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Thiol oxidation causes pulmonary vasodilation by activating K⁺ channels and inhibiting store-operated Ca²⁺ channels

Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of California, San Diego, La Jolla, California

Submitted 21 July 2006 ; accepted in final form 8 November 2006

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Cellular redox change regulates pulmonary vascular tone by affecting function of membrane and cytoplasmic proteins, enzymes, and second messengers. This study was designed to test the hypothesis that functional modulation of ion channels by thiol oxidation contributes to regulation of excitation-contraction coupling in isolated pulmonary artery (PA) rings. Acute treatment with the thiol oxidant diamide produced a dose-dependent relaxation in PA rings; the IC₅₀ was 335 and 58 µM for 40 mM K⁺- and 2 µM phenylephrine-induced PA contraction, respectively. The diamide-mediated pulmonary vasodilation was affected by neither functional removal of endothelium nor 8-bromoguanosine-3'-5'-cyclic monophosphate (50 µM) and HA-1004 (30 µM). A rise in extracellular K⁺ concentration (from 20 to 80 mM) attenuated the thiol oxidant-induced PA relaxation. Passive store depletion by cyclopiazonic acid (50 µM) and active store depletion by phenylephrine (in the absence of external Ca²⁺) both induced PA contraction due to capacitative Ca²⁺ entry. Thiol oxidation by diamide significantly attenuated capacitative Ca²⁺ entry-induced PA contraction due to active and passive store depletion. The PA rings isolated from left and right PA branches appeared to respond differently to store depletion. Although the active tension induced by passive store depletion was comparable, the active tension induced by active store depletion was 3.5-fold greater in right branches than in left branches. These data indicate that thiol oxidation causes pulmonary vasodilation by activating K⁺ channels and inhibiting store-operated Ca²⁺ channels, which subsequently attenuate Ca²⁺ influx and decrease cytosolic free Ca²⁺ concentration in pulmonary artery smooth muscle cells. The mechanisms involved in thiol oxidation-mediated pulmonary vasodilation or activation of K⁺ channels and inhibition of store-operated Ca²⁺ channels appear to be independent of functional endothelium and of the cGMP-dependent protein kinase pathway.

diamide; pulmonary artery; smooth muscle contractility; pulmonary hypertension; capacitative calcium entry; redox status

One of the unique properties of the pulmonary vasculature is the vasoconstrictive response to acute and chronic alveolar hypoxia, which is opposite of the vasodilating response of systemic vessels to hypoxia and ischemia (37, 44, 68, 81). Although the cellular and molecular mechanisms involved in hypoxic pulmonary vasoconstriction remain unclear, the redox status change in pulmonary vascular smooth muscle and endothelial cells has been implicated in regulating pulmonary vascular tone via modulation of [Ca²⁺]_cyt in PASMC (64, 71). Hypoxia may have multiple effects on cellular redox status depending on the basal status of PASMC, participating regulators and effectors in PASMC (e.g., different ion channels and receptors in the plasma membrane, capacity of intracellular Ca²⁺ stores), and interaction between smooth muscle cells and endothelial cells (1, 32).

Experiments in vitro and in vivo with isolated pulmonary arteries (PA), isolated perfused lungs, and intact animals have well demonstrated that an increase in [Ca²⁺]_cyt in PASMC is a major trigger for smooth muscle contraction and pulmonary vasoconstriction (11, 40, 67). Inhibition of Ca²⁺ influx with Ca²⁺ channel blockers (e.g., nifedipine, verapamil) improves hemodynamics in patients with pulmonary hypertension (55, 66, 72). When [Ca²⁺]_cyt rises in PASMC, Ca²⁺ binds to calmodulin, which activates myosin light-chain kinase to phosphorylate the myosin light chain. This phosphorylation increases the activity of myosin ATPase that hydrolyzes ATP, thereby releasing energy. The subsequent cycling of the myosin cross-bridges produces displacement of the myosin filament in relation to the actin filament-causing contraction (61). The excitation-contraction coupling in vascular smooth muscle takes place through two major mechanisms: 1) electromechanical coupling, the mechanism that causes contraction or relaxation through changes in membrane potential (E_m), and 2) pharmacomechanical coupling, the complex of mechanisms that can cause contraction or relaxation by mechanisms not mediated by changes in E_m (21, 62).

PASMC express multiple Ca²⁺ channels that open in response to electrical and pharmacological stimulations to increase [Ca²⁺]_cyt. There are at least three functionally distinct Ca²⁺ channels in PASMC (25, 45): 1) voltage-dependent Ca²⁺ channels (VDCC) that are activated by membrane depolarization (e.g., by raising extracellular K⁺ or inhibiting K⁺ channel activity), 2) receptor-operated Ca²⁺ channels (ROC) that are opened by agonist- or mitogen-mediated receptor activation and synthesis of phospholipase C and diacylglycerol, and 3) store-operated Ca²⁺ channels (SOC) that are opened by active and passive depletion of intracellular Ca²⁺ stores, such as the sarcoplasmic reticulum (SR) or endoplasmic reticulum.

