Chronic hypoxia augments depolarization-induced Ca2+ sensitization in pulmonary vascular smooth muscle through superoxide-dependent stimulation of RhoA

Abstract

Rho kinase (ROCK)-dependent vasoconstriction has been implicated as a major factor in chronic hypoxia (CH)-induced pulmonary hypertension. This component of pulmonary hypertension is associated with arterial myogenicity and increased vasoreactivity to receptor-mediated agonists and depolarizing stimuli resulting from ROCK-dependent myofilament Ca(2+) sensitization. On the basis of separate lines of evidence that CH increases pulmonary arterial superoxide (O(2)(-)) generation and that O(2)(-) stimulates RhoA/ROCK signaling in vascular smooth muscle (VSM), we hypothesized that depolarization-induced O(2)(-) generation mediates enhanced RhoA-dependent Ca(2+) sensitization in pulmonary VSM following CH. To test this hypothesis, we determined effects of the ROCK inhibitor HA-1077 and the O(2)(-)-specific spin trap tiron on vasoconstrictor reactivity to depolarizing concentrations of KCl in isolated lungs and Ca(2+)-permeabilized, pressurized small pulmonary arteries from control and CH (4 wk at 0.5 atm) rats. Using the same vessel preparation, we examined effects of CH on KCl-dependent VSM membrane depolarization and O(2)(-) generation using sharp electrodes and the fluorescent indicator dihydroethidium, respectively. Finally, using a RhoA-GTP pull-down assay, we investigated the contribution of O(2)(-) to depolarization-induced RhoA activation. We found that CH augmented KCl-dependent vasoconstriction through a Ca(2+) sensitization mechanism that was inhibited by HA-1077 and tiron. Furthermore, CH caused VSM membrane depolarization that persisted with increasing concentrations of KCl, enhanced KCl-induced O(2)(-) generation, and augmented depolarization-dependent RhoA activation in a O(2)(-)-dependent manner. These findings reveal a novel mechanistic link between VSM membrane depolarization, O(2)(-) generation, and RhoA activation that mediates enhanced myofilament Ca(2+) sensitization and pulmonary vasoconstriction following CH.

Fig. 1.

Chronic hypoxia (CH) increases vasoconstriction to depolarizing concentrations of KCl in isolated rat lungs (A) but attenuates KCl-mediated increases in vascular smooth muscle (VSM) intracellular Ca²⁺ concentration ([Ca²⁺]_i) in small pulmonary arteries (B). A: effect of increasing concentrations of KCl (15–60 mmol/l) on resistance (Torr·ml⁻¹·min·kg body wt⁻¹) in isolated, saline-perfused lungs from normoxic control (n = 4) and CH (n = 4) rats. B: vasoconstrictor [percent baseline internal diameter (ID); top] and VSM [Ca²⁺]_i [ratio of emission at 340 nm to emission at 380 nm for fura 2 (F₃₄₀/F₃₈₀); bottom] responses to KCl (30–120 mmol/l) in endothelium-disrupted, small pulmonary arteries from control (n = 4) and CH (n = 4) rats. Values are means ± SE. *P < 0.05 vs. control.

Fig. 2.

CH augments depolarization-induced VSM Ca²⁺ sensitization in Ca²⁺-permeabilized arteries. A: traces of ID (top) and VSM [Ca²⁺]_i (bottom) in an endothelium-disrupted artery from a CH rat after permeabilization to Ca²⁺ with ionomycin (3 μmol/l). Switching from superfusion with Ca²⁺-free physiological saline solution (PSS) to PSS containing 300 nmol/l Ca²⁺ caused an increase in F₃₄₀/F₃₈₀ and a slight constriction. Addition of KCl (30–120 mmol/l) resulted in vasoconstriction without a change in VSM [Ca²⁺]_i. B: percent vasoconstriction and VSM [Ca²⁺]_i responses to KCl in Ca²⁺-permeabilized arteries from control (n = 5) and CH (n = 5) rats. Values are means ± SE. *P < 0.05 vs. control.

Fig. 3.

Depolarization-induced vasoconstriction is Rho kinase (ROCK)-dependent in isolated lungs (A) and nonpermeabilized arteries (B) from CH rats. A: changes in resistance to KCl (15–60 mmol/l) in isolated, perfused lungs from control and CH rats in the presence of the ROCK inhibitor HA-1077 (10 μmol/l) or vehicle (n = 4 per group for each treatment). B: percent vasoconstriction to KCl (30–120 mmol/l) in endothelium-disrupted arteries from control and CH rats in the presence of HA-1077 (10 μmol/l) or vehicle (n = 4–5 per group for each treatment). Values are means ± SE. *P < 0.05 vs. control vehicle. #P < 0.05 vs. corresponding vehicle.

