Na+/Ca2+ exchanger overexpression in smooth muscle augments cytosolic Ca2+ in femoral arteries of living mice

Abstract

Plasma membrane Na⁺/Ca²⁺ exchanger-1 (NCX1) helps regulate the cytosolic Ca²⁺ concentration ([Ca²⁺]_CYT) in arterial myocytes. NCX1 mediates both Ca²⁺ entry and exit and tends to promote net Ca²⁺ entry in partially constricted arteries. Mean blood pressure (telemetry) is elevated by ≈10 mmHg in transgenic (TG) mice that overexpress NCX1 specifically in smooth muscle. We tested the hypothesis that NCX1 overexpression mediates Ca²⁺ gain and elevated [Ca²⁺]_CYT in exposed femoral arteries that also express the Ca²⁺ biosensor exogenous myosin light chain kinase. [Ca²⁺]_CYT and the NCX1-dependent (SEA0400-sensitive) component, ≈15% of total basal constriction in controls, were increased in TG arteries, but constrictions to phenylephrine and ANG II were comparable in TG and control arteries. Normalized phenylephrine dose-response curves and constriction to 30 and 300 ng/kg iv ANG II were virtually identical in control and TG arteries. ANG II-evoked constrictions, superimposed on elevated basal tone, accounted for the larger blood pressure responses to ANG II in TG arteries. TG and control mouse arteries fit the same pCa-constriction relationship over a wide range of pCa (≈125-500 nM). Vasodilation to acetylcholine, normalized to passive diameter, was also comparable in TG and control arteries, implying normal endothelial function. TG artery Na⁺ nitroprusside (nitric oxide donor)-induced dilations were, however, shifted to lower Na⁺ nitroprusside concentrations, indicating that TG myocyte vasodilator mechanisms were augmented. Maximum arterial dilation was comparable in TG and control mice, although passive diameter was ≈6-7% smaller in TG mice. The changes in TG arteries were apparently largely functional rather than structural, despite the congenital hypertension. NEW & NOTEWORTHY Smooth muscle Na⁺/Ca²⁺ exchanger-1 transgene overexpression (TG mice) increases femoral artery basal cytosolic Ca²⁺ concentration ([Ca²⁺]_CYT) and tone in vivo and raises blood pressure. Arterial constriction to phenylephrine and angiotensin II are normal but superimposed on the augmented basal [Ca²⁺]_CYT and tone (constriction) in TG mouse arteries. Similar effects in resistance arteries would explain the elevated blood pressure. Acetylcholine-induced vasodilation is unimpaired, implying a normal endothelium, but TG arteries are hypersensitive to sodium nitroprusside.

Keywords: SEA0400, sodium nitroprusside; arterial tone; vasodilation.

Fig. 1.

Na⁺/Ca²⁺ exchanger-1 (NCX1) is overexpressed in smooth muscle-specific NCX1-transgenic (TG) mice. Immunoblots show deendothelialized aortic membrane proteins from control (Ctrl) and heterozygous and homozygous TG mice that overexpress NCX1 specifically in smooth muscle (SM-NCX1-TG mice) (TG/+ and TG/TG, respectively). TG/TG mice were used for all physiological experiments in this study. The NCX1 transgene was randomly inserted into the genome, and the protein was, therefore, greatly overexpressed, as illustrated. Protein samples had to be markedly diluted so that both Ctrl and TG NCX1 bands could be visualized on the same gel (see also Supplemental Fig. S3 in Ref. 23). β-Actin was used as the loading control.

Fig. 2.

Effects of smooth muscle-specific Na⁺/Ca²⁺ exchanger-1 (NCX1) overexpression on femoral artery cytosolic Ca²⁺ concentration ([Ca²⁺]_CYT), diameter, and tone. Baseline diameter and passive diameter (PD; A), basal tone (PD − baseline diameter as %PD; B), and basal [Ca²⁺]_CYT (C) in the exposed femoral arteries of control (Ctrl) and transgenic (TG) mice in vivo under light anesthesia are shown. Baseline diameter was reduced, and basal tone and [Ca²⁺]_CYT were increased, in TG mouse arteries. *P < 0.05 vs. Ctrl mice (Student’s unpaired t-test); ***P < 0.001 vs. Ctrl mice; ###P < 0.001 vs. the respective basal diameter (two-way ANOVA with pairwise comparisons using the Holm-Sidak method). Numbers of Ctrl and TG mice are shown in parentheses at the top. The difference between mean Ctrl PD and TG PD was not significant (P = 0.085 by two-way ANOVA). When mean PD values from six independent experiments (with 51 Ctrl arteries and 51 TG arteries) were analyzed, however, the respective means of the means from each experiment (Ctrl PD: 397.2 ± 3.9 μm and TG PD: 379.5 ± 5.3 μm) were significantly different (P = 0.023 by a Student’s unpaired t-test). The 95% confidence interval for the difference of the means was 3.043–32.291; the power of the t-test was 0.617.

