Transcriptional Upregulation of α2δ-1 Elevates Arterial Smooth Muscle Cell Voltage-Dependent Ca2+ Channel Surface Expression and Cerebrovascular Constriction in Genetic HypertensionNovelty and Significance
A hallmark of hypertension is an increase in arterial myocyte voltage-dependent Ca2+ (CaV1.2) currents that induces pathological vasoconstriction. CaV1.2 channels are heteromeric complexes composed of a pore-forming CaV1.2α1 with auxiliary α2δ and β subunits. Molecular mechanisms that elevate CaV1.2 currents during hypertension and the potential contribution of CaV1.2 auxiliary subunits are unclear. Here, we investigated the pathological significance of α2δ subunits in vasoconstriction associated with hypertension. Age-dependent development of hypertension in spontaneously hypertensive rats was associated with an unequal elevation in α2δ-1 and CaV1.2α1 mRNA and protein in cerebral artery myocytes, with α2δ-1 increasing more than CaV1.2α1. Other α2δ isoforms did not emerge in hypertension. Myocytes and arteries of hypertensive spontaneously hypertensive rats displayed higher surface-localized α2δ-1 and CaV1.2α1 proteins, surface α2δ-1:CaV1.2α1 ratio, CaV1.2 current density and noninactivating current, and pressure- and depolarization-induced vasoconstriction than those of Wistar-Kyoto controls. Pregabalin, an α2δ-1 ligand, did not alter α2δ-1 or CaV1.2α1 total protein but normalized α2δ-1 and CaV1.2α1 surface expression, surface α2δ-1:CaV1.2α1, CaV1.2 current density and inactivation, and vasoconstriction in myocytes and arteries of hypertensive rats to control levels. Genetic hypertension is associated with an elevation in α2δ-1 expression that promotes surface trafficking of CaV1.2 channels in cerebral artery myocytes. This leads to an increase in CaV1.2 current-density and a reduction in current inactivation that induces vasoconstriction. Data also suggest that α2δ-1 targeting is a novel strategy that may be used to reverse pathological CaV1.2 channel trafficking to induce cerebrovascular dilation in hypertension.
See Editorial Commentary, pp 894–895
Hypertension is associated with an elevation in arterial contractility that increases systemic blood pressure and restricts organ blood flow, leading to end-organ damage.1 Hypertension is also a major predictor for a variety of cerebral diseases, including stroke, Alzheimer disease, and dementia. One characteristic pathological alteration that occurs in hypertension is an elevation in vascular smooth muscle cell (myocyte) voltage-dependent Ca2+ influx.2,3 Voltage-dependent L-type Ca2+ (CaV1.2) channels are the primary Ca2+ entry pathway in arterial myocytes and are essential for contractility regulation by a wide variety of stimuli, including intravascular pressure, membrane potential, and vasoconstrictors.4–8 A hypertension-associated elevation in CaV1.2 currents leads to an increase in intracellular Ca2+ concentration and vasoconstriction.9–11 However, molecular mechanisms that elevate arterial myocyte CaV1.2 currents in hypertension, leading to vasoconstriction, are unclear.
CaV1.2 channels are heteromeric complexes composed of a pore-forming α1 with auxiliary α2δ and β subunits.12 Four α2δ (1–4) subunit isoforms have been identified that are each encoded by different genes.13,14 α2δ subunits undergo posttranslational cleavage into a highly glycosylated extracellular α2 and a smaller δ subunit, which are subsequently coupled by a disulfide bond to form a single functional protein.14,15 α2δ subunits are membrane bound by the bilayer-spanning δ subunit. Recently, α2δ-1 was identified as being critical for functional trafficking of CaV1.2α1 subunits to the plasma membrane (surface) in arterial myocytes.16 To date, no studies have investigated pathological or disease-associated molecular changes in CaV1.2 auxiliary subunits, including α2δ subunits, in myocytes of resistance-sized arteries. In addition, it is unclear whether the subunit composition of arterial myocyte surface CaV1.2 channels is altered in disease. Given that arterial myocyte CaV1.2 currents are elevated during hypertension, leading to vasoconstriction, we determined the subunit composition of CaV1.2 channels and investigated the involvement of α2δ subunits in this pathological alteration.9–11 Elucidating molecular mechanisms governing α2δ subunit regulation of CaV1.2 channels in hypertension could lead to the development of novel approaches to treat cardiovascular diseases.
Here, we used a genetic model of hypertension, the spontaneously hypertensive rat (SHR), to investigate the pathological significance of arterial myocyte α2δ subunits in hypertension. We showed that, during hypertension, an elevation in α2δ-1 expression increases plasma membrane CaV1.2 currents in arterial myocytes, leading to vasoconstriction. We also identified α2δ-1 as a novel therapeutic target to induce cerebrovascular dilation in hypertension.
