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 Ca²⁺ sensitization. On the basis of separate lines of evidence that CH increases pulmonary arterial superoxide (O₂⁻) generation and that O₂⁻ stimulates RhoA/ROCK signaling in vascular smooth muscle (VSM), we hypothesized that depolarization-induced O₂⁻ generation mediates enhanced RhoA-dependent Ca²⁺ sensitization in pulmonary VSM following CH. To test this hypothesis, we determined effects of the ROCK inhibitor HA-1077 and the O₂⁻-specific spin trap tiron on vasoconstrictor reactivity to depolarizing concentrations of KCl in isolated lungs and Ca²⁺-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 O₂⁻ generation using sharp electrodes and the fluorescent indicator dihydroethidium, respectively. Finally, using a RhoA-GTP pull-down assay, we investigated the contribution of O₂⁻ to depolarization-induced RhoA activation. We found that CH augmented KCl-dependent vasoconstriction through a Ca²⁺ 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 O₂⁻ generation, and augmented depolarization-dependent RhoA activation in a O₂⁻-dependent manner. These findings reveal a novel mechanistic link between VSM membrane depolarization, O₂⁻ generation, and RhoA activation that mediates enhanced myofilament Ca²⁺ sensitization and pulmonary vasoconstriction following CH.

chronic hypoxia (CH) associated with high-altitude exposure and chronic obstructive pulmonary disease leads to elevated pulmonary vascular resistance and pulmonary hypertension. The vasoconstrictor component of CH-induced pulmonary hypertension is mediated, in part, by generalized hypoxic pulmonary vasoconstriction resulting from global airway hypoxia. However, hypoxic vasoreactivity is largely blunted following CH (31), suggesting that additional mechanisms provide a major contribution to the development of pulmonary hypertension. Findings that basal tone is elevated in pulmonary arterial rings from CH rats (34, 54) and that vasodilators substantially lower pulmonary vascular resistance in CH rats acutely returned to a normoxic environment (34) provide further evidence that the vasoconstrictor response to CH is multifaceted and a primary determinant of pulmonary hypertension. Consistent with these findings, recent studies from our laboratory and others have identified an effect of CH on induction of myogenic tone in small pulmonary arteries (3) and enhancement of agonist-dependent vasoconstriction through a RhoA/Rho kinase (ROCK)-mediated myofilament Ca²⁺ sensitization pathway (14, 20, 34, 54), responses that may contribute to the pathogenesis of pulmonary hypertension in this setting.

RhoA is a small GTP-binding protein that is activated in response to stimulation of many G protein-coupled receptors (45). However, RhoA-mediated vascular smooth muscle (VSM) Ca²⁺ sensitization can also be elicited by depolarizing stimuli (33, 41, 50, 55). Membrane potential (E_m) depolarization-induced VSM contraction appears to be mediated, in part, via Ca²⁺-dependent stimulation of RhoA/ROCK (33, 41, 50, 55), thus establishing a dual role for Ca²⁺ in regulation of VSM contraction: 1) activation of myosin light chain (MLC) kinase and 2) RhoA-mediated inhibition of MLC phosphatase. Interestingly, depolarization has been demonstrated to stimulate RhoA via a Ca²⁺-independent mechanism in renal tubule epithelial cells (46). Whether a similar mechanism of RhoA activation exists in VSM and whether such a mechanism contributes to enhanced depolarization-induced pulmonary vasoconstriction following exposure to CH (2, 14, 34) have not been addressed. On the basis of evidence that exogenous reactive oxygen species (ROS), including superoxide anion (O₂⁻), mediate ROCK-dependent VSM contraction (21, 22, 24) and that E_m depolarization increases ROS production in endothelial cells, macula densa, and isolated lungs (1, 28, 44), we hypothesized that depolarization-induced O₂⁻ generation mediates enhanced RhoA-dependent Ca²⁺ sensitization in pulmonary VSM following CH. This hypothesis was tested by assessment of depolarization-dependent vasoconstriction, VSM-free intracellular Ca²⁺ concentration ([Ca²⁺]_i), O₂⁻ generation, and E_m in pressurized, small pulmonary arteries from normoxic and CH rats. We additionally evaluated the contribution of O₂⁻ to depolarization-induced RhoA activation in intrapulmonary arteries and vasoreactivity in isolated perfused lungs from each group. Our findings demonstrate a unique mechanistic relationship between E_m depolarization, O₂⁻ generation, and RhoA/ROCK signaling that mediates increased myofilament Ca²⁺ sensitivity in the hypertensive pulmonary circulation.

MATERIALS AND METHODS

All protocols and surgical procedures were reviewed and approved by the Institutional Animal Care and Use Committee of the University of New Mexico Health Sciences Center.

Experimental Groups

Male Sprague-Dawley rats (250–300 g body wt; Harlan Industries) were exposed to CH by placement in a hypobaric chamber maintained at ∼380 Torr for 4 wk, as previously described (3, 20, 35, 39).

