Role of Acid-Sensing Ion Channels in Hypoxia- and Hypercapnia-Induced Ventilatory Responses

Previous reports indicate roles for acid-sensing ion channels (ASICs) in both peripheral and central chemoreception, but the contributions of ASICs to ventilatory drive in conscious, unrestrained animals remain largely unknown. We tested the hypotheses that ASICs contribute to hypoxic- and hypercapnic-ventilatory responses. Blood samples taken from conscious, unrestrained mice chronically instrumented with femoral artery catheters were used to assess arterial O2, CO2, and pH levels during exposure to inspired gas mixtures designed to cause isocapnic hypoxemia or hypercapnia. Whole-body plethysmography was used to monitor ventilatory parameters in conscious, unrestrained ASIC1, ASIC2, or ASIC3 knockout (-/-) and wild-type (WT) mice at baseline, during isocapnic hypoxemia and during hypercapnia. Hypercapnia increased respiratory frequency, tidal volume, and minute ventilation in all groups of mice, but there were no differences between ASIC1-/-, ASIC2-/-, or ASIC3-/- and WT. Isocapnic hypoxemia also increased respiratory frequency, tidal volume, and minute ventilation in all groups of mice. Minute ventilation in ASIC2-/- mice during isocapnic hypoxemia was significantly lower compared to WT, but there were no differences in the responses to isocapnic hypoxemia between ASIC1-/- or ASIC3-/- compared to WT. Surprisingly, these findings show that loss of individual ASIC subunits does not substantially alter hypercapnic or hypoxic ventilatory responses

Contribution of reactive oxygen species to the pathogenesis of pulmonary arterial hypertension.

Pulmonary arterial hypertension is associated with a decreased antioxidant capacity. However, neither the contribution of reactive oxygen species to pulmonary vasoconstrictor sensitivity, nor the therapeutic efficacy of antioxidant strategies in this setting are known. We hypothesized that reactive oxygen species play a central role in mediating both vasoconstrictor and arterial remodeling components of severe pulmonary arterial hypertension. We examined the effect of the chemical antioxidant, TEMPOL, on right ventricular systolic pressure, vascular remodeling, and enhanced vasoconstrictor reactivity in both chronic hypoxia and hypoxia/SU5416 rat models of pulmonary hypertension. SU5416 is a vascular endothelial growth factor receptor antagonist and the combination of chronic hypoxia/SU5416 produces a model of severe pulmonary arterial hypertension with vascular plexiform lesions/fibrosis that is not present with chronic hypoxia alone. The major findings from this study are: 1) compared to hypoxia alone, hypoxia/SU5416 exposure caused more severe pulmonary hypertension, right ventricular hypertrophy, adventitial lesion formation, and greater vasoconstrictor sensitivity through a superoxide and Rho kinase-dependent Ca2+ sensitization mechanism. 2) Chronic hypoxia increased medial muscularization and superoxide levels, however there was no effect of SU5416 to augment these responses. 3) Treatment with TEMPOL decreased right ventricular systolic pressure in both hypoxia and hypoxia/SU5416 groups. 4) This effect of TEMPOL was associated with normalization of vasoconstrictor responses, but not arterial remodeling. Rather, medial hypertrophy and adventitial fibrotic lesion formation were more pronounced following chronic TEMPOL treatment in hypoxia/SU5416 rats. Our findings support a major role for reactive oxygen species in mediating enhanced vasoconstrictor reactivity and pulmonary hypertension in both chronic hypoxia and hypoxia/SU5416 rat models, despite a paradoxical effect of antioxidant therapy to exacerbate arterial remodeling in animals with severe pulmonary arterial hypertension in the hypoxia/SU5416 model

An inspired CO₂ of 3.2% causes isocapnic hypoxemia in mice concurrently exposed to 7.0% O₂.

<p>A scatter plot <b>(A)</b> showing arterial CO₂ tensions (P_aCO₂; y-axis) in mice exposed to 7.0% O₂ and a range of inspired CO₂ levels (% CO₂; x-axis) was fit using linear regression (solid line, equation displayed on the graph). The dotted lines on the graph indicate the average baseline P_aCO₂ (horizontal) measured during exposure to room air, and the estimated inspired CO₂ required to maintain this P_aCO₂ level (vertical) during exposure to 7% O₂ based on the linear regression. Arterial blood samples from WT mice exposed to 7.0% O₂, 3.2% CO₂, balance N₂, were analyzed to assess P_aO₂ <b>(B)</b>, P_aCO₂ <b>(C)</b>, and pH_a <b>(D)</b>. <b>(E)</b> Representative whole-body plethysmography traces from a WT mouse show the ventilatory response to 7.0% O₂, 3.2% CO₂, balance N₂. Values are individual measurements (A) or means ± SE (B-D); n = 5–6 animals per group (B-D). *<i>P</i> < 0.05 vs. baseline (paired, two-tailed Student’s t-test).</p

Inhalation of 6% CO₂ produces reliable hypercapnia and elevation of ventilation.

