TRANSCRIPTIONAL UPREGULATION OF α₂δ-1 ELEVATES ARTERIAL SMOOTH MUSCLE CELL CA_V1.2 CHANNEL SURFACE EXPRESSION AND CEREBROVASCULAR CONSTRICTION IN GENETIC HYPERTENSION

Abstract

A hallmark of hypertension is an increase in arterial myocyte voltage-dependent Ca²⁺ (Ca_V1.2) currents that induces pathological vasoconstriction. Ca_V1.2 channels are heteromeric complexes comprising a pore forming Ca_V1.2α₁ with auxiliary α₂δ and β subunits. Molecular mechanisms that elevate Ca_V1.2 currents during hypertension and the potential contribution of Ca_V1.2 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 (SHR) was associated with an unequal elevation in α₂δ-1 and Ca_V1.2α₁ mRNA and protein in cerebral artery myocytes, with α₂δ-1 increasing more than Ca_V1.2α₁. Other α₂δ isoforms did not emerge in hypertension. Myocytes and arteries of hypertensive SHR displayed higher surface-localized α₂δ-1 and Ca_V1.2α₁ proteins, surface α₂δ-1 to Ca_V1.2α₁ ratio (α₂δ-1:Ca_V1.2α₁), Ca_V1.2 current-density and non-inactivating current, and pressure- and - depolarization-induced vasoconstriction than those of Wistar-Kyoto controls. Pregabalin, an α₂δ-1 ligand, did not alter α₂δ-1 or Ca_V1.2α₁ total protein, but normalized α₂δ-1 and Ca_V1.2α₁ surface expression, surface α₂δ-1:Ca_V1.2α₁, Ca_V1.2 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 Ca_V1.2 channels in cerebral artery myocytes. This leads to an increase in Ca_V1.2 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 Ca_V1.2 channel trafficking to induce cerebrovascular dilation in hypertension.

Introduction

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’s 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⁺ (Ca_V1.2) 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 Ca_V1.2 currents leads to an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) and vasoconstriction.^9–11 However, molecular mechanisms that elevate arterial myocyte Ca_V1.2 currents in hypertension, leading to vasoconstriction, are unclear.

Ca_V1.2 channels are heteromeric complexes comprising a pore forming α₁ with auxiliary α₂δ and β subunits.¹² Four α₂δ (1 through 4) subunit isoforms have been identified that are each encoded by different genes.^{13, 14} α₂δ subunits undergo post-translational 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 Ca_V1.2α₁ subunits to the plasma membrane (surface) in arterial myocytes.¹⁶ To date, no studies have investigated pathological or disease-associated molecular changes in Ca_V1.2 auxiliary subunits, including α₂δ subunits, in myocytes of resistance-size arteries. In addition, it is unclear whether the subunit composition of arterial myocyte surface Ca_V1.2 channels is altered in disease. Given that arterial myocyte Ca_V1.2 currents are elevated during hypertension, leading to vasoconstriction, we determined the subunit composition of Ca_V1.2 channels and investigated the involvement of α₂δ subunits in this pathological alteration.^9–11 Elucidating molecular mechanisms governing α₂δ subunit regulation of Ca_V1.2 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 show that that during hypertension, an elevation in α₂δ-1 expression increases plasma membrane Ca_V1.2 currents in arterial myocytes, leading to vasoconstriction. We also identify α₂δ-1 as a novel therapeutic target to induce cerebrovascular dilation in hypertension.

Methods

Cell isolation and tissue preparation

All 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 intraperitoneal injection of sodium pentobarbital (150 mg/Kg 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 previously described.⁴

Blood pressure measurements

Diastolic and systolic blood pressures were measured in conscious rats using a tail cuff sphygmomanometer (Kent Scientific, Torrington, Conn).

RT-PCR

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 previously described.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 (UPL). Sequences of primers and probes used and PCR reaction efficiencies are given in Table S1.

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 re-probed for Ca_V1.2α₁. 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 Ca_V1.2α₁ subunit proteins between surface and intracellular compartments, artery surface biotinylation was used, as previously described.¹⁶

Patch-clamp electrophysiology

Whole cell Ca_V1.2 currents were recorded in isolated myocytes using the whole cell patch-clamp configuration, as previously described.¹⁶

Pressurized artery myography

Endothelium-denuded artery diameter was measured over a range of intravascular pressures (20–100 mmHg) in the presence and absence of nimodipine (1 μmol/L) using edge-detection myography, as previously described.¹⁷ Diameter responses to elevating extracellular K⁺ from 6 to between 20 and 60 mmol/L at 10 mmHg in the presence of pinacidil (10 μmol/L), a K_ATP channel opener, were also recorded. Arteries treated with pregabalin for 24 h were also maintained in pregabalin throughout these experiments to inhibit Ca_V1.2 subunit membrane re-insertion.