Cell Isolation and Tissue Preparation
All of the animal protocols used were reviewed and approved by the animal care and use committee at the University of Tennessee Health Science Center. Male 6- or 12-week–old SHR and Wistar-Kyoto (WKY) rats were euthanized by IP injection of sodium pentobarbital (150 mg/kg of body weight, Vortech Pharmaceuticals, Dearborn, MI). Middle cerebral, posterior cerebral, and cerebellar arteries (≈100–200 µm diameter) were studied. Myocytes were enzymatically dissociated from dissected cerebral arteries, as described previously.4
Blood Pressure Measurements
Diastolic and systolic blood pressures were measured in conscious rats using a tail-cuff sphygmomanometer (Kent Scientific, Torrington, CT).
RT-PCR was performed on myocytes individually collected under a microscope using an enlarged patch-clamp pipette to prevent contamination from other arterial wall cell types, as described previously.4
Quantitative Real-Time PCR
Total RNA was isolated from cerebral arteries using TRIzol (Invitrogen, Grand Island, NY). cDNA was transcribed using Affinity Script Multiple temperature reverse transcriptase (Stratagene, Clara, CA). Gene specific primers and probes were designed using the Universal Probe Library. Sequences of primers and probes used and PCR reaction efficiencies are given in Table S1 (available in the online-only Data Supplement).
Protein Analysis and Biochemistry
Proteins were separated on SDS-PAGE gels and analyzed by Western blotting. Blots were cut at the 75-kDa marker to allow simultaneous probing of the upper section for α2δ-1 and lower section for actin. The upper portion of the blot was then reprobed for CaV1.2α1. Protein band intensities were determined using Quantity One (BioRad, Hercules, CA) software. For quantification, protein band intensities were first normalized to actin and then to appropriate control samples.
Artery Surface Biotinylation
To determine the distribution of α2δ-1 and CaV1.2α1 subunit proteins between surface and intracellular compartments, artery surface biotinylation was used, as described previously.16
Whole cell CaV1.2 currents were recorded in isolated myocytes using the whole cell patch clamp configuration, as described previously.16
Pressurized Artery Myography
Endothelium-denuded artery diameter was measured over a range of intravascular pressures (20–100 mm Hg) in the presence and absence of nimodipine (1 µmol/L) using edge-detection myography, as described previously.17 Diameter responses to elevating extracellular K+ from 6 to between 20 and 60 mmol/L at 10 mm Hg in the presence of pinacidil (10 µmol/L), a KATP channel opener, were also recorded. Arteries treated with pregabalin for 24 hours were also maintained in pregabalin throughout these experiments to inhibit CaV1.2 subunit membrane reinsertion.
Summary data are presented as mean±SEM. Significance was determined using paired or unpaired t tests with Welsh correction or ANOVA followed by Student-Newman-Keuls for multiple groups. P<0.05 was considered significant. Power analysis was carried out where P value was >0.05 to verify that sample size was sufficient to give a value of >0.8.
An expanded Methods section is available in the online-only Data Supplement.
Age-Dependent Development of Genetic Hypertension Is Associated With an Elevation in Arterial Myocyte α2δ-1 and CaV1.2α1 Subunit Expression
The pathological involvement of arterial myocyte CaV1.2 subunits was studied using a rat genetic model of hypertension. At 6 weeks of age, WKY and SHR diastolic, systolic, and mean arterial blood pressures were similar (Figure S1, available in the online-only Data Supplement). In contrast, at 12 weeks of age, diastolic, systolic, and mean arterial pressures were ≈63, 65, and 72 mm Hg higher in SHRs than WKY rats, respectively (Figure S1).
Four different α2δ isoforms have been described, with α2δ-1 the only isoform expressed in normotensive Sprague-Dawley (SD) rat cerebral artery myocytes.14,16 We tested the hypothesis that hypertension is associated with a shift in α2δ isoform expression in myocytes of resistance-size arteries. RT-PCR detected only α2δ-1 in pure cerebral artery myocytes from 12-week–old WKY rats and hypertensive SHRs (Figure 1A). In contrast, the same primers amplified transcripts for all of the α2δ isoforms in WKY and SHR whole brain (Figure 1A).
Quantitative PCR was performed to compare α2δ-1 and CaV1.2α1 message levels in 6- and 12-week–old WKY and SHR cerebral arteries. Eight different reference genes were screened to identify those with similar mRNA levels in cerebral arteries of WKY rats and SHRs (Table S1, available in the online-only Data Supplement). Rps5 mRNA levels were similar in WKY and SHR arteries and, thus, Rps5 was used as the reference gene for these experiments (Table S2). Quantitative PCR indicated that mean α2δ-1 and CaV1.2α1 mRNA levels were similar in 6-week–old WKY and SHR arteries (Figure 1B). In contrast, α2δ-1 and CaV1.2 mRNAs were ≈2.1- and 1.5-fold higher, respectively, in 12-week–old SHR compared with WKY arteries (Figure 1B). Age-dependent development of hypertension was also associated with a larger increase in α2δ-1 than CaV1.2α1 mRNA (Figure 1B). These data indicate that hypertension is associated with an elevation in α2δ-1 and CaV1.2α1 subunit mRNA but not with the appearance of other α2δ isoforms in arterial myocytes.