Fig. 3.

Effects of pretreatment (15 min) with 0.3 μM SEA0400 on basal cytosolic Ca²⁺ concentration ([Ca²⁺]_CYT; A) and basal tone (C) in control (Ctrl) and transgenic (TG) femoral arteries in vivo. The SEA0400-induced decrease in [Ca²⁺]_CYT (Δ[Ca²⁺]_CYT; B) and vasodilation [diameter increase as %passive diameter (PD; D) are also shown. Effects of SEA0400 were augmented in TG mouse arteries. Values are means ± SE. Statistics were by two-way ANOVA. *P < 0.05 vs. the respective Ctrl group; #P < 0.05 and ##P < 0.01 vs. the basal TG group; ΔP < 0.05 vs. the basal TG group. Numbers of mice are in parentheses.

Fig. 4.

Effects of ouabain and SEA0400 on femoral artery cytosolic Ca²⁺ concentration ([Ca²⁺]_CYT) and diameter. [Ca²⁺]_CYT increase (Δ[Ca²⁺]_CYT; A) and vasoconstriction [diameter decrease, as %passive diameter (PD); B] induced by a 15-min superfusion with 10 μM ouabain as well as the effects of 0.3 μM SEA0400 (15-min pretreatment before ouabain) on these changes. Ouabain-induced, SEA0400-sensitive effects were amplified in transgenic (TG) mouse arteries. Values are means ± SE. Statistics were by a paired t-test. #P < 0.05 vs. ouabain alone. Numbers of mice are in parentheses.

Fig. 5.

The acute and chronic angiotensin II (ANG II)-induced blood pressure elevation is augmented in transgenic (TG) mouse arteries. A: mean blood pressure (24-h MBP) in awake, mobile smooth muscle-specific NCX1 knockout mice (SM-NCX1-KO; KO), control (Ctrl) mice, and TG mice that overexpress NCX1 specifically in smooth muscle (SM-NCX1-TG; TG) mice infused with saline or ANG II (400 ng·kg⁻¹·min⁻¹ sc) for 3 wk. B: increases in aortic MBP (ΔMBP) induced by acute intravenous infusion of 30 or 300 ng/kg ANG II in lightly anesthetized KO, Ctrl, and TG mice. Values are means ± SE. Statistics were by two-way ANOVA. *P < 0.05 and ***P < 0.001 for the pairs indicated. NS, nonsignificant. Numbers of mice are in parentheses.

Fig. 6.

Phenylephrine (PE) cumulative dose-response curves for exposed femoral arteries in control (Ctrl) and transgenic (TG) mice in vivo; PE was applied by superfusion. A: PE dose versus artery diameter. Ctrl passive diameter (PD) = 404 ± 21 μm (n = 9); TG PD = 374 ± 5 μm (n = 11). B: PE-induced vasoconstriction (as %PD). C: PE-induced increase (Δ) in cytosolic Ca²⁺ concentration ([Ca²⁺]_CYT). Basal [Ca²⁺]_CYT was 221 ± 29 nM in Ctrl mouse arteries and 319 ± 36 nM in TG mouse arteries, but the normalized vasoconstriction curves (B) and Δ[Ca²⁺]_CYT curves (C) from Ctrl and TG mice were virtually superimposable (two-way ANOVA). Values are means ± SE.

Fig. 7.

Effects of α₁-adrenergic blockade [prazosin (Pra)] on femoral artery cytosolic Ca²⁺ concentration ([Ca²⁺]_CYT), diameter, and tone in femoral arteries of control (Ctrl) and smooth muscle-specific Na⁺/Ca²⁺ exchanger-1 (NCX1) overexpression [transgenic (TG)] mice. A: diameters of exposed femoral arteries of Ctrl and TG mice in vivo as well as the effects of superfusion with 300 nM Pra. Passive diameter (PD) (arteries superfused with Ca²⁺-free medium) is also shown. Ctrl PD = 403 ± 13 μm (n = 6); TG PD = 397 ± 12 μm (n = 6). B and C: [Ca²⁺]_CYT and arterial tone (as %PD), respectively, in Ctrl and TG mouse arteries under basal conditions and during superfusion with 300 nM Pra. Pra reduced [Ca²⁺]_CYT and tone to a greater extent in TG mice than in Ctrl mice. [Ca²⁺]_i, intracellular Ca²⁺ concentration. Values are means ± SE. Statistics were by two-way ANOVA. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. basal Ctrl mice; ##P < 0.01 and ###P < 0.001 vs. basal TG mice. Numbers of mice are in parentheses.

Fig. 8.