Statistical analysis

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>0.05 to verify that sample size was sufficient to give a value of >0.8.

An expanded Methods section is available as Supplemental Documentation.

Results

Age-dependent development of genetic hypertension is associated with an elevation in arterial myocyte α₂δ-1 and Ca_V1.2α₁ subunit expression

The pathological involvement of arterial myocyte Ca_V1.2 subunits was studied using a rat genetic model of hypertension. At 6 weeks of age, WKY and SHR rat diastolic, systolic, and mean arterial blood pressures were similar (Fig. S1). In contrast, at 12 weeks of age, diastolic, systolic, and mean arterial pressures were ~63, 65, and 72 mmHg higher in SHR than WKY rats, respectively (Fig 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 and hypertensive SHR rats (Fig. 1A). In contrast, the same primers amplified transcript for all α₂δ isoforms in WKY and SHR whole brain (Fig. 1A).

Figure 1.

α₂δ-1 and Ca_V1.2α₁ subunit mRNA and protein are elevated in hypertension. A, Representative gel (of 3 experiments) illustrating RT-PCR amplification of transcripts for α₂δ-1 through -4 in isolated arterial myocytes and whole brain of 12 week old WKY and SHR rats. B, Mean quantitative PCR data for α₂δ-1 and Ca_V1.2α₁ mRNA in 6 (n=8 for each) and 12 (n=6 for each) week old SHR rat arteries normalized to Rps5 and then to age-matched WKY controls. C, Exemplar Western blot illustrating α₂δ-1 and Ca_V1.2α₁ protein from 6 and 12 week old WKY and SHR rat whole arterial lysate. Blots were physically cut at 75 kDa to allow probing for actin and α₂δ-1/Ca_V1.2α₁. D, Mean data illustrating that α₂δ-1 and Ca_V1.2α₁ proteins are elevated in hypertension. * indicates P<0.05 when compared with the respective mRNA/protein at 6 weeks, # indicates P<0.05 when compared with Ca_V1.2α₁ mRNA/protein at 12 weeks (n=8 for each protein at each age).

Quantitative PCR was performed to compare α₂δ-1 and Ca_V1.2α₁ 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 and SHR (Table S1). 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 Ca_V1.2α₁ mRNA levels were similar in 6 week old WKY and SHR arteries (Fig. 1B). In contrast, α₂δ-1 and Ca_V1.2 mRNAs were ~2.1- and 1.5-fold higher, respectively, in 12 week old SHR than WKY arteries (Fig. 1B). Age-dependent development of hypertension was also associated with a larger increase in α₂δ-1 than Ca_V1.2α₁ mRNA (Fig. 1B). These data indicate that hypertension is associated with an elevation in α₂δ-1 and Ca_V1.2α₁ 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 Ca_V1.2α₁ proteins in cerebral arteries. α₂δ-1 and Ca_V1.2α₁ protein levels were similar in 6 week old WKY and SHR arteries (Fig. 1C, D). Aging between 6 and 12 weeks did not alter α₂δ-1 and Ca_V1.2α₁ protein in WKY rat arteries, but increased these proteins ~2.1- and 1.4-fold in SHR arteries (Fig. S2). At 12 weeks of age, α₂δ-1 and Ca_V1.2α₁ proteins were ~2.5- and 1.7-fold higher in SHR than age-matched WKY arteries (Fig. 1C, D). In agreement with message levels, age-dependent development of hypertension also increased α₂δ-1 more than Ca_V1.2α₁ protein (Fig. 1C, D, S2).

In summary, these data indicate that genetic hypertension is associated with transcriptional upregulation of both α₂δ-1 and Ca_V1.2α₁ in cerebral artery myocytes. α₂δ-1 and Ca_V1.2α₁ proteins are elevated more than their respective mRNAs (Fig. 1B–D, S2), suggesting that hypertension-associated changes in post-translational events also contribute to increased Ca_V1.2 channel subunit expression during hypertension. Furthermore, during hypertension there is a larger increase in mRNA and protein for α₂δ-1 than for Ca_V1.2α₁.