Next, we investigated whether age-dependent development of genetic hypertension is associated with upregulation of α2δ-1 and CaV1.2α1 proteins in cerebral arteries. α2δ-1 and CaV1.2α1 protein levels were similar in 6-week–old WKY and SHR arteries (Figure 1C and 1D). Aging between 6 and 12 weeks did not alter α2δ-1 and CaV1.2α1 protein in WKY rat arteries but increased these proteins ≈2.1- and 1.4-fold in SHR arteries (Figure S2). At 12 weeks of age, α2δ-1 and CaV1.2α1 proteins were ≈2.5- and 1.7-fold higher in SHR compared with age-matched WKY arteries (Figure 1C and 1D). In agreement with message levels, age-dependent development of hypertension also increased α2δ-1 more than CaV1.2α1 protein (Figures 1C, 1D, and S2).
In summary, these data indicate that genetic hypertension is associated with transcriptional upregulation of both α2δ-1 and CaV1.2α1 in cerebral artery myocytes. α2δ-1 and CaV1.2α1 proteins are elevated more than their respective mRNAs (Figures 1B through 1D and S2), suggesting that hypertension-associated changes in posttranslational events also contribute to increased CaV1.2 channel subunit expression during hypertension. Furthermore, during hypertension there is a larger increase in mRNA and protein for α2δ-1 than for CaV1.2α1.
Hypertension Is Associated With an Elevation in Surface α2δ-1 and CaV1.2α1 Proteins in Arteries
α2δ-1 induces membrane trafficking of CaV1.2α1 subunits in SD rat arterial myocytes.16 Therefore, we tested the hypothesis that an increase in α2δ-1 contributes to elevated surface CaV1.2 expression in hypertension. Surface (plasma membrane) and intracellular α2δ-1 and CaV1.2α1 proteins were measured in age-matched WKY and hypertensive SHR cerebral arteries using biotinylation. Surface-localized α2δ-1 and CaV1.2α1 proteins were ≈2.6- and 2-fold higher, respectively, in SHR compared with WKY rat arteries (Figure 2A and 2B). A larger percentage of total α2δ-1 and CaV1.2α1 was located at the plasma membrane in SHR compared with WKY arteries (Figure 2A and 2C). In WKY arteries, more of the total amount of α2δ-1 (≈85%) than CaV1.2α1 (≈77%) was located at the surface. In contrast, in SHR arteries, the percentages of total α2δ-1 (≈93%) and CaV1.2α1 (≈92%) located at the surface were similar (Figure 2A and 2C). These data indicate that, during hypertension, an elevation in α2δ-1 and CaV1.2α1 total protein translates to an increase in surface expression of these subunits in arterial myocytes. Furthermore, hypertension is associated with an alteration in the distribution of α2δ-1 and CaV1.2α1 proteins between intracellular and surface compartments.
Pregabalin Reduces Surface Trafficking of CaV1.2 Channel Subunits More Effectively in Hypertensive Than Normotensive Rat Arteries
Pregabalin, an α2δ-1/2 ligand, reduces surface trafficking of CaV1.2, 2.1, and 2.2 channels in neurons and arterial myocytes.14,16,18–20 Next, we studied pregabalin regulation of α2δ-1 and CaV1.2α1 subunit surface expression and subunit cellular distribution in WKY and SHR cerebral arteries. For these experiments, arteries were incubated for 24 hours with or without pregabalin. Pregabalin (24 hours) did not alter total protein of α2δ-1 (% control: WKY, 115 ± 9; SHR, 118 ± 20) or CaV1.2α1 (% control: WKY, 116 ± 10; SHR, 109 ± 14; Figure 3A; WKY, n=4–5; SHR, n=5; P>0.05 for each). In contrast, pregabalin reduced surface α2δ-1 and CaV1.2 and increased intracellular levels of these proteins in both WKY and SHR arteries (Figure 3A through 3C and Figure S3). Pregabalin reduced plasma membrane α2δ-1 and CaV1.2α1 ≈3.1 and 1.9-fold more, respectively, in hypertensive SHR compared with WKY control arteries (Figure 3C). To evaluate pregabalin regulation of α2δ-1 and CaV1.2 cellular distribution, surface:intracellular protein ratios were calculated. Consistent with data shown in Figure 2C, 2a larger proportion of α2δ-1 and CaV1.2 subunits were present at the plasma membrane in SHR compared with WKY arteries (Figure 3D). Pregabalin induced a larger reduction in surface:intracellular α2δ-1 and CaV1.2 in SHR compared with WKY arteries (Figure 3D).
Hypertension was associated with a larger increase in surface α2δ-1 than CaV1.2α1 protein in arteries (Figure 2A and 2B). We calculated the band intensity ratio of surface α2δ-1: CaV1.2α1 and regulation by pregabalin. Although this methodology cannot determine subunit stoichiometry, total protein loaded in each lane is identical, allowing for comparison of this ratio in SHR and WKY arteries from the same blot. The mean surface α2δ-1:CaV1.2α1 band intensity ratio was ≈1.38 in SHR arteries and ≈1.06 in WKY arteries, or ≈1.3-fold higher in SHRs (Figure 3E). Pregabalin reduced the surface α2δ-1:CaV1.2α1 band intensity ratio to ≈0.91 in SHR arteries but did not change the ratio in WKY rat arteries (Figure 3E).
