeling. We found βENaC immunolabeling in cerebral VSMCs of βENaC m/m mice was suppressed 46% compared with +/+ controls (Fig. 1). Images reflect βENaC immunolabeling near the surface of the VSMC.

Fig. 1.

Cerebral artery vascular smooth muscle cells (VSMC) epithelial Na⁺ channel (βENaC) immunolabeling signal is downregulated in βENaC m/m mice. A: representative images of βENaC immunolabeling in cerebral artery VSMCs in +/+ and m/m mice. B: group data demonstrate βENaC immunolabeling is reduced 46% in freshly dissociated cerebral artery VSMCs. n = number of VSMCs examined. *Statistical significance, P < 0.05.

Agonist-induced constrictor responses in middle cerebral artery.

Changes in inner diameter (μm) in response to KCl (4–80 mM; n = 9 per group) and phenylephrine (10⁻⁷–10⁻⁴ M; n = 7 per group) are shown in Fig. 2, A and B, respectively. MCAs from βENaC+/+ and m/m mice vasoconstricted identically to KCl. With the exception of the response to 10⁻⁶ M PE, MCAs from βENaC+/+ and m/m mice vasoconstricted similarly to phenylephrine. Vasoconstriction to maximal doses of PE (10⁻⁵ and 10⁻⁴) were similar between βENaC+/+ and m/m mice. Two-way repeated-measures ANOVA showed an effect of KCl dose (P < 0.001) and PE dose (P < 0.001) but no effect of genotype (P = 0.17, P = 0.35) for KCl and PE, respectively] or interaction between genotype and KCl or PE dose (P = 0.93, P = 0.30). Post hoc analysis indicates a significant difference between +/+ and m/m PE responses only at 10⁻⁶ M PE (P < 0.05). Log EC₅₀ values for PE, also presented in Fig. 2B, were −5.9 ± 0.4 and −5.2 ± 0.2 M for +/+ and m/m groups, respectively (P = 0.09).

Fig. 2.

Vasoconstriction to depolarizing agonist KCl and α-agonist phenylephrine (PE) is similar in middle cerebral arteries (MCAs) from (βENaC+/+) and m/m mice. A: vasoconstriction to KCl is identical in MCAs from +/+ (n = 9) and m/m (n = 9) mice. B: vsoconstriction to PE is similar in +/+ (n = 7) and m/m (n = 7) mice. Using a post hoc analysis, only the response at 10⁻⁶ M PE is significantly different between genotypes. Log of EC₅₀ for each genotype is shown, P = 0.09. *Statistically significant difference, P < 0.05.

Pressure-induced constriction.

Middle cerebral artery vasoconstrictor responses to stepwise increases in luminal pressure are shown in Fig. 3; changes in inner diameter under normal PSS (A) and Ca²⁺-free conditions (B) in βENaC+/+ and m/m mice are shown. The arteries passively dilated (Ca²⁺-free) to the same extent (+/+, 79 ± 4 μm; m/m, 76 ± 4 μm at 90 mmHg). The relationship between intraluminal pressure and myogenic tone is shown in Fig. 3C. In βENaC+/+ mice, myogenic tone increased from 1% at 15 mmHg to 10% at 90 mmHg (P < 0.001). However, in the m/m mice, myogenic tone did not increase from 15 to 90 mmHg (P = 0.512). At 90 mmHg, middle cerebral arteries from βENaC+/+ animals developed significantly more myogenic tone than those from βENaC m/m (10 ± 4 vs. 4 ± 1%). To further quantify the effect of βENaC on the slope of the pressure-myogenic tone relationship, we plotted pressure on a log scale (Fig. 3D) and obtained the mean slope for both groups. As shown in Fig. 3E, the slope of the pressure (log)-myogenic tone relationship in βENaC m/m mice was significantly less than βENaC+/+ [11 ± 2 vs. 3 ± 2% tone/mmHg (log), Fig. 3E]. The slope of the line for βENaC m/m log of pressure vs. myogenic tone was not different than the slope of a horizontal line, where slope = 0 (P = 0.10), suggesting MCAs from βENaC m/m mice failed to develop myogenic tone.

Fig. 3.

Pressure-induced constriction in the MCA is suppressed in βENaC m/m mice. Relationship between intraluminal pressure and intravascular diameter in MCAs from +/+ (n = 5) (A) and m/m (n = 5) (B) mice under active (Ca²⁺ containing, +Ca²⁺) and passive (Ca²⁺ free, -Ca²⁺) conditions. C: MCAs from −/− mice do not develop myogenic tone when intraluminal pressure is increased. Two-way repeated-measures ANOVA showed an effect of intraluminal pressure (P < 0.001) and genotype (P = 0.020) and an interaction between genotype and intraluminal pressure (P = 0.012). D: relationship between the log of pressure and myogenic tone. Log of pressure and myogenic tone relationship was linearized to obtain the slope, or sensitivity, of the myogenic response. E: slope of the relationship between log of pressure and myogenic tone.