Isolated Lung Protocols: Contribution of ROCK and O₂⁻ to KCl-Dependent Vasoconstriction

The following protocols were performed to determine the contribution of ROCK and O₂⁻ to depolarization-mediated pulmonary vasoconstriction in lungs from control and CH rats. Lungs were isolated from rats, perfused with a physiological salt solution (PSS) containing (in mmol/l) 129.8 NaCl, 5.4 KCl, 0.5 NaH₂PO₄, 0.83 MgSO₄, 19 NaHCO₃, 1.8 CaCl₂, and 5.5 glucose, with 4% bovine serum albumin (wt/vol) added as a colloid (all from Sigma), and prepared for experimentation using established procedures (18, 38, 39, 52). After stabilization of baseline pressures, we assessed a cumulative concentration-response relationship to KCl in lungs from control and CH rats by switching the perfusate to PSS containing depolarizing concentrations of KCl (15–60 mmol/l). This PSS had the same osmolality as the control solution and is identical in composition, except it contained more KCl and less NaCl than normal PSS. All experiments were conducted in the presence of the ROCK inhibitor HA-1077 (10 μmol/l; Sigma) (3, 5), the O₂⁻-specific spin trap 4,5-dihydroxy-1,3-benzene disulfonic acid (tiron, 10 mmol/l; Sigma) (18, 20, 25), or vehicle (PSS). To eliminate influences of endogenous nitric oxide (NO) and cyclooxygenase (COX) products on depolarization-dependent vasoconstriction, we conducted parallel experiments after administration of the NO synthase inhibitor N^ω-nitro-l-arginine (l-NNA, 300 μmol/l; Sigma) and the COX inhibitor meclofenamate (160 μmol/l; Sigma). These concentrations of l-NNA and meclofenamate have been shown to effectively inhibit NO and prostaglandin synthesis in this preparation (10, 52).

Isolated Small Pulmonary Artery Preparation: Assessment of Vasoreactivity, VSM [Ca²⁺]_i, E_m, and O₂⁻ Levels

Endothelial disruption and cannulation of small pulmonary arteries for dimensional analysis.

Rats were anesthetized with pentobarbital sodium (200 mg/kg ip), and the left lung was removed and immediately placed in ice-cold PSS. A pulmonary artery [100–200 μm internal diameter (ID), ∼1 mm long] without side branches was dissected free and transferred to a vessel chamber (model CH-1, Living Systems) containing cold PSS. After the proximal end of the vessel was cannulated with a tapered glass pipette and blood was flushed out, the vessel lumen was rubbed with a strand of moose mane to disrupt the endothelium, thereby allowing assessment of depolarization-induced myofilament Ca²⁺ sensitization independent of complicating influences of the endothelium. Arteries were then cannulated at the distal end and pressurized to 12 or 35 Torr using a servo-controlled peristaltic pump (Living Systems). These pressures are estimates of in vivo pressures in conscious rats from each group, as determined previously (38). Arteries were required to hold a steady pressure when the servo-control function was switched off to verify the absence of leaks. Any vessels with apparent leaks were discarded. The vessel chamber was transferred to the stage of a Nikon Eclipse TS100 microscope, and the preparation was superfused with PSS (37°C) equilibrated with 10% O₂-6% CO₂-balance N_2. A vessel chamber cover was positioned to permit this same gas mixture to flow over the top of the chamber bath. This gas mixture yields an approximate superfusate pH 7.40, 57 Torr Po₂, and 31 Torr Pco₂ (18). Bright-field images of vessels were obtained with a charge-coupled device camera (model CCD100M, IonOptix), and dimensional analysis was performed by IonOptix Sarclen software to measure ID. The effectiveness of endothelial disruption was verified by the lack of a vasodilatory response to ACh (1 μmol/l) in UTP-constricted vessels. These methods have been described in previous studies from our laboratory (3, 7, 19, 20, 35).

Measurement of VSM [Ca²⁺]_i.

Pressurized, endothelium-disrupted arteries were loaded abluminally with the cell-permeant, ratiometric, Ca²⁺-sensitive fluorescent indicator fura 2-AM (2 μmol/l fura 2-AM + 0.05% pluronic acid; Molecular Probes) for 45 min at room temperature in darkness, as described previously (3, 7, 19, 20, 35). Vessels were rinsed for 20 min with PSS (37°C; equilibrated with 10% O₂-6% CO₂) after the loading period to wash out excess dye and to allow for hydrolysis of acetoxymethyl groups by intracellular esterases. Fura 2-loaded arteries were alternately excited at 340 and 380 nm at a frequency of 10 Hz with an IonOptix Hyperswitch dual-excitation light source, and the respective 510-nm emissions were collected with a photomultiplier tube. Background-subtracted ratio of emission at 340 nm to emission at 380 nm was calculated with IonOptix Ion Wizard software and recorded continuously throughout the experiment, with simultaneous measurement of ID from red-wavelength bright-field images, as described above. VSM [Ca²⁺]_i is expressed as the mean ratio of fluorescence at 340 nm to fluorescence at 380 nm (F₃₄₀/F₃₈₀) from the background-subtracted 510-nm signal.

VSM Ca²⁺ permeabilization.

To directly assess mechanisms of myofilament Ca²⁺ sensitization independent of changes in VSM [Ca²⁺]_i, we clamped VSM [Ca²⁺]_i in some experiments by permeabilization with the Ca²⁺ ionophore ionomycin, as previously described (19, 20). Briefly, arteries were equilibrated in Ca²⁺-free PSS containing 3 mmol/l EGTA (Sigma) and 3 μmol/l ionomycin (Sigma) to permeabilize the VSM to Ca²⁺. Vessels were then equilibrated in PSS with a calculated free Ca²⁺ concentration of 300 nmol/l [containing (in mmol/l) 129.8 NaCl, 5.4 KCl, 0.5 NaH₂PO₄, 1.3 MgSO₄, 19 NaHCO₃, 6.8 CaCl₂, 5.5 glucose, 8.2 EGTA, and 0.003 ionomycin (Sigma)]. This Ca²⁺ concentration was calculated using the K_d of EGTA for Ca²⁺ of 43.7 nmol/l and the K_d of EGTA for Mg²⁺ of 3.33 mmol/l at 37°C and pH 7.4, as described previously (19, 20). The addition of 300 nmol/l Ca²⁺-containing PSS provided optimal vasoconstrictor responses to KCl with minimal effects on basal tone in Ca²⁺-permeabilized arteries in preliminary experiments.