Collectively, these data indicate that pregabalin blocks surface expression of α2δ-1 and CaV1.2α1 subunits more effectively in hypertensive than in normotensive rat arteries. During hypertension, surface α2δ-1 protein is elevated more so than CaV1.2α1 protein, leading to an increase in the ratio of plasma membrane α2δ-1:CaV1.2α1 subunits. Pregabalin reverses this elevation in surface α2δ-1:CaV1.2α1 subunits. These data also indicate that α2δ-1 is essential for upregulation of surface CaV1.2 channels in arterial myocytes during genetic hypertension.
α2δ-1 Targeting Reverses Hypertension-Associated Modifications in CaV1.2 Current Density and Inactivation in Arterial Myocytes
To investigate the functional impact of elevated α2δ-1 expression and effects of α2δ-1 targeting, CaV1.2 currents were measured in age-matched WKY and hypertensive SHR cerebral artery myocytes. Mean peak CaV1.2 current density (Ba2+ as charge carrier) was ≈5.3 pA/pF in hypertensive SHR cells compared with ≈2.4 pA/pF in WKY cells or ≈2.2-fold larger (Figure 4A and 4B and Table). Pregabalin (24 hours) reduced peak CaV1.2 current density in SHR cells to ≈2.2 pA/pF, or by ≈59%, and to ≈1.6 pA/pF in WKY cells, or by ≈32% (Figure 4A and 4B and Table). Pregabalin reduced peak CaV1.2 current density in SHR myocytes to the current density of untreated WKY cells (Figure 4A and 4B and Table). The relationship between cell capacitance and peak CaV1.2 current was investigated (Figure 4C). When data were fit with a linear function, the slope was −5.41 for SHR cells and −2.40 for WKY cells, or 2.3-fold higher (Figure 4C). Pregabalin reduced slopes by ≈58% and 25% in SHR and WKY cells, respectively (Figure 4C). Slopes were similar for untreated WKY cells and pregabalin-treated SHR cells (Figure 4C; P>0.05). Mean cell capacitance for WKY (16.3 ± 0.8 pF) and SHR (17.1 ± 1.3 pF) cells were similar and were not altered by pregabalin (WKY, 18.8 ± 1 pF; SHR, 15.8 ± 0.8 pF; P>0.05 when comparing all), indicating that current density and slope increased because of changes in CaV1.2 channels (Figure 4B and 4C).
The voltage-dependence of half-maximal CaV1.2 current activation and slope were similar in untreated control and pregabalin-treated WKY and SHR arterial myocytes (Figure 4D and Table). The voltage dependence of half-maximal inactivation and slope were also similar in untreated control and pregabalin-treated WKY and SHR cells (Figure 4E and Table). In contrast, untreated SHR cells displayed a noninactivating CaV1.2 current that was ≈2-fold larger than in WKY cells (Figure 4A and 4E). Pregabalin (24 hours) reduced the noninactivating current in SHR cells such that it was similar to WKY cells (Figure 4A and 4E). CaV1.2 current inactivation rates (τ) were similar in control and pregabalin-treated WKY and SHR cells (Figure S4).
In addition to acting as an inhibitor of α2δ-1-induced CaV1.2 channel trafficking, pregabalin is a weak CaV1.2 channel pore blocker that does not directly alter CaV1.2 current voltage dependence in normotensive SD rat arterial myocytes.16 To determine whether the reduction in CaV1.2 current amplitude in pregabalin-treated WKY and SHR myocytes was because of CaV1.2 pore block, we measured CaV1.2 current regulation in untreated cells by acute bath application of pregabalin. Acute pregabalin reduced CaV1.2 currents in WKY cells by ≈12% (Figure 4F). In contrast, pregabalin reduced CaV1.2 currents in SHR myocytes by ≈23%, or ≈1.9-fold more than in WKY cells (Figure 4F). Acute pregabalin-induced CaV1.2 current inhibition was significantly smaller than that induced by 24-hour pregabalin treatment in both WKY (≈32% inhibition) and SHR (≈59% inhibition) cells (Figure 4B, 4C, and 4F). When combined with the biochemical data illustrated in Figure 3, these data indicate that acute and chronic pregabalin inhibit CaV1.2 currents through distinct mechanisms in arterial myocytes.
Collectively, data indicate that genetic hypertension is associated with an elevation in α2δ-1 expression that stimulates surface expression of CaV1.2α1 subunits, leading to a CaV1.2 current elevation and an increase in noninactivating current. α2δ-1 targeting reduces the hypertension-associated α2δ-1-induced elevation in CaV1.2α1 surface expression, leading to a reduction in CaV1.2 current density. α2δ-1 targeting also restores CaV1.2 current inactivation.