Passive properties and dimensional analysis.

To determine whether changes in passive properties might account for changes in myogenic tone in βENaC m/m mice, we evaluated pressure-induced dilation, circumferential strain, and circumferential stress under Ca²⁺-free conditions in βENaC+/+ and m/m mice. As shown in Fig. 4, we found that pressure-induced dilation (Fig. 4A), circumferential strain (Fig. 4B), and circumferential stress (Fig. 4C) under Ca²⁺-free conditions were identical. To determine whether structural remodeling occurs in βENaC m/m mice, we evaluated wall thickness and the wall-to-lumen ratio and found them to be identical among the βENaC genotypes (Table 2). These findings suggest βENaC m/m mice do not have changes in passive vascular properties or structural remodeling, at least at 6–8 wk of age.

Fig. 4.

Passive properties of the MCA in βENaC+/+ and m/m mice are identical. Internal vessel diameter (A), circumferential strain (B) and circumferential stress (C) under Ca²⁺-free conditions are identical in +/+ (n = 5) and m/m (n = 5) mice.

Table 2.

Inner and outer diameter, wall thickness, and wall-to-lumen ratio at 50 mmHg in middle cerebral arteries of βENaC mice

	Genotype		P
+/+	m/m
Inner diameter, μm	67±3	65±3	0.64
Outer diameter, μm	80±2	77±3	0.40
Wall thickness, μm	13±1	12±1	0.34
Wall-to-lumen ratio	0.20±0.02	0.20±0.02	0.96
n	9	9	9

Data are expressed as means ± SE.

DISCUSSION

Myogenic constriction is a mechano-dependent response inherent to certain blood vessels. It is characterized by vasoconstriction in response to stretch-induced by an increase in luminal pressure and vasodilation to decreases in luminal pressure (3, 8). The molecular identity of the signaling complex that transduces vessel stretch into vasoconstriction is unknown. The discovery of a family of mechanosensory proteins found in neurons and muscle tissue of the nematode led to the hypothesis that related mammalian proteins might also act as mechanosensors in vascular smooth muscle (5, 6). ENaC proteins are evolutionarily related to degenerins, sharing amino acid homology and protein structure. In lung, kidney, and colon epithelial tissue, α, β, and γENaC proteins associate to form a Na⁺ channel that plays a critical role in salt and water transport. Previous studies suggest βENaC and γENaC are the predominant ENaC subunits found in cerebral VSMCs (4, 10, 11). Early studies using transient gene silencing approaches (siRNA/dominant-negative constructs) suggested silencing of βENaC alone is sufficient to nearly abolish the myogenic response (10). The importance of βENaC in the long-term regulation of myogenic constriction is unknown; therefore, the purpose of the current investigation is to determine whether long-term suppression of βENaC inhibits myogenic constriction in middle cerebral arteries. Our findings demonstrate that pressure-induced, but not agonist-induced constriction, is abolished in MCAs from a mouse model of reduced βENaC.

βENaC m/m model.

The mouse model used in the current study is a model of βENaC downregulation. The model was generated in the attempt to create a model of Liddle's syndrome by insertion of a premature stop codon near the coding region for the C terminus (13). However, the presence of the premature stop codon destabilizes message and reduces βENaC expression and function (13). Reduced levels of ENaC expression and function are supported by findings of 1) reduced expression of βENaC mRNA (<4%) in the kidney, lung, and colon; 2) undetectable tubular and bronchiolar βENaC immunolabeling; 3) reduced amiloride-sensitive short-circuit current in lung explants; 4) reduced amiloride-sensitive transport in colon explants; and 5) a twofold increase in serum aldosterone level (13). Consistent with this, we found that βENaC immunolabeling signal in cerebral VSMCs is also inhibited in βENaC m/m mice. These findings indicate the βENaC m/m mouse is a mouse with βENaC downregulation in VSMCs. Thus, the model was used to determine the importance of βENaC in pressure-induced constriction in MCAs.

Importance of βENaC in pressure-induced constriction.