Measurement of VSM E_m.

VSM cell E_m values were recorded using intracellular sharp electrodes from pressurized, endothelium-disrupted, Ca²⁺-permeabilized pulmonary arteries from control and CH rats prepared as described above. VSM cells were impaled with microelectrodes (50- to 100-MΩ tip resistance) containing 1 M KCl and filled with Lucifer Yellow dye (Sigma) for subsequent identification of cell type. A Neuroprobe amplifier (model 1600, A-M Systems) was used to record E_m. Analog output from the amplifier was low-pass filtered at 1 kHz and routed to a Tektronix RM502A oscilloscope and a data acquisition system (Dataq Instruments). Criteria for acceptance of E_m recordings were as follows: 1) an abrupt negative deflection in potential as the microelectrode is advanced into the cell, 2) stable E_m for ≥1 min, and 3) an abrupt change in potential to ∼0 mV after the electrode is retracted from the cell. After completion of E_m recordings, fluorescent imaging of dye-loaded cells was used to verify the cell type for each recording. The mean E_m of all VSM cells recorded for an individual rat was considered a single replicate for statistical purposes. We described these methods in earlier studies (7, 35).

Measurement of O₂⁻.

Fluorescence detection of dihydroethidium (DHE; Molecular Probes) oxidation was used as a measure of O₂⁻ levels in pressurized, endothelium-disrupted, Ca²⁺-permeabilized arteries from control and CH rats, as reported previously (20). Cells are permeable to DHE, which is converted to the fluorescent products ethidium and 2-hydroxyethidium in an O₂⁻-dependent manner (6, 57). Arteries were prepared for experimentation as described above and transferred to the stage of a Nikon Diaphot microscope. After 30 min of equilibration, arteries were loaded with DHE (10 μmol/l DHE and 0.05% pluronic acid). Vessels were incubated in this solution for 30 min at room temperature in darkness and then rinsed for 5 min with PSS (37°C) to wash out excess dye. We obtained fluorescent images using a standard tetramethylrhodamine isothiocyanate filter before (for background subtraction) and after loading the vessel with DHE. Images (1 per minute) were generated with a charge-coupled device camera (Photometrics SenSys 1400) and processed with MetaFluor software (Molecular Devices). Normalized fluorescence intensity is defined as average gray-scale values for all pixels in the field above background.

Experimental Protocols Assessing Vasoreactivity, VSM [Ca²⁺]_i, E_m, and O₂⁻ Levels in Pressurized Small Pulmonary Arteries

KCl-induced vasoconstrictor and VSM [Ca²⁺]_i responses in nonpermeabilized arteries: role of ROCK.

To characterize effects of CH on depolarization-mediated vasoconstriction and the contribution of ROCK to this response, we assessed ID and VSM [Ca²⁺]_i responses to depolarizing concentrations of KCl (30–120 mmol/l) in nonpermeabilized arteries from control and CH rats in the presence or absence of the selective ROCK inhibitor HA-1077 (10 μmol/l) (3, 5, 26, 40, 49).

Role of ROCK, PKC, L-type Ca²⁺ channels, and O₂⁻ in KCl-dependent VSM Ca²⁺ sensitization.

The contribution of ROCK to KCl-induced myofilament Ca²⁺ sensitization was established by measurement of vasoconstrictor responses to increasing concentrations of KCl in the presence of HA-1077 (10 μmol/l) or vehicle (PSS) in Ca²⁺-permeabilized control and CH arteries. F₃₄₀/F₃₈₀ values were measured continuously to verify that VSM [Ca²⁺]_i remained clamped for the duration of the experiment.

Since VSM Ca²⁺ sensitization can additionally be mediated by PKC (19), we assessed responses to KCl in separate sets of Ca²⁺-clamped arteries from each group after administration of the broad-spectrum PKC inhibitor GF-109203X (1 μmol/l; Biomol) (3, 19, 47) or vehicle (dimethylsulfoxide). We previously reported that these concentrations of HA-1077 (3) and GF-109203X (3, 19) selectively inhibit ROCK and PKC, respectively, in this preparation.

To verify that L-type Ca²⁺ channels do not contribute to KCl-induced constriction in Ca²⁺-permeabilized arteries, parallel experiments were conducted in arteries pretreated with the L-type Ca²⁺ channel inhibitor diltiazem (50 μmol/l) or vehicle (PSS). This concentration of diltiazem inhibits KCl-mediated increases in VSM [Ca²⁺]_i in this preparation (17).

The role of O₂⁻ in mediating KCl-dependent Ca²⁺ sensitization was determined in Ca²⁺-permeabilized small pulmonary arteries from control and CH rats. Vasoconstrictor responses to increasing concentrations of KCl (30–120 mmol/l) were assessed in arteries in the presence of the O₂⁻ scavenger tiron (10 mmol/l; Sigma), a combination of tiron and HA-1077 (10 mmol/l and 10 μmol/l, respectively), or vehicle (PSS). This concentration of tiron inhibits xanthine oxidase-induced ROS generation in isolated pulmonary arteries, as determined in previous studies by our laboratory (18).

Effects of CH and ROCK inhibition on basal VSM E_m and KCl-mediated depolarization.

These experiments were performed to determine whether pulmonary VSM E_m is persistently depolarized in CH arteries compared with control vessels after administration of depolarizing concentrations of KCl and to assess potential influences of HA-1077 on basal E_m. The endothelium of arteries from each group was disrupted, pressurized, permeabilized to Ca²⁺, and prepared for experimentation as described above. VSM E_m was assessed using sharp electrodes under basal conditions and after administration of KCl (30 and 60 mmol/l). Basal VSM E_m was measured in the presence or absence of HA-1077 (10 μmol/l) in similar experiments.