This study was designed to investigate whether oxidation of pulmonary vascular smooth muscle and endothelial cells by the thiol oxidant diamide affects electromechanical and pharmacomechanical coupling in isolated PA and what mechanisms are potentially involved in the thiol oxidant-mediated regulation of pulmonary vascular tone.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Isolation of PA rings and tension measurement. Use of animal tissues for this study was approved by the Institutional Review Board at the University of California, San Diego. The right and left branches (2nd division) of the main PA and the intrapulmonary arteries (3rd and 4th division) were isolated from male Sprague-Dawley rats (100–250 g). The adipose, connective tissues, and adventitia were carefully removed, and the remaining muscular arteries were cut into 2-mm-long rings. For some of the experiments, the endothelium of PA rings was removed by gently rubbing the inner lumen of the vessels with a rough wooden stick. Functional removal of the endothelium was confirmed by the loss of relaxant response of PA rings to ACh (10 µM). This procedure did not damage the vessels because phenylephrine (PE)- or high K⁺-mediated active tension was actually enhanced in PA rings with denuded endothelium.

Two stainless steel hooks (0.1 mm in diameter) were inserted through the lumen of PA rings. One hook was mounted in a perfusion chamber (1 ml in volume), and the other hook was connected to an isometric force transducer (Harvard Apparatus). Isometric tension was continuously monitored and recorded with DATAQ data acquisition software (DATAQ Instruments). Resting passive tension, i.e., that offering maximal tension in rings exposed to 40 mM K⁺ (40K), was 600 mg. The rings were equilibrated for 1 h at resting (or basal) tension and then challenged three times with 40K perfusate to obtain a stable contractile response. After experiments, each PA ring was weighed using a fine balance. The active tension (mg) was normalized by wet tissue weight (mg) and expressed in milligram tension per milligram weight (mg/mg).

Isolated PA rings were superfused with modified Krebs solution (MKS) consisting of (in mM) 138 NaCl, 1.8 CaCl₂, 4.7 KCl, 1.2 MgSO₄, 1.2 NaH₂PO₄, 5 HEPES, and 10 glucose (pH 7.4, at 37°C). In Ca²⁺-free solution, CaCl₂ was replaced by equimolar MgCl₂ and 1 mM EGTA was added to chelate residual Ca²⁺. In the high-K⁺ solution, NaCl was replaced by equimolar KCl to maintain osmolarity.

Cell preparation and culture. Rat PASMC were prepared from PA of male Sprague-Dawley rats. Briefly, the isolated PA were incubated for 20 min in HBSS containing 1.5 mg/ml collagenase (Worthington Biochemical, Lakewood, NJ). Adventitia and endothelium were carefully removed after the incubation. The remaining smooth muscle was digested for 45–50 min with 1.5 mg/ml collagenase and 0.5 mg/ml elastase (Sigma, St. Louis, MO) at 37°C. PASMC were sedimented by centrifugation, resuspended in fresh medium, and plated. In addition, the embryonic rat heart-derived myogenic cell line H9c2 (12), purchased from the American Type Culture Collection, was used for some experiments. The H9c2 cells were cultured in DMEM containing 10% FBS. Subconfluent H9c2 cells were then plated onto coverslips and cultured in 10% FBS-DMEM for 3–5 days before patch-clamp experiments.

Electrophysiological measurements. Whole cell K⁺ currents were recorded at room temperature (22–24°C) from PASMC with an Axopatch one-dimensional amplifier and a DigiData 1200 interface (Axon Instruments, Union City, CA). Cells were plated on glass coverslips, mounted on a plastic glass cell-perfusion chamber on a Nikon inverted microscope, and bathed in physiological saline solution. Borosilicate patch pipettes (2–4 M) were fabricated on a model P-97 electrode puller (Sutter Instruments, Novato, CA) and polished with a MF-63 microforge (Narashige Scientific Instruments Laboratories, Tokyo, Japan). Step-pulse protocols and data acquisition were performed with pCLAMP software. Currents were filtered at 1–2 kHz (–3 dB) and digitized at 2–4 kHz. Series resistance compensation was performed in all whole cell experiments. Leak and capacitative currents were subtracted with the P/4 protocol in pCLAMP software.