Hypertension is associated with an elevation in surface α₂δ-1 and Ca_V1.2α₁ proteins in arteries

α₂δ-1 induces membrane trafficking of Ca_V1.2α₁ subunits in SD rat arterial myocytes.¹⁶ Therefore, we tested the hypothesis that an increase in α₂δ-1 contributes to elevated surface Ca_V1.2 expression in hypertension. Surface (plasma membrane) and intracellular α₂δ-1 and Ca_V1.2α₁ proteins were measured in age-matched WKY and hypertensive SHR cerebral arteries using biotinylation. Surface-localized α₂δ-1 and Ca_V1.2α₁ proteins were ~2.6- and 2-fold higher, respectively, in SHR than WKY rat arteries (Fig. 2A, B). A larger percentage of total α₂δ-1 and Ca_V1.2α₁ was located at the plasma membrane in SHR than WKY arteries (Fig. 2A, C). In WKY arteries, more of the total amount of α₂δ-1 (~85 %) than Ca_V1.2α₁ (~77%) was located at the surface. In contrast, in SHR arteries the percentage of total α₂δ-1 (~93 %) and Ca_V1.2α₁ (~92 %) located at the surface were similar (Fig. 2A, C). These data indicate that during hypertension, an elevation in α₂δ-1 and Ca_V1.2α₁ 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 Ca_V1.2α₁ proteins between intracellular and surface compartments.

Figure 2.

Arterial surface α₂δ-1 and Ca_V1.2α₁ subunits are elevated in hypertension. A, Representative Western blot illustrating increased surface expression of both α₂δ-1 and Ca_V1.2α₁ proteins in 12 week old WKY and SHR arteries. Blot was physically cut at 75 kDa to allow probing for α₂δ-1 and Ca_V1.2α₁. B, Mean data illustrate that surface levels of α₂δ-1 and Ca_V1.2α₁ subunits are higher in arteries during hypertension. C, Mean data illustrating the percentage of total (surface + intracellular) α₂δ-1 and Ca_V1.2α₁ proteins located at the surface * indicates P<0.05 versus same protein in age-matched WKY rat arteries, # indicates P<0.05 when compared with WKY α₂δ-1 (n=5 – 9 each for WKY and SHR).

Pregabalin reduces surface trafficking of Ca_V1.2 channel subunits more effectively in hypertensive than normotensive rat arteries

Pregabalin, an α₂δ-1/2 ligand, reduces surface trafficking of Ca_V1.2, 2.1, and 2.2 channels in neurons and arterial myocytes.^{14, 16, 18–20} Next, we studied pregabalin regulation of α₂δ-1 and Ca_V1.2α₁ subunit surface expression and subunit cellular distribution in WKY and SHR cerebral arteries. For these experiments, arteries were incubated for 24 h with or without pregabalin. Pregabalin (24 h) did not alter total protein of α₂δ-1 (% control: WKY, 115 ± 9; SHR, 118 ± 20) or Ca_V1.2α₁ (% control: WKY, 116 ± 10; SHR, 109 ± 14) (Fig. 3A, WKY n= 4–5, SHR n= 5, P>0.05 for each). In contrast, pregabalin reduced surface α₂δ-1 and Ca_V1.2 and increased intracellular levels of these proteins in both WKY and SHR arteries (Fig. 3A, B, C, S3). Pregabalin reduced plasma membrane α₂δ-1 and Ca_V1.2α₁ ~3.1 and 1.9-fold more, respectively, in hypertensive SHR than WKY control arteries (Fig. 3C). To evaluate pregabalin regulation of α₂δ-1 and Ca_V1.2 cellular distribution, surface:intracellular protein ratios were calculated. Consistent with data shown in figure 2C, a larger proportion of α₂δ-1 and Ca_V1.2 subunits were present at the plasma membrane in SHR than WKY arteries (Fig. 3D). Pregabalin induced a larger reduction in surface:intracellular α₂δ-1 and Ca_V1.2 in SHR than in WKY arteries (Fig. 3D).

Figure 3.

Pregabalin reduces surface expression of α₂δ-1 and Ca_V1.2α₁ channel proteins more effectively in arteries of hypertensive rats than in controls. A, Representative Western blot illustrating that pregabalin does not change total (whole arterial) α₂δ-1 and Ca_V1.2α₁ proteins in WKY and SHR arteries. B, Representative Western blots illustrating pregabalin (24 h)-induced changes in surface and intracellular α₂δ-1 and Ca_V1.2α₁ proteins. Blots were cut at 75 kDa to allow probing for α₂δ-1 and Ca_V1.2α₁. C, Pregabalin reduced surface α₂δ-1 and Ca_V1.2α₁ proteins more in SHR than WKY arteries. D, Mean data illustrating α₂δ-1 and Ca_V1.2α₁ subunit distribution in WKY and SHR arteries and regulation by pregabalin. E, Surface α₂δ-1 to Ca_V1.2α₁ and modulation by pregabalin. Pregabalin concentration in all figures was 100 μmol/L. * indicates P<0.05 compared with untreated WKY and # indicates P<0.05 versus untreated SHR rat arteries (n=4–5 each for untreated and pregabalin-treated WKY and SHR).