α2δ-1 Targeting Reverses Elevated Pressure- and Depolarization-Induced Vasoconstriction in Hypertension
The functional significance of hypertension-associated alterations in α2δ-1 signaling was studied by measuring arterial contractility. Diameter regulation by intravascular pressure (20–100 mm Hg) was measured in WKY and SHR cerebral arteries that had been incubated for 24 hours with or without pregabalin. SHR arteries developed more myogenic tone than WKY arteries over the entire pressure range (Figure 5A, 5B). Pregabalin reduced myogenic tone in WKY and SHR arteries, decreased tone more in SHR than in WKY arteries (eg, % reduction in myogenic tone at 60 mm Hg: SHR, ≈41%; WKY, ≈28%), and reduced tone in SHR arteries to levels in untreated WKY arteries (Figure 5). Nimodipine (1 µmol/L), a voltage-dependent Ca2+ channel blocker, fully dilated control and pregabalin-treated WKY and SHR arteries at all pressures (20–100 mm Hg) studied, indicating that myogenic tone occurred because of CaV1.2 channel activation (Figure 5B). Passive arterial diameters were similar for WKY (249 ± 11 µm) and hypertensive SHR (242 ± 9 µm) cerebral arteries (values given at 60 mm Hg; n=10 for each; P>0.05).
Elevating extracellular K+ induces depolarization, activation of voltage-gated Ca2+ channels, Ca2+ influx, and vasoconstriction.4 As an alternative approach to investigate the functional impact of α2δ-1 targeting, we studied K+-induced vasoconstriction in WKY and SHR arteries. Increasing extracellular K+ from 6 to 20, 40, or 60 mmol/L induced graded vasoconstriction that was larger in SHR than in WKY cerebral arteries (Figure 6). Pregabalin reduced K+-induced vasoconstriction more in SHR than in WKY arteries (Figure 6). For example, pregabalin reduced the mean 60-mmol/L K+-induced constriction by ≈54% in SHRs and ≈37% in WKY rats (Figure 6B). These data indicate that α2δ-1 targeting reduces pressure- and depolarization-induced vasoconstriction more effectively in hypertensive SHR arteries than in control WKY rat arteries.
To date, no studies have investigated involvement of CaV1.2 channel auxiliary subunits in the pathological elevation of arterial myocyte CaV1.2 currents and vasoconstriction in hypertension. Here, we demonstrated for the first time that genetic hypertension is associated with transcriptional and posttranslational upregulation of α2δ-1 subunits in myocytes of resistance-size arteries. The additional α2δ-1 subunits increase surface trafficking of CaV1.2α1 subunits, which are also elevated in hypertension. The consequent increase in surface α2δ-1 and CaV1.2α1 proteins elevates CaV1.2 current density and generates a noninactivating current, leading to vasoconstriction. We also demonstrated that α2δ-1 targeting normalizes myocyte α2δ-1 and CaV1.2α1 surface expression, re-establishes CaV1.2 current density and inactivation, and reduces hypertensive rat artery contractility to levels in controls. These data indicate that α2δ-1 elevates CaV1.2 currents and CaV1.2-dependent vasoconstriction during hypertension and demonstrate that α2δ-1 targeting is a viable therapeutic strategy to reverse these pathological alterations and induce cerebrovascular dilation.
Our data indicate that the development of genetic hypertension is associated with a transcriptional and posttranslational increase in α2δ-1 and CaV1.2α1 in arterial myocytes. In contrast, other α2δ isoforms did not emerge during hypertension, an alteration that could have contributed to pathological CaV1.2 current modifications. Previous studies have described that CaV1.2α1 mRNA and protein are higher in mesenteric arteries and aorta of hypertensive SHRs than WKY rat controls.21,22 In contrast, angiotensin II– and hypoxia-induced hypertension did not alter CaV1.2α1 mRNA but elevated CaV1.21 protein in cultured mesenteric arteries and neonatal piglet pulmonary arteries.23,24 These findings lead to the proposal that hypertension may not be associated with an increase in CaV1.2α1 message but posttranslational upregulation of CaV1.2α1 protein.21–24 Here, we used both age-dependent development of hypertension in SHRs and comparison with WKY rat controls to investigate relative changes in α2δ-1 and CaV1.2α1 mRNA and protein. Our data indicate that the increase in α2δ-1 (≈2.1-fold) and CaV1.2α1 (≈1.5-fold) mRNA cannot fully account for the elevation in α2δ-1 (≈2.5-fold) and CaV1.21 (≈1.7-fold) proteins during hypertension. These data indicate that both transcriptional and posttranslational mechanisms elevate α2δ-1 and CaV1.2α1 proteins in cerebral artery myocytes during hypertension.