For the recording of optimal whole cell voltage-gated K⁺ (Kv) currents, cells were superfused with a standard extracellular solution containing (in mM) 141 NaCl, 4.7 KCl, 3.0 MgCl₂, 10 HEPES, 1 EGTA, and 10 glucose (pH 7.4). The pipette (intracellular) solution contained (in mM) 135 KCl, 4 MgCl₂, 10 HEPES, 10 EGTA, and 5 Na₂ATP (pH 7.2). Under these conditions, the contribution of ATP-sensitive K⁺ (K_ATP) channels and Ca²⁺-activated K⁺ (K_Ca) channels to the whole cell currents was minimized because of high concentrations of ATP (5 mM) and EGTA (10 mM) in the pipette (intracellular) solution.

Solutions and chemicals. Cyclopiazonic acid (CPA; Sigma), nifedipine (Sigma), and diamide (Sigma) were dissolved in DMSO to make stock solutions of 50, 100, and 500 mM, respectively; aliquots of the stock solutions were then diluted in MKS or Ca²⁺-free MKS on the day of use to their final concentrations. ACh (Sigma), 8-bromoguanosine-3'-5'-cyclic monophosphate (8-BrcGMP; Fluka), cromakalim (Sigma), N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride (HA-1004; Sigma), DL-DTT, 4-aminopyridine (Sigma), hydrogen peroxide (H₂O₂; Sigma), and PE (Sigma) were dissolved in distilled water to make stock solutions of 10–100 mM; aliquots of the stock solutions were then diluted in superfusate to various final concentrations for experimentation. pH values of solutions were measured after addition of the drugs and readjusted to 7.4.

Statistical analysis. Data are expressed as means ± SE. Statistical analysis was performed with the paired or unpaired Student's t-test or ANOVA and post hoc tests (Student-Newman-Keuls) as indicated. Differences were considered to be significant when P < 0.05.

	RESULTS

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Characterization of 40K- and PE-induced contraction in isolated rat PA rings. Raising extracellular K⁺ concentration ([K⁺]_o) from 4.7 to 40 mM (40K), by shifting the K⁺ equilibrium potential (E_K) from –85 to –31 mV and causing membrane depolarization in PASMC, increased active tension in isolated left and right branches of extrapulmonary arteries (Fig. 1A). Furthermore, extracellular application of the -adrenergic-receptor agonist PE (2 µM) also increased active tension in isolated PA rings (Fig. 1A). The 40K-induced active tension appeared to be slightly greater in the right branch (1,253.6 ± 54.8 mg/mg wt) than in the left branch (1,092.7 ± 61.2 mg/mg, n = 18; P = 0.06). However, the PE-mediated active tension, normalized as percentage of the amplitude of 40K-induced tension, was 15.6% smaller in the left branch (66.0 ± 1.9%) than in the right branch (78.2 ± 1.8%; n = 18, P < 0.001) (Fig. 1B).

View larger version (19K): [in this window] [in a new window]   Fig. 1. Comparison of amplitude of high K+- and phenylephrine (PE)-mediated vasoconstriction in left and right pulmonary artery (PA) branches with intact endothelium. A: representative tracings showing active tension induced by 40 mM K+ (40K) or 2 µM PE in left (top) and right (bottom) PA branches. B: summarized data showing amplitude of the 40K-induced active tension (left) and PE-induced active tension normalized to 40K-mediated tension (right). ***P < 0.001 vs. left (n = 18 rings).

View larger version (22K): [in this window] [in a new window]   Fig. 2. Excitation-contraction coupling in PA depends on Ca2+ influx. A: representative tracings (left) and summarized data (means ± SE; right) showing active tension induced by 40K before (Cont), during, and after (wash) removal of extracellular Ca2+ (0Ca; top) (n = 6) or addition of 0.1 µM nifedipine (Nif; bottom) (n = 12). ***P < 0.001 vs. control and washout. B: representative tracings (left) and summarized data (means ± SE; right) showing active tension induced by 2 µM PE before, during, and after removal of extracellular Ca2+ (top) (n = 6) or addition of 0.1 µM nifedipine (bottom) (n = 10). ***P < 0.001 vs. control and washout. All PA rings were obtained from right PA branches with intact endothelium.