Previous reports suggest that suppression of renal VSMC βENaC using transient gene-silencing approaches leads to a 1) 50% reduction in protein expression, 2) total loss of pressure-induced constriction, and 3) intact vasoconstriction to depolarizing and α-adrenergic agonists (10). Consistent with previous findings using transient suppression of βENaC, we found pressure-induced constriction is abolished in MCAs from βENaC m/m mice in the current investigation. Furthermore, the slope of the log of pressure-myogenic tone relationship in βENaC m/m MCAs is not different from a horizontal line (slope = 0), suggesting pressure-induced constriction is abolished. These findings demonstrate that βENaC is critical to pressure-induced constriction.

There are now three lines of evidence suggesting βENaC may be a key component of the vascular mechanosensor. First, blotting and immunolabeling in VSMCs more easily detect βENaC protein than γENaC and ASIC2 using multiple antibodies (4, 10–12). Second, transient silencing of βENaC using siRNA or dominant-negative isoforms has a greater impact on myogenic responsiveness than silencing of γENaC (10). Third, ASIC2 homozygous null mice have modest alterations in cerebral myogenic constrictor responses (7), while the responses are abolished in βENaC m/m mice, as shown in the current investigation. The findings of the current investigation confirm that βENaC is an important mediator of the myogenic response in cerebral vessels.

The role of other DEG/ENaC proteins.

Although our findings support an important role for βENaC, it is likely that βENaC is not the only DEG/ENaC protein involved in transduction of the myogenic response. In previous studies, we have detected expression of γENaC and a closely related family member ASIC2, both colocalize with βENaC (4, 11). However, we have been unable to detect expression of αENaC message or protein in cerebral vessels, suggesting αENaC is not highly expressed in this tissue (4). Thus, ASIC2, βENaC, and γENaC may associate to form the pore of a heteromultimeric mechanosensor in VSMCs (5, 15). We speculate that in the absence of βENaC, the remaining ENaC/ASIC subunits may not assemble properly to form a functional ion channel pore.

Is there a generalized suppression of vasoconstrictor ability in βENaC m/m mice?

Similar to our previous studies, KCl-induced vasoconstriction is identical following inhibition of degenerin channel activity or expression (7, 10, 11). In contrast, we found that vasoconstriction to PE was suppressed at lower concentrations; raising the possibility that vasoconstrictor responsiveness may be modestly altered in βENaC m/m mice. However, the underlying cause and physiological significance of the altered response to PE is not clear. One potential cause may be related to the sequence in which they are administered in the protocol. KCl is administered at the beginning of the protocol to ensure the vessels constrict to a depolarizing stimulus. This is an important control because the myogenic response may be initiated by a membrane depolarization-induced activation of L-type Ca²⁺ channels (3, 8, 9). PE, which uses a G protein-coupled pathway, is administered following the pressure ramp to ensure the vessels have not been injured during the pressure ramp, typically by passage of air bubbles. Although depolarization induced L-type Ca²⁺ channels and downstream components of G protein-coupled signaling contribute to the myogenic response, KCl may be a more appropriate control for the ability of a vessel to constrict to depolarizing stimulus such as pressure. However, we cannot rule out the possibility that the modest loss of PE-induced constriction contributes to the loss of myogenic constriction in the βENaC m/m mice. The underlying cause and physiological significance of the altered response to PE remains to be determined.

Lack of evidence for structural remodeling in βENaC m/m vessels.

Myogenic tone is a function of the difference between vessel diameters under active and passive conditions; disease processes, such as hypertension, which limit the distensibility of a vessel, can suppress myogenic tone (1, 2). Two lines of evidence suggest it is unlikely that hypertension-induced changes vascular structure account for the loss of myogenic tone at 6–8 wk. First, acute measurements suggest BP is not different in adult βENaC+/+ and m/m mice (13). Second, indicators of vascular remodeling, such as wall thickness, wall-to-lumen ratio, and passive vascular properties, including pressure-induced vasodilation, circumferential stress, and strain, are identical between βENaC+/+ and m/m mice. Another potential factor is aldosterone. Plasma aldosterone levels are elevated in adult βENaC m/m mice, and mineralocorticoid receptor activation can induce vascular remodeling (16). However, a lack of evidence for vascular remodeling at 6–8 wk of age does not support a role for elevated aldosterone. Taken together, these findings suggest the loss of myogenic tone is not due to changes in the passive properties or structural remodeling of MCAs in βENaC m/m mice, but rather a change in the ability of the vessel to constrict to a pressure stimulus.

In summary, the findings of the current investigation demonstrate that pressure-induced constriction in middle cerebral arteries is abolished in a mouse model of βENaC downregulation, while constriction to a depolarizing agonist is unchanged, and constriction to α-adrenergic agonist is mildly suppressed. These data demonstrate that βENaC is an important mediator of myogenic constriction. These findings are consistent with the evolutionary conservation of function of DEG/ENaC proteins as mechanosensors.