Effects of intravenous injection of 3 mg/kg hexamethonium on arterial diameter (representative original recording; A) and mean arterial tone (B) and on cytosolic Ca²⁺ concentration ([Ca²⁺]_CYT) representative original data (C) and mean data (D) in exposed femoral arteries of control (Ctrl) and smooth muscle-specific Na⁺/Ca²⁺ exchanger-1 transgenic (TG) mice in vivo. [Ca²⁺]_i, intracellular Ca²⁺ concentration. Hexamethonium redued [Ca²⁺]_CYT to ≪50 nM and virtually abolished tone in both Ctrl and TG arteries. Ctrl passive diameter (PD) = 382 ± 14 μm (n = 8); TG PD = 386 ± 12 μm (n = 8). Values are means ± SE. Statistics were by two-way ANOVA. *P < 0.05 and ***P < 0.001 vs. basal Ctrl mice; ###P < 0.001 vs. basal TG mice. Numbers of mice are in parentheses.

Fig. 9.

Effects of superfusion of 100 nM angiotensin II (ANG II) with or without 100 nM losartan on the diameter (A), cytosolic Ca²⁺ concentration ([Ca²⁺]_CYT; B), and tone [as %passive diameter (PD); C] of exposed femoral arteries in control (Ctrl) and transgenic (TG) mice in vivo. Losartan abolished nearly all of the ANG II-induced increases in [Ca²⁺]_CYT and tone in both Ctrl and TG arteries. Values are means ± SE. Statistics were by two-way ANOVA. ***P < 0.001 vs. the respective basal Ctrl or TG mice; ##P < 0.01 vs. ANG II alone; ¶P < 0.05 vs. basal Ctrl mice. Numbers of mice are in parentheses.

Fig. 10.

Effects of superfusion with 100 nM losartan on the diameter (A), cytosolic Ca²⁺ concentration ([Ca²⁺]_CYT; B), and tone [as %passive diameter (PD); C] on exposed femoral arteries in control (Ctrl) and transgenic (TG) mice in vivo. Losartan had a negligible effect on [Ca²⁺]_CYT and tone in both Ctrl and TG arteries. Values are means ± SE. Statistics were by two-way ANOVA. *P < 0.05 vs. basal Ctrl mice. Numbers of mice are in parentheses.

Fig. 11.

Acetylcholine (ACh) cumulative dose-response curves for exposed femoral arteries in control (Ctrl) and transgenic (TG) mice in vivo; ACh was applied by superfusion. A: ACh dose versus artery diameter. Ctrl passive diameter (PD) = 390 ± 19 μm (n = 6); TG PD = 361 ± 16 μm (n = 10). B: phenylephrine-induced vasodilation (as %PD). C: ACh-induced decrease (Δ) in cytosolic Ca²⁺ concentration ([Ca²⁺]_CYT). Basal [Ca²⁺]_CYT was 230 ± 23 nM in Ctrl mouse arteries and 335 ± 10 nM in TG mouse arteries, but the normalized vasodilation curves (B) and [Ca²⁺]_CYT curves (C) from Ctrl and TG mice were virtually superimposable. Values are means ± SE.

Fig. 12.

Sodium nitroprusside (SNP) cumulative dose-response curves for exposed femoral arteries in control (Ctrl) and transgenic (TG) mice in vivo; SNP was applied by superfusion. A: SNP dose versus artery diameter. Ctrl passive diameter (PD) = 407 ± 23 μm (n = 6); TG PD = 387 ± 13 μm (n = 6). B: SNP-induced decrease (Δ) in cytosolic Ca²⁺ concentration ([Ca²⁺]_CYT). Basal [Ca²⁺]_CYT was 219 ± 31 nM in Ctrl mouse arteries and 309 ± 27 nM in TG mouse arteries. C: SNP-induced vasodilation (as %PD). TG curves were all significantly shifted to the left (indicating higher affinity and lower SNP EC₅₀ values) relative to the respective Ctrl curves (*P < 0.05). Values are means ± SE. Statistics were by two-way ANOVA. Numbers of mice are in parentheses.

Fig. 13.

Femoral artery constriction [%passive diameter (PD)] in vivo graphed as a function of pCa (−log[Ca²⁺]_CYT; cytosolic Ca²⁺ concentration). Data were taken from Figs. 2–12 [○, control (Ctrl); ▼, transgenic (TG)] and from Wang et al. (58) (Δ). The regression lines for the three sets of data for [Ca²⁺]_CYT = 100–500 nM (R = 0.91, 0.69 and, 0.97, respectively) were indistinguishable and correspond to a constriction of ~10% of PD per 100 nM increase in [Ca²⁺]_CYT. For comparison, the myogenic constriction versus pCa data in isolated, pressurized (60 mmHg) rat small posterior cerebral arteries taken from Fig. 10 of Knot and Nelson (29) are also shown (■); the constriction, as %PD, was calculated based on a PD of 210 μm. The graph also contains the normalized exogenous myosin light chain kinase transgene Förster resonance energy transfer (FRET) ratio curve (●; where 0 = “0” [Ca²⁺]_CYT; 100 = saturating [Ca²⁺]_CYT) from Fig. 1B of Wang et al. (58).