Using a novel application of biotinylation, we recently determined the surface to intracellular distribution of arterial α2δ-1 and CaV1.2α1 proteins in normotensive rats.16 Essentially all (>95%) α2δ-1 and CaV1.2α1 proteins locate to the surface in cerebral artery myocytes of normotensive SD rats.16 Here, a smaller percentage of total α2δ-1 (≈85%) and CaV1.2α1 (≈77%) was located in the plasma membrane in WKY rat arteries. Explanations for slight differences in α2δ-1 and CaV1.2α1 distribution between SD and WKY rats include the different rat strains and animal age (7 weeks in Reference 16 versus 12 weeks here). To determine the cellular distribution of α2δ-1 and CaV1.2α1 subunit proteins in SHR and WKY cerebral arteries, we compared the percentage of total protein expressed at the surface and the surface:intracellular protein ratio in both SHR and WKY cerebral arteries. Both of these analysis methods indicate that a higher proportion of α2δ-1 and CaV1.2α1 is located at the plasma membrane in hypertensive rat arteries compared with controls. The net result of both the transcription and translational increase in α2δ-1 and CaV1.2α1 protein and higher relative surface expression elevates plasma membrane levels of these proteins. Our data also indicate that there is a fractional shift in surface α2δ-1:CaV1.2α1 during hypertension, a change that occurs because of a larger elevation in surface α2δ-1 than in CaV1.2α1. These results provide evidence that an elevation in α2δ-1:CaV1.2α1 subunit ratio can modify native CaV1.2 current properties and that there may not be rigid α2δ-1:CaV1.2α1 subunit stoichiometry in arterial myocytes. Also possible is that, in normotension, a proportion of arterial myocyte CaV1.2 channel complexes may not contain α2δ-1 subunits. During hypertension, the higher elevation in surface α2δ-1 than CaV1.2α1 may increase the proportion of channels that contain α2δ-1. Future studies should be designed to further investigate native CaV1.2 channel stoichiometry in arterial myocytes and changes that occur in cardiovascular disease. Collectively, these results indicate that α2δ-1 increases surface expression and functionality of CaV1.2α1 subunits in arterial myocytes during hypertension.
Pregabalin is a gabapentinoid drug used to treat neuropathic pain, fibromyalgia, and epileptic seizures.25,26 Of all 4 α2δ isoforms, only α2δ-1 and -2 contain complete metal ion adhesion site and RRR motifs, which are required for gabapentanoid drug binding.14,20 Gabapanetin reduced α2δ subunit recycling from Rab-11–positive recycling endosomes.27 Pregabalin also reduced surface expression of both α2δ-1 and CaV1.2α1 proteins in cerebral artery myocytes of normotensive SD rats.16 Here, pregabalin did not alter total α2δ-1 or CaV1.2α1 protein. Rather, pregabalin reduced surface α2δ-1 and CaV1.2α1 more in hypertensive arteries than in control rat arteries, essentially normalizing surface levels of these proteins compared with those in WKY rats. Pregabalin also reduced the α2δ-1:CaV1.2α1 subunit ratio in hypertensive rat arteries compared with that in WKY controls. Given that pregabalin normalized elevated CaV1.2 current density and the proportion of noninactivating current to those in WKY cells, our data indicate that upregulated α2δ-1 functionality contributes to the increase in CaV1.2 currents in arterial myocytes in hypertension.
Voltage-dependent Ca2+ currents are elevated in myocytes from vasculature, including renal, cerebral, and mesenteric arteries, when studying a variety of different hypertension models, such as SHR, angiotensin II–induced, aortic banding, stroke-prone SHR, hypoxia-induced pulmonary hypertension, and Osborne-Mendel rats on a high fat diet.2,3,7,9,21,23,24,28–30 CaV1.2 current density measured here is consistent with that reported previously in cerebral artery myocytes when using WKY and SHR models.2,30 Our data indicate that CaV1.2 current density was ≈2.2-fold larger in hypertensive than in control rat arterial myocytes. In contrast, half-maximal CaV1.2 current activation and voltage dependence of half-maximal inactivation were similar in WKY and SHR cells, consistent with previous reports.2,9,28,30 A noninactivating CaV1.2 current in myocytes of hypertensive rats was double that in controls, a modification that would significantly increase Ca2+ influx at steady-state membrane potentials, thereby stimulating vasoconstriction. Pregabalin (24 hours) reduced elevated CaV1.2 current density and noninactivating current to levels in controls, suggesting that these pathological modifications occurred because of an increase in α2δ-1 surface expression. Our data are consistent with pregabalin acting as both a weak CaV1.2 channel pore blocker and an effective chronic inhibitor of α2δ-1 surface expression in hypertensive rat arterial myocytes. In our previous study, acute pregabalin reduced CaV1.2 currents by ≈33% in SD rat cerebral artery myocytes.16 Here, the same acute pregabalin concentration reduced CaV1.2 currents by ≈12% in WKY rat myocytes. Our previous study used 7-week–old SD rats, whereas here acute pregabalin effects were measured in 12-week–old WKY rat myocytes. Our data indicate that CaV1.2 channel properties in 12-week–old WKY myocytes are not identical to those in 7-week–old SD rat myocytes, including the percentage of CaV1.2α1 protein that is located at the surface. Data here indicate that acute pregabalin is a more effective inhibitor of CaV1.2 currents in SHR compared with WKY myocytes. This may be because of the higher number of surface α2δ-1 subunits and the higher α2δ-1:CaV1.2 ratio in SHR myocytes. Acute gabapentin also inhibited voltage-dependent Ca2+ currents in pyramidal neocortical cells but did not alter currents generated by recombinant CaV2.1 channels or endogenous Ca2+ channels in dorsal root ganglia neurons.31,32 In a model of neuropathic pain in which α2δ-1 is upregulated, chronic pregabalin inhibited α2δ-1 trafficking to presynaptic terminals, thereby inhibiting Ca2+ channel function.19 Our data indicate that chronic pregabalin inhibits α2δ-1–induced trafficking of CaV1.2α1 channel subunits, thereby reducing CaV1.2 currents in arterial myocytes during hypertension.
