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. 2013 Jul 15;305(2):L154-64.
doi: 10.1152/ajplung.00313.2012. Epub 2013 May 17.

Chronic Hypoxia Selectively Enhances L- And T-type Voltage-Dependent Ca2+ Channel Activity in Pulmonary Artery by Upregulating Cav1.2 and Cav3.2

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Chronic Hypoxia Selectively Enhances L- And T-type Voltage-Dependent Ca2+ Channel Activity in Pulmonary Artery by Upregulating Cav1.2 and Cav3.2

Jun Wan et al. Am J Physiol Lung Cell Mol Physiol. .
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Abstract

Hypoxia-induced pulmonary hypertension (HPH) is characterized by sustained pulmonary vasoconstriction and vascular remodeling, both of which are mediated by pulmonary artery smooth muscle cell (PASMC) contraction and proliferation, respectively. An increase in cytosolic Ca²⁺ concentration ([Ca²⁺]cyt) is a major trigger for pulmonary vasoconstriction and an important stimulus for cell proliferation in PASMCs. Ca²⁺ influx through voltage-dependent Ca²⁺ channels (VDCC) is an important pathway for the regulation of [Ca²⁺]cyt. The potential role for L- and T-type VDCC in the development of HPH is still unclear. Using a hypoxic-induced pulmonary hypertension mouse model, we undertook this study to identify if VDCC in pulmonary artery (PA) are functionally upregulated and determine which type of VDCC are altered in HPH. Mice subjected to chronic hypoxia developed pulmonary hypertension within 4 wk, and high-K⁺- and U-46619-induced contraction of PA was greater in chronic hypoxic mice than that in normoxic control mice. Additionally, we demonstrate that high-K⁺- and U-46619-induced Ca²⁺ influx in PASMC is significantly increased in the hypoxic group. The VDCC activator, Bay K8864, induced greater contraction of the PA of hypoxic mice than in that of normoxic mice in isometric force measurements. L-type and T-type VDCC blockers significantly attenuated absolute contraction of the PA in hypoxic mice. Chronic hypoxia did not increase high-K⁺- and U-46619-induced contraction of mesenteric artery (MA). Compared with MA, PA displayed higher expression of calcium channel voltage-dependent L-type α1C-subunit (Cav1.2) and T-type α1H-subunit (Cav3.2) upon exposure to chronic hypoxia. In conclusion, both L-type and T-type VDCC were functionally upregulated in PA, but not MA, in HPH mice, which could result from selectively increased expression of Cav1.2 and Cav3.2.

Keywords: calcium channel voltage-dependent L-type α1C-subunit; calcium channel voltage-dependent T-type α1H-subunit; hypoxia; mouse; pulmonary artery; voltage-dependent calcium ion channel.