<p>WT mice were exposed to a range of inspired CO₂ levels to determine optimal conditions for testing hypercapnic ventilatory responses. <b>(A)</b> Representative traces of whole-body plethysmography illustrate frequency and depth of breathing in conscious, unrestrained mice exposed to normal air at baseline (21% O₂, 0% CO₂, balance N₂), and to increasing levels of hypercapnia (3.7, 6.0, and 9.8% CO₂). Summary data shows <b>(B)</b> respiratory frequency (breaths/min), <b>(C)</b> tidal volume (μL/breath/g), and <b>(D)</b> minute ventilation (mL/min/g) at each inspired CO₂ level. Arterial blood samples taken from WT mice at baseline (normal air) or during exposure to 6% CO₂ were analyzed for <b>(E)</b> P_aO_2, <b>(F)</b> P_aCO₂, and <b>(G)</b> pH_a. Values are means ± SE; n = 4–8 animals/group. *<i>P</i> < 0.05 vs. baseline (1-way ANOVA; Dunnett’s post-hoc test (B) or two-tailed, paired Student’s t-test (C-E)).</p

Blood gas levels were not different between paired WT and knockout groups at baseline, during hypercapnia, or during isocapnic hypoxemia.

<p>Blood gas levels were not different between paired WT and knockout groups at baseline, during hypercapnia, or during isocapnic hypoxemia.</p

ASIC2, but not ASIC1 or ASIC3, contributes to the hypoxic ventilatory response.

<p>Ventilatory responses to isocapnic hypoxemia (7.0% O₂, 3.2% CO₂, bal N₂) were assessed using whole-body plethysmography and compared between <b>(A-C)</b> ASIC1^-/-, <b>(D-F)</b> ASIC2^-/-, and <b>(G-I)</b> ASIC3^-/- and WT mice. Values are means ± SE; n = 8 (A-F) or 11–12 (G-I) animals/group. *<i>P</i> < 0.05 vs. baseline; ^#<i>P</i> < 0.05 vs. WT (2-way, repeated measures ANOVA; Sidak’s post-hoc test). Displayed P value corresponds to the comparison between WT and KO (panel D).</p

The hypercapnic ventilatory response does not require ASIC1, 2, or 3.

<p>Whole-body plethysmography was used to determine respiratory frequency (breaths/min; A, D, G), tidal volume (μL/breath/g; B, E, H), and minute ventilation (ml/min/g body wt; C, F, I) at baseline and during exposure to hypercapnia (6% CO₂) in WT, ASIC1^-/- (A-C), ASIC2^-/- (D-F), and ASIC3^-/- mice (G-I). Values are means ± SE; n = 8 (A-C), 7–8 (D-F), or 11–12 (G-I) animals/group. *<i>P</i> < 0.05 vs. baseline (2-way, repeated measures ANOVA; Sidak’s post-hoc test). Displayed P value corresponds to comparison between WT and KO (panel D).</p

Enhanced basal tone and ET-1-induced pulmonary VSM Ca²⁺ sensitization following hypoxia/SU5416 is mediated by Rho kinase (ROK).

<p>(A) HA-1077-mediated change in vessel diameter (% baseline diameter) in non-permeabilized, endothelium-disrupted, pressurized small pulmonary arteries. (B) Vasoconstriction (% baseline diameter) to endothelin-1 (ET-1; 10⁻¹⁰ to 10⁻⁷ M) in the presence of HA-1077 (10 μM) in Ca²⁺-permeabilized, endothelium-disrupted, pressurized small pulmonary arteries from normoxic and hypoxic rats treated with vehicle or SU5416. (C) Effect of HA-1077 on ET-1 mediated vasoconstriction in each group compared to vehicle-treated arteries (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180455#pone.0180455.g009" target="_blank">Fig 9</a>). Values are means ± SE; n = 4–5 animals/group; * p < 0.05 vs. vehicle-treated arteries; analyzed by two-way ANOVA and individual groups compared with the Student-Newman-Keuls test.</p

Right ventricular hypertrophy is not attenuated by TEMPOL.

<p>Representative AZAN trichrome-stained whole heart sections (A) and higher magnification images of the right ventricle (B) from rats treated with vehicle, SU5416, and/or TEMPOL and exposed to normoxia or hypoxia. AZAN trichrome shows cell nuclei (dark red), collagen (blue) and orange-red in cytoplasm. C) Summary data showing Fulton’s index [ratio of RV to left ventricular plus septal (LV + S) heart weight] Values are means ± SE; n = animals/group (indicated in bars). *P < 0.05 vs. the normoxia group; # P < 0.05 vs. the corresponding SU5416 vehicle group; analyzed with multiple two-way ANOVA and individual groups compared with the Student-Newman-Keuls test.</p

TEMPOL increases H₂O₂-specific oxidative stress.

<p>A) H₂O₂ levels assessed by Amplex Red Assay in control PASMC in the absence or presence of PEG-catalase (PEG-CAT; 250 U/ml) or TEMPOL (1 mM). SOTS-1 (0.01 mM) is a superoxide donor and used to stimulate increased oxidative stress. Values are means ± SE; <i>n</i> = 5 animals per group; *P ≤ 0.05 vs. vehicle-treated group; # P < 0.05 vs. baseline; analyzed by two-way ANOVA and individual groups compared with the Student-Newman-Keuls test. Representative western blot and summary data showing B) 4-HNE and C) S-sulfenylated proteins in whole lung homogenates from normoxic and hypoxic rats treated with SU5416 and/or TEMPOL. Values are means ± SE; <i>n</i> = 6 animals per group; *P ≤ 0.05 vs. normoxic group; τ p < 0.05 vs. TEMPOL-vehicle group; analyzed by multiple two-way ANOVA and individual groups compared with the Student-Newman-Keuls test.</p