Hypertension was associated with a larger increase in surface α₂δ-1 than Ca_V1.2α₁ protein in arteries (Fig. 2A, B). We calculated the band intensity ratio of surface α₂δ-1 to Ca_V1.2α₁ and regulation by pregabalin. While this methodology cannot determine subunit stoichiometry, total protein loaded in each lane is identical, allowing comparison of this ratio in SHR and WKY arteries from the same blot. The mean surface α₂δ-1:Ca_V1.2α₁ band intensity ratio was ~1.38 in SHR arteries and ~1.06 in WKY arteries, or ~1.3-fold higher in SHR (Fig. 3E). Pregabalin reduced the surface α₂δ-1 to Ca_V1.2α₁ band intensity ratio to ~0.91 in SHR rat arteries, but did not change the ratio in WKY rat arteries (Fig. 3E).

Collectively, these data indicate that pregabalin blocks surface expression of α₂δ-1 and Ca_V1.2α₁ subunits more effectively in hypertensive than normotensive rat arteries. During hypertension, surface α₂δ-1 protein is elevated more so than Ca_V1.2α₁ protein, leading to an increase in the ratio of plasma membrane α₂δ-1 to Ca_V1.2α₁ subunits. Pregabalin reverses this elevation in surface α₂δ-1 to Ca_V1.2α₁ subunits. These data also indicate that α₂δ-1 is essential for upregulation of surface Ca_V1.2 channels in arterial myocytes during genetic hypertension.

α₂δ-1 targeting reverses hypertension-associated modifications in Ca_V1.2 current density and inactivation in arterial myocytes

To investigate the functional impact of elevated α₂δ-1 expression and effects of α₂δ-1 targeting, Ca_V1.2 currents were measured in age-matched WKY and hypertensive SHR cerebral artery myocytes. Mean peak Ca_V1.2 current density (Ba²⁺ as charge carrier) was ~5.3 pA/pF in hypertensive SHR compared with ~2.4 pA/pF in WKY cells, or ~2.2-fold larger (Fig. 4A, B and Table 1). Pregabalin (24 h) reduced peak Ca_V1.2 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 % (Fig. 4A, B and Table 1). Pregabalin reduced peak Ca_V1.2 current density in SHR myocytes to the current density of untreated WKY cells (Fig 4A, B and Table 1). The relationship between cell capacitance and peak Ca_V1.2 current was investigated (Fig. 4C). When data were fit with a linear function, the slope was −5.41 for SHR and −2.40 for WKY cells, or 2.3-fold higher (Fig. 4C). Pregabalin reduced slopes by ~58 and 25 % in SHR and WKY cells, respectively (Fig. 4C). Slopes were similar for untreated WKY and pregabalin-treated SHR cells (Fig. 4C, P>0.05). Mean cell capacitance for WKY (16.3 ± 0.8 pF) and SHR (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 due to changes in Ca_V1.2 channels (Fig. 4B, C).

Figure 4.

Pregabalin reverses elevated Ca_V1.2 currents in hypertensive rat arterial smooth muscle cells. A, Representative Ca_V1.2 current density recordings from control and pregabalin-treated WKY and SHR arterial smooth muscle cells (10 mmol/L Ba²⁺ as charge carrier). B, Mean current density-voltage relationships of WKY (n=17), pregabalin-treated WKY (n=13), SHR (n=16) and pregabalin-treated SHR (n=18) cells. C, Scatter plot with linear fit for peak Ca_V1.2 current versus cell capacitance in WKY (n=17), pregabalin-treated WKY (n=13), SHR (n=16) and pregabalin-treated SHR (n=18) cells. WKY: slope=−2.40, r=−0.76, p=3.3×10-4. WKY+pregabalin: slope=−1.79, r=−0.90, p=7.5×10-4. SHR: slope=−5.41, r=−0.77, p=3.5×10-4. SHR+pregabalin: slope=−2.25, r=−0.72, p=1.1×10-4. D, Voltage-dependent Ca_V1.2 current activation in WKY (n=13), pregabalin-treated WKY (n=9), SHR (n=12) and pregabalin-treated SHR (n=6) cells. E, Voltage-dependent current inactivation of WKY (n=17), pregabalin-treated WKY (n=13), SHR (n=16) and pregabalin-treated SHR (n=18) cells. *indicates significance from WKY at indicated potentials (P<0.05). F, Graph illustrating the time course of Ca_V1.2 currents (at +20 mV) and inhibition by acute pregabalin (100 μmol/L). WKY (control n=8, pregabalin n=9), SHR (control n=14, pregabalin n=6) cells. The arrow (not applicable for controls) indicates where pregabalin was added. Pregabalin concentration in all figures was 100 μmol/L. * indicates P<0.05 when compared to untreated WKY, # indicates P<0.05 when compared to pregabalin-treated WKY and § indicates P<0.05 when compared to untreated SHR.