Intravascular pressure and depolarization both stimulated a larger vasoconstriction in arteries of hypertensive rats than in controls. Consistent with our findings, pressure-induced Ca2+ influx and associated vasoconstriction were larger in arteries from animal models with both genetic and induced hypertension.7,21,23,24 We show that nimodipine abolished myogenic tone at all pressures, indicating that CaV1.2 channel activity was essential to generate tone in hypertensive and control rat arteries. Chronic pregabalin (24 hours) was a more effective vasodilator of hypertensive than control rat arteries, effectively reversing the pathological vasoconstriction. Pregabalin is also a weak CaV1.2 channel pore blocker, which induces a small vasodilation.16 Thus, pregabalin-induced CaV1.2 pore block may also have contributed to vasodilation in both WKY and SHR arteries. Although unlikely, pregabalin could have caused vasodilation through additional mechanisms, including through inducing membrane hyperpolarization. Our data are inconsistent with this possibility, because pregabalin similarly reduced surface CaV1.2 subunits, CaV1.2 currents, myogenic tone, and K+-induced constriction, and inhibitions of pressure- and depolarization-induced vasoconstriction were equivalent. Thus, data demonstrate that pregabalin dilates hypertensive rat arteries primarily by reducing surface expression of CaV1.2 subunits in myocytes.
Hypertension is associated with increased risk for cerebral diseases, including stroke, Alzheimer disease, and dementia. Cerebral blood flow is reduced in hypertensive humans and 12-week–old SHRs when compared with normotensive controls.33,34 Voltage-dependent Ca2+ channel blockers have been used for >2 decades to treat hypertension.35 However, Ca2+ channel blockers inhibit CaV1.2 channels in multiple cell types in vivo and induce multiple adverse effects, including sweating, edema, and nausea.35,36 Therefore, the development of alternative approaches to target CaV1.2 channels in arterial myocytes could provide significant benefits over current inhibitors. Here we used pregabalin as an in vitro tool to test the concept that α2δ-1 targeting induces vasodilation in cerebral arteries of hypertensive animals. Data here provide a foundation for future studies aimed at developing novel approaches to target α2δ-1 in arterial myocytes. All of the data in our study were obtained by studying cerebral arteries that regulate brain regional blood flow but do not control systemic blood pressure. Clinical pregabalin does not appear to modify systemic blood pressure in normotensive humans at doses used to treat neuropathic pain, fibromyalgia, and epileptic seizures.26 There are several explanations for this observation. First, there are a large number of distinct mechanisms that control cerebral and systemic artery contractility. To date, no studies have examined the molecular identity or physiological functions of α2δ subunits in systemic artery myocytes that regulate diastolic and systolic blood pressures. α2δ-1 may not be the principal α2δ isoform or α2δ subunits may not regulate CaV1.2 channel activity in systemic artery myocytes. Pregabalin is an α2δ-1/2 ligand. If α2δ-1 or α2δ-2 is not expressed or does not regulate CaV1.2 channels in systemic artery myocytes, pregabalin should not induce systemic vasodilation or alter blood pressure. Second, clinical doses of pregabalin that are used to treat neuropathic pain, fibromyalgia, and epileptic seizures may be insufficient to induce vasodilation in vivo. Gabapentin, a lower affinity pregabalin analog, enters cells through system-L neutral amino acid transporters.32 Arterial myocytes may not uptake pregabalin as effectively as neurons. In vivo, intracellular myocyte pregabalin concentrations may be less than those obtained in vitro that alter CaV1.2 function. Third, many in vivo mechanisms, including those mediated by baroreceptors or the renin-angiotensin system, may compensate for pregabalin-induced systemic vasodilation, leading to no net change in blood pressure. Fourth, our data indicate that pregabalin is more effective at inhibiting CaV1.2α1 subunit trafficking in cerebral artery myocytes of hypertensive compared with normotensive rats. In vivo, pregabalin may be a more effective vasodilator in hypertensive subjects and have a smaller effect in normotensive subjects in which clinical systemic blood pressure measurements have been obtained. Our study provides the first evidence that arterial myocyte α2δ-1 functionality is upregulated in hypertension and that α2δ-1 targeting is a novel approach for reducing pathological vasoconstriction in hypertension. Data also indicate that α2δ-1 targeting can modify cerebral artery contractility, setting the stage for future studies to use a variety of other α2δ-1 targeting strategies, including RNA interference and genetic models, to investigate physiological and pathological involvement of α2δ subunits in arteries of other vascular beds and in vivo.
