Transcriptional upregulation of α2δ-1 elevates arterial smooth muscle cell voltage-dependent Ca2+ channel surface expression and cerebrovascular constriction in genetic hypertension

Abstract

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.

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).

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).

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).

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.

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.

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.