Table 1.

Properties of arterial myocyte Ca_V1.2 currents. Numbers in parentheses indicate experimental number.

	WKY	WKY + pregabalin	SHR	SHR + pregabalin
IV relationship
Peak current density (pA/pF)	2.40±0.15 (17)	1.65±0.13 (13)*	5.33±0.38 (16)*,#	2.23±0.14 (16)
Peak voltage (mV)	10.25±0.74 (17)	12.31±1.45 (13)	11.09±1.03 (16)	11.26±0.89 (18)
Voltage-dependent activation
V1/2act (mV)	9.24±1.88 (12)	10.70±3.72 (9)	11.62±1.80 (12)	11.18±3.29 (5)
Slope	13.09±1.55 (12)	14.56±1.66 (9)	12.74±2.84 (12)	10.51±1.09 (5)
Voltage-dependent inactivation
V1/2inact (mV)	−14.23±1.57 (8)	−17.20±1.28 (13)	−11.33±1.90 (16)	−14.71±1.15 (11)
Slope	6.44±0.76 (8)	8.58±1.16 (13)	7.86±0.70 (16)	8.09±0.69 (11)

^*indicates P<0.05 compared to WKY and

^# indicates P<0.05 versus SHR + pregabalin.

The voltage-dependence of half-maximal Ca_V1.2 current activation (V_1/2act) and slope (k) were similar in untreated control and pregabalin-treated WKY and SHR arterial myocytes (Fig. 4D and Table 1). The voltage-dependence of half-maximal inactivation (V_1/2inact) and k were also similar in untreated control and pregabalin-treated WKY and SHR cells (Fig. 4E and Table 1). In contrast, untreated SHR cells displayed a non-inactivating Ca_V1.2 current that was ~2-fold larger than in WKY cells (Fig. 4A, E). Pregabalin (24 h) reduced the non-inactivating current in SHR cells such that it was similar to WKY cells (Fig. 4A, E). Ca_V1.2 current inactivation rates (τ) were similar in control and pregabalin-treated WKY and SHR cells (Fig. S4).

In addition to acting as an inhibitor of α₂δ-1-induced Ca_V1.2 channel trafficking, pregabalin is a weak Ca_V1.2 channel pore blocker that does not directly alter Ca_V1.2 current voltage-dependence in normotensive SD rat arterial myocytes.¹⁶ To determine whether the reduction in Ca_V1.2 current amplitude in pregabalin-treated WKY and SHR rat myocytes was due to Ca_V1.2 pore block, we measured Ca_V1.2 current regulation in untreated cells by acute bath application of pregabalin. Acute pregabalin reduced Ca_V1.2 currents in WKY cells by ~12 % (Fig. 4F). In contrast, pregabalin reduced Ca_V1.2 currents in SHR myocytes by ~23 %, or ~1.9-fold more than in WKY cells (Fig. 4F). Acute pregabalin-induced Ca_V1.2 current inhibition was significantly smaller than that induced by 24 h pregabalin treatment in both WKY (~32 % inhibition) and SHR (~59 % inhibition) cells (Figs. 4B, C, F). When combined with the biochemical data illustrated in Figure 3, these data indicate that acute and chronic pregabalin inhibit Ca_V1.2 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 Ca_V1.2α₁ subunits, leading to a Ca_V1.2 current elevation and an increase in non-inactivating current. α₂δ-1 targeting reduces the hypertension-associated α₂δ-1-induced elevation in Ca_V1.2α₁ surface expression, leading to a reduction in Ca_V1.2 current density. α₂δ-1 targeting also restores Ca_V1.2 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 mmHg) was measured in WKY and SHR cerebral arteries that had been incubated for 24 h with or without pregabalin. SHR arteries developed more myogenic tone than WKY arteries over the entire pressure range (Fig. 5A, B). Pregabalin reduced myogenic tone in WKY and SHR arteries, decreased tone more in SHR than in WKY arteries (e.g., % reduction in myogenic tone at 60 mmHg: SHR, ~41 %; WKY, ~28 %), and reduced tone in SHR arteries to levels in untreated WKY arteries (Fig. 5A, B). 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 mmHg) studied, indicating that myogenic tone occurred due to Ca_V1.2 channel activation (Fig. 5B). Passive arterial diameters were similar for WKY (249 ± 11 μm) and hypertensive SHR (242 ± 9μm) cerebral arteries (values given at 60 mmHg, n=10 for each, P>0.05).