In summary, we identify for the first time that a hypertension-associated increase in α2δ-1 elevates CaV1.2α1 surface expression in arterial myocytes leading to pressure- and depolarization-induced vasoconstriction. Our data also indicate that α2δ-1 targeting is a novel approach to reverse elevated CaV1.2 channel surface expression in arterial myocytes and vasoconstriction in hypertension.
A hallmark of hypertension is an increase in voltage-dependent CaV1.2 currents in arterial myocytes that induces vasoconstriction.1–3 Molecular mechanisms that elevate arterial myocyte CaV1.2 currents in hypertension and the significance of auxiliary subunits in this pathological alteration are unclear. We show that the development of genetic hypertension is associated with a transcriptional and posttranslational upregulation of α2δ-1 subunits in cerebral artery myocytes. This increase in α2δ-1 subunits elevates CaV1.2 channel surface expression, CaV1.2 current density, and vasoconstriction. α2δ-1 targeting using pregabalin, an α2δ-1 ligand, reduced α2δ-1 and CaV1.2 surface expression and CaV1.2 current density in myocytes. Pregabalin also dilated cerebral arteries of hypertensive rats. Our study provides the first evidence that α2δ-1 subunits are upregulated in cerebral artery myocytes during hypertension and contribute to the pathological elevation in myocyte CaV1.2 currents and vasoconstriction. We also identified α2δ-1 as a potential novel therapeutic target for inducing cerebrovascular dilation in hypertension.
We thank Dr Marie Dennis Leo for critical reading of the article.
Sources of Funding
This work was supported by National Institutes of Health grants to J.H.J.
The online-only Data Supplement is available with this article at http://hyper.ahajournals.org/lookup/suppl/doi:10.1161/HYPERTENSIONAHA.112.199661/-/DC1.
- Received May 30, 2012.
- Revision received June 19, 2012.
- Accepted July 29, 2012.
- © 2012 American Heart Association, Inc.
- Wilde DW,
- Furspan PB,
- Szocik JF.
- Cox RH,
- Lozinskaya IM.
- Jaggar JH.
- Navedo MF,
- Amberg GC,
- Westenbroek RE,
- Sinnegger-Brauns MJ,
- Catterall WA,
- Striessnig J,
- Santana LF.
- Wamhoff BR,
- Bowles DK,
- Owens GK.
- Pesic A,
- Madden JA,
- Pesic M,
- Rusch NJ.
- Bannister JP,
- Adebiyi A,
- Zhao G,
- Narayanan D,
- Thomas CM,
- Feng JY,
- Jaggar JH.
- Adebiyi A,
- McNally EM,
- Jaggar JH.
- Bauer CS,
- Nieto-Rostro M,
- Rahman W,
- Tran-Van-Minh A,
- Ferron L,
- Douglas L,
- Kadurin I,
- Sri RY,
- Fernandez-Alacid L,
- Millar NS,
- Dickenson AH,
- Lujan R,
- Dolphin AC.
- Pratt PF,
- Bonnet S,
- Ludwig LM,
- Bonnet P,
- Rusch NJ.
- Wang WZ,
- Saada N,
- Dai B,
- Pang L,
- Palade P.
- Hirenallur-S DK,
- Haworth ST,
- Leming JT,
- Chang J,
- Hernandez G,
- Gordon JB,
- Rusch NJ.
- Tran-Van-Minh A,
- Dolphin AC.
- Simard JM,
- Li X,
- Tewari K.
- Wilde DW,
- Massey KD,
- Walker GK,
- Vollmer A,
- Grekin RJ.
- Xie MJ,
- Zhang LF,
- Ma J,
- Cheng HW.
- Hendrich J,
- Van Minh AT,
- Heblich F,
- Nieto-Rostro M,
- Watschinger K,
- Striessnig J,
- Wratten J,
- Davies A,
- Dolphin AC.
Novelty and Significance
What Is New?
We demonstrate for the first time that genetic hypertension is associated with a transcriptional and posttranslational upregulation of α2δ-1 subunits in myocytes of resistance-sized cerebral arteries that increase CaV1.2α1 subunit surface trafficking, thereby elevating CaV1.2 current density and arterial contractility.
Pharmacological targeting of α2δ-1 inhibits the pathological increase in CaV1.2 current density and cerebral artery contractility during hypertension. This study identifies α2δ-1 as a novel therapeutic target for inducing cerebrovascular vasodilation in hypertension.
What Is Relevant?
Upregulation of α2δ-1 subunits is essential for the elevation in CaV1.2 current density and cerebrovascular tone in genetic hypertension.
Pharmacological targeting of α2δ-1 can reverse the pathological elevation in surface CaV1.2 channels, CaV1.2 current density, and vasoconstriction in cerebral artery myocytes.
Upregulation of α2δ-1 subunits during genetic hypertension increases CaV1.2 channel surface expression and CaV1.2 current density, leading to vasoconstriction. α2δ-1 targeting reverses this pathological increase in CaV1.2 channel surface expression, CaV1.2 current density, and contractility in cerebral arteries.