Figures

Fig. 1.
Fig. 1.
Chronic hypoxia induces pulmonary hypertension in mice. A: representative records of right ventricular pressure (RVP, left) and summarized data (mean ± SE) of right ventricular systolic pressure (RVSP, right) in normoxic mice (Nor, n = 6) and mice exposed to hypoxia for 4 wk (Hyp, n = 13). ***P < 0.001 vs. Nor. B: hematoxylin and eosin (H&E) staining of lung sections (left, horizontal bar = 10 μm) and calculated pulmonary arterial (PA) wall thickness (determined by the percentage ratio of vascular wall area to total area; right) in Nor (n = 10) and Hyp (n = 10) mice. **P < 0.01 vs. Nor. C: summarized data (mean ± SE) of the ratio of right ventricle (RV) to left ventricle (LV) plus septum (S) weight [RV/(LV + S)] in Nor (n = 7) and Hyp (n = 7) mice. **P < 0.01 vs. Nor.
Fig. 2.
Fig. 2.
Assessment of endothelium function in PA rings. A: representative tracing showing changes in phenylephrine (PE, 100 nM)-induced contraction before, during, and after application of acetylcholine (ACh, 10 μM) in pulmonary artery (PA) ring preparations. B: summarized data (means ± SE) showing change in absolute tension in the presence or absence of ACh. Application of ACh did not significantly decrease PE-induced contraction in PA rings (n = 4, not significant).
Fig. 3.
Fig. 3.
Chronic hypoxia enhances active tension induced by high K+ or U-46619 in pulmonary arteries. A and B: representative tracings showing changes in tension before, during, and after application of various concentrations of extracellular K+ (10–120 mM; A) or U-46619 (0.3–300 nM; B) in isolated PA from normoxic mice (Normoxia) and mice exposed to chronic hypoxia for 4 wk (Hypoxia). Horizontal bars depict 5 min. C: summarized data (means ± SE) showing the dose-response curves of high-K+-induced absolute active tension (left) and U-46619-induced absolute active tension (right) in isolated pulmonary arterial rings from Nor (n = 8, open circles) and Hyp (n = 8, closed squares) mice. **P < 0.01, Nor vs. Hyp.
Fig. 4.
Fig. 4.
Chronic hypoxia enhances 40 mM K+ (40K)-induced Ca2+ influx in mouse pulmonary arterial smooth muscle cells (PASMC). A and B: representative records showing changes in cytosolic Ca2+ concentration ([Ca2+]cyt) before, during, and after application of 40K (A) or 100 nM U-46619 (B) in freshly dissociated PASMC from mice exposed to normoxia or hypoxia for 4 wk. C: summarized data (means ± SE) showing the amplitudes of 40K-mediated increase in [Ca2+]cyt (left) and U-46619-mediated increase in [Ca2+]cyt (right) in PASMC from Nor (n = 14 for 40K and n = 37 for U-46619) and Hyp (n = 23 for 40K and n = 69 for U-46619) mice. **P < 0.01 vs. Nor.
Fig. 5.
Fig. 5.
Chronic hypoxia enhances Bay K8644-mediated pulmonary vasoconstriction. A: representative tracings showing isometric tension before, during, and after application of the voltage-dependent Ca2+ channels (VDCC) agonist Bay K8644 (0.1–10 μM) in isolated pulmonary arterial rings from mice exposed to normoxia and hypoxia (for 4 wk). B: summarized data (means ± SE) showing the dose-response curves of Bay K8644-induced active tension (depicted as increase in absolute active tension, left, and tension increase normalized to the maximal tension) in PA rings isolated from Nor (open circles, n = 4) and Hyp (solid squares, n = 8) mice. **P < 0.01, vs. Nor.
Fig. 6.
Fig. 6.
L- and T-type VDCC blockers inhibit high-K+-induced active tension in PA rings from hypoxic mice to a greater extent than in PA rings from normoxic mice. A–C: representative tracings (a) and summarized data expressed as absolute decrease (b) and percent decrease (c) to 40K-mediated active tension in the presence of various concentrations of nifedipine (0.03–3 μM; A), mibefradil (0.03–3 μM; B), and ω-agatoxin (0.1–30 nM; C) in PA rings isolated from Nor (left, and open circles, n = 4) and Hyp (right, and solid circles, n = 4) mice. *P < 0.05 and **P < 0.01 vs. Nor. The inhibitory effects of nifedipine, a L-type VDCC blocker, and mibefradil, a T-type VDCC blocker, on 40K-induced vasoconstriction is greater in PA rings from hypoxic mice than in PA rings from normoxic mice.
Fig. 7.
Fig. 7.
L- and T-type VDCC blockers inhibit U-46619-induced active tension in PA rings from hypoxic mice to a greater extent than in PA rings from normoxic mice. A–C: representative tracings (a) and summarized data expressed as absolute decrease (b) and percentage decrease (c) to 100 nM U-46619-mediated active tension in the presence of various concentrations of nifedipine (0.03–3 μM; A), mibefradil (0.03–3 μM; B), or ω-agatoxin (0.1–30 nM; C) in PA rings isolated from Nor (left, and open circles, n = 4) and Hyp (right, and solid circles, n = 4) mice. *P < 0.05 and **P < 0.01 vs. Nor. The inhibitory effects of high-dose nifedipine, an L-type VDCC blocker, on U-46619-induced vasoconstriction is greater in PA rings from hypoxic mice than in PA rings from normoxic mice.
Fig. 8.
Fig. 8.
Chronic hypoxia does not affect 40K- and U46619-induced contraction of mesenteric arteries (MA) in mice. A: representative tracings showing isometric tension before, during, and after application of 40K and various concentrations (1–100 nM) of U-46619 (a thromboxane A2 analog) in isolated MA rings from mice exposed to normoxia (top) and hypoxia (bottom). B: summarized data (means ± SE) showing the amplitudes of 40K-induced active tension in MA rings isolated from Nor (n = 4) and Hyp (n = 4) mice. C: summarized data showing the dose-response curves of U-46619-induced active tension (depicted as increase in absolute active tension, left, and tension increase normalized to the maximal tension) in PA rings isolated from Nor (open circles, n = 4) and Hyp (solid squares, n = 4) mice. D: representative tracings showing isometric tension before, during, and after application of various concentrations (0.1–10 μM) of Bay K8644 (a VDCC activator) in MA rings isolated from Nor (left) and Hyp (right) mice. E: summarized data showing the dose-response curves of Bay K8644-induced changes in active tension (depicted as changes in absolute active tension, left, and tension change normalized to the maximal tension) in MA rings isolated from Nor (open circles, n = 4) and Hyp (solid squares, n = 4) mice.
Fig. 9.
Fig. 9.
Chronic hypoxia upregulates mRNA and protein expression of calcium channel voltage-dependent L-type α1C-subunit (Cav1.2) and T-type α1H-subunit (Cav3.2) in PA but not in MA from mice. A: relative mRNA expression levels of Cav1.2, Cav2.1, Cav3.1, and Cav3.2 in PA (a) and MA (b) isolated from Nor (open bars, n = 6) and Hyp (solid bars, n = 5) mice. **P < 0.01 vs. Nor. c, Comparison of hypoxia-mediated changes in mRNA expression of Cav1.2, Cav2.1, Cav3.1, and Cav3.2 in MA (open bars) and PA (solid bars). **P < 0.01 vs. MA. B: a, immunohistochemical images (×60) showing Cav1.2 and Cav3.2 in PA (left) and MA (right) isolated from Nor (top) and Hyp (bottom) mice. b, Summarized data (means ± SE) showing mRNA expression level of Cav1.2 and Cav3.2 in PA and MA isolated from Nor (open bars; n = 8) and Hyp (solid bars; n = 8) mice. *P < 0.05 and **P < 0.01 vs. Nor. C: a, representative Western blot image of protein lysates from normoxic and hypoxic mouse lung tissues probed for Cav1.2 (top) and Cav3.2 (bottom). Normoxic and hypoxic samples were loaded on the same membrane. Brain tissues where probed as a positive control. b, Summarized data (means ± SE) showing protein expression level normalized to β-actin for Cav1.2 and Cav3.2 in normoxic and hypoxic mouse lung homogenates (n = 3, *P < 0.05 vs. Nor).

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