Effects of CH and ROCK inhibition on KCl-induced O₂⁻ generation.

To assess the association between depolarization-induced RhoA/ROCK signaling and O₂⁻ levels, we evaluated the effects of KCl on DHE oxidation products by fluorescence microscopy as a measure of O₂⁻ generation in Ca²⁺-permeabilized arteries from control and CH rats. After washout of DHE, KCl (60 mmol/l) was added to the superfusate, and DHE fluorescence was measured over a 12-min period. In separate experiments, vessels were pretreated with tiron (10 mmol/l) before administration of KCl to verify the contribution of O₂⁻ to the fluorescent signal. In parallel protocols, we measured KCl-induced changes in DHE fluorescence intensity after administration of HA-1077 (10 μmol/l) or vehicle in arteries from CH rats.

Western Blotting: Effects of CH and O₂⁻ on Depolarization-Mediated RhoA Activation

Intrapulmonary arteries from control and CH rats were dissected from accompanying airways and surrounding lung tissue in a HEPES-based PSS containing (in mmol/l) 130 NaCl, 4 KCl, 1.2 MgSO₄, 4 NaHCO₃, 1.8 CaCl₂, 10 HEPES, 1.18 KH₂PO₄, 6 glucose, and 0.03 EDTA, with pH adjusted to 7.4 with NaOH. Arteries were incubated at 37°C for 30 min with ionomycin (3 μmol/l) to clamp VSM [Ca²⁺]_i, stimulated with KCl (60 mmol/l) or vehicle for 5 min, and then snap-frozen in liquid N₂. Separate sets of arteries were treated with KCl (60 mmol/l) in the presence of tiron (10 mmol/l) or vehicle for analysis of O₂⁻-dependent activation of RhoA. Each sample was homogenized in 10 mmol/l Tris·HCl homogenization buffer containing 255 mmol/l sucrose, 2 mmol/l EDTA, 12 μmol/l leupeptin, 1 μmol/l pepstatin A, 0.3 μmol/l aprotinin, and 1 mmol/l phenylmethylsulfonyl fluoride (Sigma). Samples were centrifuged at 10,000 g for 10 min at 4°C to remove insoluble debris. The supernatant was collected, and sample protein concentrations were determined by the Bradford method (Bio-Rad Protein Assay). RhoA activity was assessed using a Rho activation assay kit (Cytoskeleton) that detects levels of GTP-bound RhoA, as previously reported (19, 20). Levels of GTP-bound RhoA were normalized to levels of total RhoA protein determined from separate Western blots (25 μg/lane).

Calculations and Statistics

Pulmonary vascular resistance in isolated lung studies was calculated as the difference between arterial and venous pressure divided by flow. Vasoconstrictor responses to KCl were calculated as a change in resistance from baseline. Vasoconstrictor responses in isolated arteries were calculated as a percentage of baseline ID. VSM [Ca²⁺]_i is represented as ratio of emission at 340 nm to emission at 380 nm for fura 2 (F₃₄₀/F₃₈₀). GTP-bound RhoA was normalized to total RhoA expression for each sample. Values are means ± SE; n refers to the number of animals in each group. A t-test, two-way ANOVA, or two-way repeated-measures ANOVA was used to make comparisons when appropriate. If differences were detected by ANOVA, individual groups were compared with the Student-Newman-Keuls test. P < 0.05 was accepted as significant for all comparisons.

RESULTS

CH rats exhibited polycythemia, as indicated by a significantly greater hematocrit (66.2 ± 0.4%, n = 28) than in normoxic control rats (45.8 ± 0.5%, n = 29). This model of CH additionally produces many other cardiopulmonary changes observed in chronic obstructive pulmonary disease and prolonged residence at high altitude, including right ventricular hypertrophy, pulmonary hypertension, and arterial remodeling (38).

CH Enhances Depolarization-Induced Vasoconstriction in Isolated Lungs but Attenuates the VSM [Ca²⁺]_i Response to KCl in Small Pulmonary Arteries

Basal vascular resistances were significantly elevated in lungs isolated from CH rats compared with normoxic controls (see supplemental Table 1S in the online version of this article), as previously reported (38). Vasoconstrictor responses to KCl were greater in isolated, saline-perfused lungs from CH rats than in control lungs (Fig. 1A). Because pulmonary vasoreactivity to KCl may be modified by endothelial factors, we additionally examined vasoconstriction to KCl in isolated lungs pretreated with the NO synthase inhibitor l-NNA and the COX inhibitor meclofenamate. Similar to effects of CH in untreated lungs, vasoconstrictor responses to KCl were greater in lungs from CH than control rats after combined NO synthase and COX inhibition (see supplemental Fig. 1S).

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.

To assess effects of CH on VSM reactivity and Ca²⁺ mobilization in response to a depolarizing stimulus, we additionally performed concentration-response curves to KCl in fura 2-loaded small pulmonary arteries from each group. All arteries in these and subsequent protocols were studied at 12 Torr transmural pressure, unless otherwise noted, and the endothelium was disrupted to eliminate influences of endothelium-derived factors on depolarization-induced intracellular Ca²⁺ mobilization and vasoconstriction. Basal ID and VSM [Ca²⁺]_i were similar between groups (see supplemental Table 2S). VSM [Ca²⁺]_i responses to KCl were markedly blunted in arteries from CH rats compared with controls, despite similar or moderately increased vasoconstriction in CH arteries (Fig. 1B), indicating that CH increases depolarization-induced VSM Ca²⁺ sensitization.