Fig. 4.

Enhanced depolarization-induced VSM Ca²⁺ sensitization following CH is dependent on ROCK, but not PKC or L-type Ca²⁺ channels, in Ca²⁺-permeabilized arteries. Vasoconstriction to KCl (30–120 mmol/l) in Ca²⁺-permeabilized, endothelium-disrupted arteries from control and CH rats in the presence of HA-1077 (10 μmol/l; A), the broad-spectrum PKC inhibitor GF-109203X (1 μmol/l; B), the L-type Ca²⁺ channel antagonist diltiazem (50 μmol/l; C), or vehicle (n = 5 per group for each treatment). Values are means ± SE. *P < 0.05, CH vehicle vs. control vehicle. #P < 0.05, CH HA-1077 vs. CH vehicle.

Fig. 5.

CH-induced pulmonary VSM membrane potential (E_m) depolarization persists on exposure to depolarizing concentrations of KCl (A) and is unaltered by ROCK inhibition (B) in Ca²⁺-permeabilized arteries. A: VSM E_m responses to increasing concentrations of KCl [5.4 (basal), 30, and 60 mmol/l] in pressurized, endothelium-disrupted, Ca²⁺-permeabilized arteries from control and CH rats (n = 4 per group at each KCl concentration). B: VSM E_m in arteries from control and CH rats prepared as described in A and treated with HA-1077 (10 μmol/l) or vehicle (n = 3 per group for each treatment). Values are means ± SE. *P < 0.05 vs. control. #P < 0.05 vs. basal. †P < 0.05 vs. 30 mM KCl.

Fig. 6.

CH augments depolarization-induced pulmonary arterial RhoA activation but does not alter RhoA expression. A: mean densitometric data for total RhoA normalized to Coomassie-stained total protein in homogenates of intrapulmonary arteries from control (n = 14) and CH (n = 15) rats. B: mean densitometric data for activated (GTP-bound) RhoA normalized to total RhoA in homogenates of Ca²⁺-permeabilized intrapulmonary arteries from control (n = 7 per group for each treatment) and CH (n = 7–8 per group for each treatment) rats under basal conditions (5.4 mmol/l KCl) or in response to 60 mmol/l KCl. Values are means ± SE. *P < 0.05 vs. control KCl. #P < 0.05 vs. CH basal.

Fig. 7.

O₂⁻ mediates increased KCl-induced vasoconstriction and associated VSM Ca²⁺ sensitization following CH in isolated perfused lungs (A) and Ca²⁺-permeabilized arteries (B and C). A: changes in resistance to KCl (15–60 mmol/l) in isolated, perfused lungs from control and CH rats in the presence of the O₂⁻-specific spin trap tiron (10 mmol/l) or vehicle (n = 5 per group for each treatment). B and C: vasoconstrictor responses to KCl (30–120 mmol/l) in Ca²⁺-permeabilized, endothelium-disrupted arteries from control (n = 5 per treatment; B) and CH (n = 5 per treatment; C) rats in the presence of tiron (10 mmol/l), tiron + HA-1077 (10 μmol/l), or vehicle. Values are means ± SE. *P < 0.05 vs. control vehicle. #P < 0.05, CH tiron and CH tiron + HA-1077 vs. CH vehicle.

Fig. 8.

CH increases basal and KCl-stimulated O₂⁻ levels in Ca²⁺-permeabilized arteries (A and B). O₂⁻ mediates CH-induced increases in KCl-stimulated RhoA activity in intrapulmonary arteries (C). A: dihydroethidium (DHE, 10 μmol/l) fluorescence [arbitrary units (AU)] as a function of time in pressurized, endothelium-disrupted, Ca²⁺-permeabilized arteries from control and CH rats (n = 5 per group for each treatment) in the presence of vehicle or tiron (10 mmol/l). Horizontal bar indicates addition of KCl (60 mmol/l). B: mean baseline and KCl-stimulated DHE fluorescence for control and CH arteries in the presence of vehicle or tiron. C: representative images of Western blots for GTP-bound (activated) RhoA and mean densitometric data for GTP-bound RhoA normalized to total RhoA in homogenates of intrapulmonary arteries from control (C) and CH rats (n = 5 per group for each treatment) in the presence of KCl (60 mmol/l) + tiron (10 mmol/l) or KCl (60 mmol/l) + vehicle. Values are means ± SE. *P < 0.05 vs. control vehicle. #P < 0.05 vs. CH vehicle. †P < 0.05 vs. baseline.