Figure 5.

Pregabalin reverses elevated pressure-induced vasoconstriction in hypertension. A, Representative traces illustrating steady-state myogenic tone in response to increasing intravascular pressure in a WKY (black) and SHR (red) artery. Horizontal black bars indicate an increase in bath K⁺ from 6 to 60 mmol/L. B Pregabalin (24 h, 100 μmol/L) reduced pressure (20 – 100 mmHg)-induced myogenic tone (filled symbols) more so in arteries from hypertensive rats than in controls. Myogenic tone was abolished by nimodipine (1 μmol/L, empty symbols). Mean data (n: WKY, 6–10; WKY + pregabalin, 6–9; SHR, 6–10; SHR+pregabalin, 6–7). * indicates P<0.05 when compared with untreated WKY and # indicates P<0.05 for SHR+pregabalin when compared with untreated SHR.

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 WKY cerebral arteries (Fig. 6A, B). Pregabalin reduced K⁺-induced vasoconstriction more in SHR than WKY arteries (Fig. 6A, B). For example, pregabalin reduced the mean 60 mmol/L K⁺-induced constriction by ~54 % in SHR and ~37 % in WKY (Fig. 6B). These data indicate that α₂δ-1 targeting reduces pressure- and depolarization-induced vasoconstriction more effectively in hypertensive SHR than in control WKY rat arteries.

Figure 6.

Pregabalin reverses elevated depolarization-induced vasoconstriction in hypertension. A, Representative traces illustrating diameter responses to increasing extracellular K⁺. B, Pregabalin (24 h, 100 μmol/L) reduced depolarization (20, 40, and 60 mmol/L K⁺)-induced vasoconstriction more so in arteries from hypertensive rats than controls. Mean data (n: WKY, 6; WKY + pregabalin, 6; SHR, 6; SHR + pregabalin, 6). * indicates P<0.05 when compared with untreated WKY, and # indicates P<0.05 for SHR + pregabalin when compared with untreated SHR.