CH Augments E_m Depolarization-Induced Vasoconstriction Independent of Changes in VSM [Ca²⁺]_i

In further experiments, we assessed concentration-response relationships to KCl in arteries in which VSM [Ca²⁺]_i had been clamped using the Ca²⁺ ionophore ionomycin to directly evaluate whether CH augments depolarization-induced myofilament Ca²⁺ sensitization through a Ca²⁺-independent signaling pathway. Figure 2A depicts traces of ID and the ratio of emission at 340 nm to emission at 380 nm vs. increasing concentrations of KCl from a Ca²⁺-permeabilized CH artery. Switching from superfusion with Ca²⁺-free PSS to PSS containing 300 nmol/l Ca²⁺ produced an increase in VSM [Ca²⁺]_i [from 0.576 ± 0.035 (control, n = 34) to 0.609 ± 0.027 (CH, n = 33)] and a modest vasoconstriction [18.7 ± 3.2% (control, n = 34) and 20.6 ± 2.5% (CH, n = 33)], resulting in similar ID and [Ca²⁺]_i between groups (see supplemental Table 2S). After stabilization of vessel ID and VSM [Ca²⁺]_i, a concentration-response curve to KCl was performed. Interestingly, KCl caused constriction without increasing VSM [Ca²⁺]_i (Fig. 2, A and B), thus providing direct evidence that E_m depolarization mediates vasoconstriction in hypertensive pulmonary arteries through a Ca²⁺-independent signaling mechanism. Consistent with enhancement of myofilament Ca²⁺ sensitivity in nonpermeabilized arteries by CH (Fig. 1B), KCl-induced constriction was greater in CH than control arteries (Fig. 2B, top), whereas VSM [Ca²⁺]_i remained stable in both groups (Fig. 2B, bottom). Similar results were obtained from arteries pressurized to 35 Torr (see supplemental Fig. 2S).

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.

Increased Depolarization-Induced VSM Ca²⁺ Sensitization Following CH Is Dependent on ROCK, but not PKC or L-Type Ca²⁺ Channels

Because depolarization stimulates RhoA/ROCK signaling independent of changes in [Ca²⁺]_i in renal tubular epithelium (46), we next evaluated the contribution of ROCK to KCl-induced vasoconstriction in isolated lungs and nonpermeabilized arteries. Whereas the selective ROCK inhibitor HA-1077 modestly attenuated reactivity to KCl in control lungs, vasoconstriction was nearly abolished after ROCK inhibition in lungs from CH rats (Fig. 3A). Parallel responses to ROCK inhibition were noted in l-NNA- and meclofenamate-pretreated lungs from each group (see supplemental Fig. 1S).

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.

A similar inhibitory influence of HA-1077 on depolarization-induced vasoconstriction was demonstrated in nonpermeabilized arteries from CH rats (Fig. 3B), although considerable VSM tone remained after ROCK inhibition. Furthermore, in contrast to the response to ROCK inhibition in isolated lungs (Fig. 3A), HA-1077 had no effect on reactivity to KCl in arteries from control rats. These findings support a pivotal role for ROCK in mediating enhanced KCl-mediated vasoconstriction in the hypertensive pulmonary circulation.

To determine the contribution of ROCK and PKC to increased depolarization-induced VSM Ca²⁺ sensitization following CH, we examined effects of HA-1077 or the broad-spectrum PKC antagonist GF-109203X (30, 47) on KCl-mediated vasoconstriction in Ca²⁺-clamped arteries. In previous studies, we verified the efficacy and specificity of GF-109203X for PKC vs. ROCK in this preparation (3, 20). HA-1077 significantly attenuated vasoconstrictor responses to KCl only in arteries from CH rats (Fig. 4A), effectively normalizing reactivity to that of control arteries. In contrast, GF-109203X did not alter constriction to KCl in arteries from control or CH rats (Fig. 4B), suggesting that PKC does not contribute to enhanced depolarization-induced VSM Ca²⁺ sensitization following CH. As expected in Ca²⁺-clamped arteries, blockade of L-type Ca²⁺ channels with diltiazem was also without effect on vasoconstriction to KCl in both groups (Fig. 4C).

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.

VSM E_m Is Depolarized in CH Compared With Control Arteries, and This Separation of E_m Persists After Exposure to Depolarizing Concentrations of KCl

VSM cell basal E_m was depolarized in pressurized, Ca²⁺-permeabilized small pulmonary arteries from CH animals relative to control vessels, as previously reported in nonpermeabilized arteries (Fig. 5A) (35). Furthermore, this separation of VSM E_m between control and CH vessels was maintained during exposure to depolarizing concentrations of KCl.

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.

VSM Cell E_m Is Unaltered by ROCK Inhibition

Although ROCK has been reported to cause VSM E_m depolarization through inhibition of voltage-sensitive K⁺ channels (29), we observed no effect of HA-1077 on VSM E_m in pressurized, Ca²⁺-permeabilized pulmonary arteries from either group (Fig. 5B). These data suggest that inhibitory effects of HA-1077 on KCl-induced Ca²⁺ sensitization are not related to alterations in E_m.

CH Augments Depolarization-Induced RhoA Activation in Pulmonary Arteries

To determine whether RhoA activation induces depolarization-dependent ROCK signaling in pulmonary arteries from CH rats, we assessed effects of KCl (60 mmol/l) on levels of GTP-bound (activated) RhoA in Ca²⁺-permeabilized pulmonary arteries from control and CH rats. Total RhoA expression was unaltered by CH (Fig. 6A), as demonstrated previously (20). Although we observed a tendency for CH to elevate basal RhoA activity, as described in earlier studies (20), this did not achieve statistical significance (Fig. 6B). Consistent with vasoactive responses to KCl in pressurized arteries (Fig. 4A), KCl increased GTP-bound RhoA levels in arteries from CH, but not control, rats (Fig. 6B). Because VSM [Ca²⁺]_i was clamped in these experiments, our results demonstrate that depolarization stimulates RhoA through a Ca²⁺-independent signaling pathway in arteries from pulmonary hypertensive rats.

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.

O₂⁻ Mediates Enhanced Depolarization-Induced VSM Ca²⁺ Sensitization Following CH

Since CH increases pulmonary arterial O₂⁻ generation (11, 20, 27, 56) and O₂⁻ has been implicated in RhoA activation in the systemic (21, 22) and pulmonary (20, 24) circulations, we next used isolated lungs and Ca²⁺-clamped pulmonary arteries to examine whether O₂⁻ mediates CH-induced increases in vasoreactivity to KCl. Consistent with this possibility, the O₂⁻-specific spin trap tiron attenuated vasoconstriction to KCl in isolated lungs from CH, but not control, animals, thus normalizing reactivity between groups (Fig. 7A).

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.

Similar to effects of ROCK inhibition (Fig. 4A), tiron significantly inhibited KCl-mediated vasoconstriction in Ca²⁺-permeabilized CH arteries, but not control vessels (Fig. 7, B and C). However, the combination of tiron and HA-1077 did not provide greater attenuation of KCl-dependent reactivity than scavenging of O₂⁻ alone, again resulting in similar vasoconstriction between groups.

CH Increases Basal and KCl-Stimulated O₂⁻ Levels in Pressurized Pulmonary Arteries

Basal DHE fluorescence was significantly greater in pressurized, Ca²⁺-permeabilized arteries from CH rats than controls (Fig. 8, A and B). Interestingly, KCl (60 mmol/l) markedly increased DHE fluorescence in arteries from CH rats but had no effect in control vessels. As expected, tiron abolished the CH-induced elevation of basal and KCl-stimulated O₂⁻ production without altering DHE fluorescence in arteries from normoxic rats. Control experiments revealed no time-dependent changes in fluorescence in CH arteries (see supplemental Fig. 3SA). ROCK inhibition with HA-1077 had no effect on basal or KCl-induced O₂⁻ generation in CH arteries (see supplemental Fig. 3SB), which argues against a potential role for ROCK in enhancement of basal O₂⁻ levels or depolarization-dependent Ca²⁺ sensitization through stimulated O₂⁻ production.

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.

Contribution of O₂⁻ to KCl-Stimulated RhoA Activity in Pulmonary Arteries

We next sought to further define the relationship between O₂⁻ and RhoA activation following CH by assessing effects of tiron on KCl-induced RhoA activation in pulmonary arteries from control and CH rats. In accordance with the inhibitory influence of O₂⁻ scavenging on ROCK-dependent myofilament Ca²⁺ sensitization in CH arteries (Fig. 7C), tiron abolished the KCl-induced elevation of GTP-bound RhoA in pulmonary arteries from CH rats but had no effect in control arteries (Fig. 8C). These results provide direct evidence that depolarization-induced O₂⁻ generation elicits RhoA activation.

DISCUSSION

The major findings of this study are as follows: 1) E_m depolarization mediates constriction of small pulmonary arteries through a novel Ca²⁺-independent signaling pathway in VSM; 2) CH augments depolarization-induced myofilament Ca²⁺ sensitization through stimulation of RhoA/ROCK; 3) CH increases O₂⁻ generation in response to KCl in small pulmonary arteries; and 4) O₂⁻ mediates depolarization-dependent RhoA activation through a mechanism that does not involve Ca²⁺ as a second messenger. Collectively, these results indicate that CH increases E_m depolarization-induced Ca²⁺ sensitization in pulmonary VSM through O₂⁻-dependent activation of the RhoA/ROCK signaling cascade.

Studies of chronic ROCK inhibition reveal a major contribution of this enzyme to the development of pulmonary hypertension (9, 16). Furthermore, acute reversal of pulmonary arterial pressure and total pulmonary resistance following administration of ROCK inhibitors in CH rats (32, 34) suggests that vasoconstrictor responses involving ROCK signaling are of primary importance in mediating CH-induced pulmonary hypertension. Recent studies have begun to address the component mechanisms of this vasoconstriction, revealing that CH enhances spontaneous arterial tone and vasoconstriction to receptor-mediated agonists through a ROCK-dependent Ca²⁺ sensitization pathway in pulmonary VSM (2, 3, 20, 34, 54).

In addition to its role in augmentation of basal and agonist-induced vasoconstriction, CH has been demonstrated to increase reactivity to depolarizing concentrations of KCl (2, 14, 34), although this is not a universal finding (35, 42). We have confirmed that CH enhances KCl-dependent vasoconstriction in isolated perfused lungs. However, consistent with earlier results from endothelium-intact pulmonary arteries (35), KCl-dependent increases in pulmonary VSM [Ca²⁺]_i were markedly blunted after CH in endothelium-disrupted arteries. Although the mechanism by which CH impairs K⁺-mediated increases in VSM [Ca²⁺]_i is not clear, these findings nevertheless implicate myofilament Ca²⁺ sensitization in mediating vasoconstriction to a depolarizing stimulus in hypertensive arteries.

A contribution of ROCK to depolarization-induced vasoconstriction after CH is supported by our findings that HA-1077 markedly blunted responsiveness to KCl in isolated lungs as well as pulmonary arteries from CH animals. Although we cannot exclude potential nonspecific actions of HA-1077 in this preparation, the specificity of this compound at the concentration used in the present study (10 μmol/l) is supported by evidence that the potency of HA-1077 for inhibition of ROCKI and ROCKII (IC₅₀ = 1.2 and 0.82 μmol/l, respectively) is ∼30–100 times greater than for inhibition of PKC or MLC kinase (5, 26, 40, 49). Further supporting the specificity of HA-1077 for ROCK vs. PKC is our recent finding that 10 μmol/l HA-1077 does not alter vasoconstriction to the PKC agonist PMA in isolated pulmonary arteries (3). Inhibitory effects of HA-1077 on depolarization-induced constriction in hypertensive pulmonary arteries are also consistent with our finding that KCl preferentially activates RhoA in these same vessels, thus supporting a role for RhoA/ROCK signaling in the enhanced vasoconstrictor reactivity following CH.