Discussion

To date, no studies have investigated involvement of Ca_V1.2 channel auxiliary subunits in the pathological elevation of arterial myocyte Ca_V1.2 currents and vasoconstriction in hypertension. Here, we demonstrate for the first time that genetic hypertension is associated with transcriptional and post-translational upregulation of α₂δ-1 subunits in myocytes of resistance-size arteries. The additional α₂δ-1 subunits increase surface trafficking of Ca_V1.2α₁ subunits, which are also elevated in hypertension. The consequent increase in surface α₂δ-1 and Ca_V1.2α₁ proteins elevates Ca_V1.2 current density and generates a non-inactivating current, leading to vasoconstriction. We also demonstrate that α₂δ-1 targeting normalizes myocyte α₂δ-1 and Ca_V1.2α₁ surface expression, re-establishes Ca_V1.2 current density and inactivation, and reduces hypertensive rat artery contractility to levels in controls. These data indicate that α₂δ-1 elevates Ca_V1.2 currents and Ca_V1.2-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 post-translational increase in α₂δ-1 and Ca_V1.2α₁ in arterial myocytes. In contrast, other α₂δ isoforms did not emerge during hypertension, an alteration that could have contributed to pathological Ca_V1.2 current modifications. Previous studies have described that Ca_V1.2α₁ mRNA and protein is higher in mesenteric arteries and aorta of hypertensive SHR than WKY rat controls.^{21, 22} In contrast, angiotensin II- and hypoxia-induced hypertension did not alter Ca_V1.2α₁ mRNA, but elevated Ca_V1.2α₁ 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 Ca_V1.2α₁ message, but post-translational upregulation of Ca_V1.2α₁ protein.^21–24 Here, we used both age-dependent development of hypertension in SHR and comparison to WKY rat controls to investigate relative changes in α₂δ-1 and Ca_V1.2α₁ mRNA and protein. Our data indicate that the increase in α₂δ-1 (~2.1-fold) and Ca_V1.2α₁ (~1.5-fold) mRNA cannot fully account for the elevation in α₂δ-1 (~2.5-fold) and Ca_V1.2α₁ (~1.7-fold) proteins during hypertension. These data indicate that both transcriptional and post-translational mechanisms elevate α₂δ-1 and Ca_V1.2α₁ 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 Ca_V1.2α₁ proteins in normotensive rats.¹⁶ Essentially all (>95 %) α₂δ-1 and Ca_V1.2α₁ proteins locate to the surface in cerebral artery myocytes of normotensive SD rats.¹⁶ Here, a smaller percentage of total α₂δ-1 (~85%) and Ca_V1.2α₁ (~77 %) was located in the plasma membrane in WKY rat arteries. Explanations for slight differences in α₂δ-1 and Ca_V1.2α₁ distribution between SD and WKY rats include the different rat strains and animal age (7 weeks in ref. ¹⁶ vs 12 weeks here). To determine the cellular distribution of α₂δ-1 and Ca_V1.2α₁ 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 Ca_V1.2α₁ is located at the plasma membrane in hypertensive rat arteries than in controls. The net result of both the transcription and translational increase in α₂δ-1 and Ca_V1.2α₁ 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:Ca_V1.2α₁ during hypertension, a change that occurs due to a larger elevation in surface α₂δ-1 than Ca_V1.2α₁. These results provide evidence that an elevation in α₂δ-1 to Ca_V1.2α₁ subunit ratio can modify native Ca_V1.2 current properties and that there may not be rigid α₂δ-1:Ca_V1.2α₁ subunit stoichiometry in arterial myocytes. Also possible is that in normotension, a proportion of arterial myocyte Ca_V1.2 channel complexes may not contain α₂δ-1 subunits. During hypertension, the higher elevation in surface α₂δ-1 than Ca_V1.2α₁ may increase the proportion of channels that contain α₂δ-1. Future studies should be designed to further investigate native Ca_V1.2 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 Ca_V1.2α₁ subunits in arterial myocytes during hypertension.

Pregabalin is a gabapentinoid drug used to treat neuropathic pain, fibromyalgia, and epileptic seizures.^{25, 26} Of all four 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 Ca_V1.2α₁ proteins in cerebral artery myocytes of normotensive SD rats.¹⁶ Here, pregabalin did not alter total α₂δ-1 or Ca_V1.2α₁ protein. Rather, pregabalin reduced surface α₂δ-1 and Ca_V1.2α₁ more in hypertensive arteries than in control rat arteries, essentially normalizing surface levels of these proteins to those in WKY. Pregabalin also reduced the α₂δ-1 to Ca_V1.2α₁ subunit ratio in hypertensive rat arteries to that in WKY controls. Given that pregabalin normalized elevated Ca_V1.2 current density and the proportion of non-inactivating current to those in WKY cells, our data indicate that upregulated α₂δ-1 functionality contributes to the increase in Ca_V1.2 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} Ca_V1.2 current density measured here is consistent with that previously reported in cerebral artery myocytes when using WKY and SHR rat models.^{2, 30} Our data indicate that Ca_V1.2 current density was ~2.2-fold larger in hypertensive than control rat arterial myocytes. In contrast, Ca_V1.2 current V_1/2act and V_1/2inact were similar in WKY and SHR cells, consistent with previous reports.^{2, 9, 28, 30} A non-inactivating Ca_V1.2 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 h) reduced elevated Ca_V1.2 current density and non-inactivating current to levels in controls, suggesting that these pathological modifications occurred due to an increase in α₂δ-1 surface expression. Our data are consistent with pregabalin acting as both a weak Ca_V1.2 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 Ca_V1.2 currents by ~33 % in SD rat cerebral artery myocytes.¹⁶ Here, the same acute pregabalin concentration reduced Ca_V1.2 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 Ca_V1.2 channel properties in 12 week old WKY myocytes are not identical to those in 7 week old SD rat myocytes, including the percentage of Ca_V1.2α₁ protein that is located at the surface. Data here indicate that acute pregabalin is a more effective inhibitor of Ca_V1.2 currents in SHR than WKY myocytes. This may be due to the higher number of surface α₂δ-1 subunits and the higher α₂δ-1:Ca_V1.2 ratio in SHR myocytes. Acute gabapentin also inhibited voltage-dependent Ca²⁺ currents in pyramidal neocortical cells, but did not alter currents generated by recombinant Ca_V2.1 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 pre-synaptic terminals, thereby inhibiting Ca²⁺ channel function.¹⁹ Our data indicate that chronic pregabalin inhibits α₂δ-1-induced trafficking of Ca_V1.2α₁ channel subunits, thereby reducing Ca_V1.2 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 Ca_V1.2 channel activity was essential to generate tone in hypertensive and control rat arteries. Chronic pregabalin (24 h) was a more effective vasodilator of hypertensive than control rat arteries, effectively reversing the pathological vasoconstriction. Pregabalin is also a weak Ca_V1.2 channel pore blocker, which induces a small vasodilation.¹⁶ Thus, pregabalin-induced Ca_V1.2 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 as pregabalin similarly reduced surface Ca_V1.2 subunits, Ca_V1.2 currents, myogenic tone and K⁺-induced constriction and inhibition of pressure- and depolarization-induced vasoconstriction were equivalent. Thus, data demonstrate that pregabalin dilates hypertensive rat arteries primarily by reducing surface expression of Ca_V1.2 subunits in myocytes.