In is noteworthy that KCl-dependent vasoconstriction was more sensitive to ROCK inhibition in isolated lungs than in endothelium-disrupted arteries in each group of rats. Potentiation of ROCK-mediated vasoconstriction to depolarizing stimuli by the epithelium may account for these differential responses between preparations and could explain our present findings that CH more potently augmented KCl-dependent vasoconstriction in isolated lungs than in endothelium-disrupted pulmonary arteries. Consistent with this possibility are previous studies indicating that vasoreactivity to KCl is attenuated by inhibitors of ROCK in isolated perfused lungs (9, 14, 34) and endothelium-intact arteries from CH rats (14, 34, 54), whereas no such inhibition was observed in endothelium-denuded vessels from these animals (2, 54). ROCK additionally contributes to endothelium-dependent contractions in rat aorta (4). Furthermore, Homma and colleagues (14) found that the augmented KCl-induced vasoconstriction in lungs from pulmonary hypertensive rats was reduced by endothelin or serotonin receptor antagonism, suggesting that endogenous endothelin-1 and serotonin mediate enhanced KCl-dependent Ca²⁺ sensitization following hypoxic acclimation. Nevertheless, our observation that HA-1077 attenuated reactivity to KCl in endothelium-disrupted arteries from CH, but not control, rats indicates that CH plays an additional role in the increase in depolarization-induced VSM Ca²⁺ sensitization through a mechanism inherent to the VSM. Therefore, the major objective of the present study was to evaluate VSM signaling mechanisms responsible for this induction of ROCK-mediated vasoconstriction following CH.

Whereas RhoA stimulation in VSM is well established to occur secondary to activation of G protein-coupled receptors (45), ROCK-mediated VSM Ca²⁺ sensitization can also be elicited by depolarizing stimuli (33, 41, 50), presumably via Ca²⁺-dependent stimulation of RhoA (33, 41, 50) (i.e., Ca²⁺-induced Ca²⁺ sensitization). However, depolarization has been demonstrated to activate this pathway via a novel Ca²⁺-independent mechanism in renal tubular epithelium (46). In the present study, we addressed the hypothesis that a similar mechanism of RhoA activation exists in VSM that contributes to enhanced depolarization-induced pulmonary vasoconstriction following CH. We tested this hypothesis by assessing KCl-mediated constriction in arteries in which VSM [Ca²⁺]_i had been clamped with the Ca²⁺ ionophore ionomycin (20). This approach eliminates any contribution of Ca²⁺ mobilization/influx to depolarization-induced vasoconstriction and controls for previously reported effects of ROCK inhibitors to lower VSM [Ca²⁺]_i (53). Our finding that KCl elicited constriction in Ca²⁺-permeabilized arteries provides direct support for membrane depolarization-induced vasoconstriction independent of changes in VSM [Ca²⁺]_i. In addition, this response to KCl was greater in CH than control arteries, indicating that CH augments depolarization-mediated vasoconstriction through a previously undescribed Ca²⁺-independent signaling pathway in VSM. Whereas this response to depolarization was independent of ROCK in normotensive pulmonary arteries, HA-1077-mediated attenuation of KCl-dependent vasoconstriction and normalization of reactivity between vessels from normoxic and CH rats demonstrate a primary role for ROCK signaling in enhanced depolarization-induced vasoconstriction following CH. Interestingly, substantial vasoconstriction remained after ROCK or PKC inhibition in both groups, suggesting that additional Ca²⁺ sensitization pathways (13) or, perhaps, actin polymerization (12) contributes to depolarization-dependent constriction in the pulmonary circulation. However, inasmuch as we cannot be certain that GF-109203X effectively inhibited all isoforms of PKC, we cannot exclude a potential role for PKC-dependent Ca²⁺ sensitization in mediating the persistent vasoconstriction in the presence of GF-109203X or HA-1077. RhoA activity assays in Ca²⁺-permeabilized arteries provided direct evidence that CH promotes Ca²⁺-independent, depolarization-induced RhoA activation. Although such RhoA activation could result from L-type Ca²⁺ channels acting as metabotropic receptors linking depolarization to stimulation of a heterotrimeric G protein, as described in basilar arterial myocytes (51), this is not a likely explanation, since L-type Ca²⁺ channel inhibition was without effect on KCl-mediated Ca²⁺ sensitization in the present study. Collectively, our present observations demonstrate that CH increases depolarization-induced Ca²⁺ sensitization in pulmonary VSM through stimulation of RhoA signaling. Furthermore, activation of RhoA by membrane depolarization can occur through a distinct Ca²⁺-independent mechanism in hypertensive arteries, similar to that previously described in renal tubular epithelial cells (46). If we consider that isolated pulmonary arteries exhibit pressure-dependent depolarization that is augmented following CH (35), such a mechanism of depolarization-induced RhoA activation could contribute to the ROCK-dependent myogenicity observed in these vessels (3).

Although ROS generation has been reported to be reduced in isolated lungs from CH rats (37), substantial evidence supports a contribution of ROS to enhanced agonist-mediated pulmonary vasoconstriction and pulmonary hypertension following CH (8, 11, 15, 20, 27). However, few studies have investigated a potential link between ROS and VSM Ca²⁺ sensitivity in this response. This idea is supported by evidence that O₂⁻ mediates ROCK-dependent VSM contraction in ductus arteriosus (22), aorta (21), and pulmonary arteries (20, 24). Furthermore, a mechanistic relationship between endothelin receptor stimulation, O₂⁻ generation, and RhoA-induced myofilament Ca²⁺ sensitization was recently demonstrated in hypertensive rat pulmonary arteries (20). Depolarizing stimuli have additionally been associated with increased ROS generation in endothelial cells, macula densa, and isolated mouse and rat lungs (1, 44). Thus it is feasible that O₂⁻ functions as an intracellular second messenger linking pulmonary VSM depolarization to RhoA activation and associated vasoconstriction.

The effect of CH in promotion of depolarization-dependent O₂⁻ generation in the present study appears to be a key determinant of enhanced pulmonary VSM Ca²⁺ sensitivity and associated vasoconstriction following CH, since the O₂⁻-specific spin trap tiron selectively attenuated KCl-induced vasoconstriction in isolated lungs and Ca²⁺-clamped pulmonary arteries from CH rats. Although the effect of tiron to inhibit vasoconstriction in isolated lungs from CH rats could reflect an increase in NO bioavailability by limiting O₂⁻-dependent NO scavenging, this seems unlikely, when we consider that a similar response was not observed in control lungs and that tiron has no effect on endothelium-derived NO-dependent vasodilation in isolated lungs from control or CH rats ventilated with normoxic gas (18). Our finding that the combination of O₂⁻ and ROCK did not provide greater inhibition of KCl-dependent reactivity than scavenging of O₂⁻ alone provides additional evidence that O₂⁻ signals in series with RhoA/ROCK to increase myofilament Ca²⁺ sensitivity in CH arteries. The causal link between depolarization-induced O₂⁻ generation and RhoA stimulation is demonstrated by the effect of tiron to selectively abolish the KCl-dependent elevation of RhoA activity in pulmonary arteries from CH rats. However, the distal signaling mechanisms by which O₂⁻-dependent RhoA activation leads to vasoconstriction remain to be established. Whereas RhoA may signal through the well-established pathway of ROCK-dependent inhibition of MLC phosphatase, it is additionally possible that this response is mediated by direct effects of ROCK to phosphorylate the MLC or, rather, to activate other Ca²⁺-independent MLC kinases, including ZIP kinase or integrin-linked kinase, to increase myofilament Ca²⁺ sensitivity (13). An alternative possibility is that RhoA activation promotes actin polymerization and the formation of stress fibers to mediate contraction (12) and, thus, does not represent a classical Ca²⁺ sensitization phenomenon. Although this study also does not address whether O₂⁻ per se or, rather, a product of O₂⁻ metabolism (e.g., H₂O₂ or hydroxyl radical) is directly responsible for RhoA activation, earlier studies have indicated an exclusive role for O₂⁻ in this response (21, 24). These results, together with data from Ca²⁺-permeabilized arteries and DHE experiments, support a novel link between membrane depolarization, O₂⁻ generation, and RhoA/ROCK signaling that mediates enhanced myofilament Ca²⁺ sensitivity in the hypertensive pulmonary circulation. These findings may additionally establish a mechanistic basis for previous studies that have established a crucial role for ROS in the development of hypoxic pulmonary hypertension (8, 15).

Our present finding that CH causes basal E_m depolarization in endothelium-disrupted, Ca²⁺-permeabilized pulmonary arteries is consistent with previous results from isolated, endothelium-intact arteries (35) and isolated pulmonary arterial myocytes (36, 42, 43). Interestingly, we found that this separation of VSM E_m between control and CH vessels persisted with increasing concentrations of KCl, presumably because of decreased membrane K⁺ permeability associated with downregulation of VSM K⁺ channels following CH (36, 37). Although these results may account for greater depolarization-induced VSM Ca²⁺ sensitization following CH, an additional possibility is that CH augments KCl-induced vasoconstriction for a given level of E_m. Indeed, our observation that KCl increased DHE fluorescence only in CH arteries suggests that the mechanism linking depolarization to O₂⁻ generation and RhoA activation is not functional in control tissue but, rather, reflects a pathophysiological response to CH. Whether this mechanism results from an increase in expression or functional coupling of a O₂⁻-generating enzyme to E_m or, rather, a reduction of antioxidant system activity (e.g., superoxide dismutase) remains to be determined.

In conclusion, this study establishes a novel signaling axis involving Ca²⁺-independent E_m depolarization and O₂⁻-induced activation of the RhoA/ROCK pathway in pulmonary VSM. Future studies will be aimed at identifying the contribution of depolarization-induced ROS generation to myogenic and receptor-mediated vasoconstriction in the hypertensive pulmonary vasculature, establishing the source(s) of O₂⁻ that mediates enhanced RhoA-dependent Ca²⁺ sensitization following CH, and determining the signaling mechanism linking membrane depolarization to RhoA stimulation. Such a mechanism may involve the influence of CH on the activity or cellular trafficking of components of the ROS-RhoA signaling axis through alterations in the composition of signaling platforms within the lipid bilayer or possible effects of E_m to directly regulate enzymatic activity of relevant second messengers. The concept of depolarization as a Ca²⁺-independent effector of ROS signaling has potentially broad physiological implications for not only RhoA-induced VSM contraction, but also for regulation of cytoskeletal organization, motility, proliferation, and apoptosis in pulmonary hypertension.

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grants HL-92598 (N. L. Jernigan), HL-77876 and HL-88192 (T. C. Resta), and HL-58124 and HL-07736 (B. R. Walker) and American Heart Association Grants 0755775Z (T. C. Resta) and 0625647Z (B. R. S. Broughton).

DISCLOSURES

No conflicts of interest are declared by the authors.