Hypertension is associated with increased risk for cerebral diseases, including stroke, Alzheimer’s disease, and dementia. Cerebral blood flow is reduced in hypertensive humans and 12 week old SHR rats, when compared to normotensive controls.^{33, 34} Voltage-dependent Ca²⁺ channel blockers have been used for over two decades to treat hypertension.³⁵ However, Ca²⁺ channel blockers inhibit Ca_V1.2 channels in multiple cell types in vivo and induce multiple side effects, including sweating, edema, and nausea.^{35, 36} Therefore, the development of alternative approaches to target Ca_V1.2 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 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 pressure. α₂δ-1 may not be the principal α₂δ isoform, or α₂δ subunits may not regulate Ca_V1.2 channel activity in systemic artery myocytes. Pregabalin is an α₂δ-1/2 ligand. If α₂δ-1 or α₂δ-2 are not expressed or do not regulate Ca_V1.2 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 analogue, 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 Ca_V1.2 function. Third, many in vivo mechanisms, including those mediated by baroreceptors or the renin-angiotensin sytem, 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 Ca_V1.2α₁ subunit trafficking in cerebral artery myocytes of hypertensive than 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 Ca_V1.2α₁ 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 Ca_V1.2 channel surface expression in arterial myocytes and vasoconstriction in hypertension.

Perspectives.

A hallmark of hypertension is an increase in voltage-dependent Ca_V1.2 currents in arterial myocytes that induces vasoconstriction. ^1–3 Molecular mechanisms that elevate arterial myocyte Ca_V1.2 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 post-translational upregulation of α₂δ-1 subunits in cerebral artery myocytes. This increase in α₂δ-1 subunits elevates Ca_V1.2 channel surface expression, Ca_V1.2 current density, and vasoconstriction. α₂δ-1 targeting using pregabalin, an α₂δ-1 ligand, reduced α₂δ-1 and Ca_V1.2 surface expression and Ca_V1.2 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 Ca_V1.2 currents and vasoconstriction. We also identify α₂δ-1 as a potential novel therapeutic target for inducing cerebrovascular dilation in hypertension.

Novelty and Significance.

1) What is new?

• We demonstrate for the first time that genetic hypertension is associated with a transcriptional and post-translational upregulation of α₂δ-1 subunits in myocytes of resistance-size cerebral arteries that increase Ca_V1.2α₁ subunit surface trafficking, thereby elevating Ca_V1.2 current density and arterial contractility.

• Pharmacological targeting of α₂δ-1 inhibits the pathological increase in Ca_V1.2 current density and cerebral artery contractility during hypertension. This study identifies α₂δ-1 as a novel therapeutic target for inducing cerebrovascular vasodilation in hypertension.

2) What Is Relevant?

• Upregulation of α₂δ-1 subunits is essential for the elevation in Ca_V1.2 current density and cerebrovascular tone in genetic hypertension.

• Pharmacological targeting of α₂δ-1 can reverse the pathological elevation in surface Ca_V1.2 channels, Ca_V1.2 current density and vasoconstriction in cerebral artery myocytes.

3) Summary

• Upregulation of α₂δ-1 subunits during genetic hypertension increases Ca_V1.2 channel surface expression and Ca_V1.2 current density, leading to vasoconstriction. α₂δ-1 targeting reverses this pathological increase in Ca_V1.2 channel surface expression, Ca_V1.2 current density and contractility in cerebral arteries.

Non-standard Abbreviations and Acronyms

None