In an isolated PA ring (from the rat main PA) superfused with a Ca²⁺-free solution, activation of the -adrenergic receptor by PE caused a transient contraction (Fig. 3A), which was caused by a rise in [Ca²⁺]_cyt resulting from Ca²⁺ mobilization from the SR. After 5–10 min of treatment with the agonist (PE) in the absence of extracellular Ca²⁺ (which would be sufficient to deplete Ca²⁺ from the SR), 1 µM of phentolamine was used to block -adrenergic receptors and to inhibit ROC. Restoration of extracellular Ca²⁺ under these conditions (i.e., the intracellular store is depleted because of PE-mediated Ca²⁺ release, and the receptors and ROC are both inactivated by phentolamine) caused a contraction due to CCE through SOC. The CCE-mediated contraction (Fig. 3A, shaded area) accounted for 20–50% of total contraction induced by PE in the presence of extracellular Ca²⁺. Removal of phentolamine, or reactivation of the receptor and ROC, caused a further contraction caused by Ca²⁺ entry through ROC (Fig. 3A). These results indicate that the rise in [Ca²⁺]_cyt responsible for agonist-mediated PA contraction results from three major sources: Ca²⁺ release from intracellular store (mainly IP₃-sensitive SR), Ca²⁺ influx through ROC, and Ca²⁺ influx through SOC (or CCE).

View larger version (21K): [in this window] [in a new window]   Fig. 3. Pulmonary vasoconstriction induced by capacitative Ca2+ entry (CCE) due to active depletion of intracellular stores in different branches of PA. A and B: representative tracings showing active tension induced by 2 µM PE in the presence and absence (0Ca) of extracellular Ca2+ in the main PA (A) and the left and right PA branches (B). Phentolamine (Phen, 1 µM), an -adrenergic-receptor blocker, was applied to the vessels after PE-mediated transient PA contraction and before restoration of extracellular Ca2+. Shadowed areas indicate the increase in active tension due to a rise in cytosolic free Ca2+ concentration ([Ca2+]cyt) via CCE. B: representative (Ba) and summarized data (Bb, means ± SE) showing the amplitude of PE-induced active tension due to Ca2+ release (in the absence of extracellular Ca2+) and to CCE (when extracellular Ca2+ was restored in the presence of phentolamine) in left and right PA branches (n = 12). *P < 0.05 vs. right branches. C: representative tracing showing PE-induced oscillatory contraction in a PA ring isolated from right PA branches.

Studies in vitro (28, 39, 70) have demonstrated that intracellular Ca²⁺ stores in PASMC can also be depleted by blockade of the SR Ca²⁺-Mg²⁺-ATPase (SERCA) using CPA (5–10 µM), which subsequently causes CCE or Ca²⁺ influx through SOC. The passive store depletion-mediated CCE elicited a transient contraction in isolated PA rings (right and left branches), whose amplitude was 30% of the 40K-induced contraction (Fig. 4A). Although the amplitude of CCE-mediated contraction because of active store depletion (by PE) differed dramatically between right and left branches, the amplitude of CCE-mediated PA contraction due to passive store depletion (by CPA) was comparable (Fig. 4).

View larger version (21K): [in this window] [in a new window]   Fig. 4. Comparison of CCE-mediated pulmonary vasoconstriction induced by active and passive store depletion in left and right PA branches. A: representative tracings showing tension changes induced by 40K, cyclopiazonic acid (CPA; 50 µM, in the presence and absence of extracellular Ca2+), and PE (2 µM, in the presence and absence of extracellular Ca2+) in left (top) and right (bottom) PA branches. Phentolamine (Phen, 1 µM) was applied to the vessels after PE-mediated transient PA contraction and before restoration of extracellular Ca2+. Shadowed areas indicate the increase in active tension due to a rise in [Ca2+]cyt via CCE mediated by CPA-induced passive store depletion and by PE-induced active store depletion. B: summarized data (means ± SE) showing the amplitude of CPA- and PE-induced active tension mediated by CCE (left; n = 12–17) and the amplitude of PE-induced active tension after treatment with phentolamine (right; n = 12) in left and right PA branches. **P < 0.01 and ***P < 0.001 vs. left branches.

The thiol oxidant diamide inhibits PA contraction induced by 40K and PE with different IC₅₀. In isolated PA rings (from right branches) preconstricted by 40K (Fig. 5A) or 2 µM PE (Fig. 5B), diamide (1–1,000 µM) caused a dose-dependent inhibition of the active tension. The summarized data shown in Fig. 5C indicate that the PE-mediated PA contraction was much more sensitive to diamide than the 40K-mediated contraction. The IC₅₀ of diamide was 335 µM for 40K-mediated contraction, whereas it was 58 µM for PE-induced PA contraction (Fig. 5C). These results suggest that diamide may affect 40K- and PE-induced PA contraction through different mechanisms.

View larger version (24K): [in this window] [in a new window]   Fig. 5. Dose-dependent relaxing effect of diamide on pulmonary vasoconstriction induced by 40K and PE (2 µM). A and B: representative tracings showing tension changes in response to 1–1,000 µM diamide in PA rings constricted by 40K (A) and PE (B). Diamide was applied to the vessels when 40K- and PE-mediated active tension reached plateau. C: dose-response curves (means ± SE) showing 40K- and PE-induced active tension (normalized to the maximal tension; n = 4) in PA rings (right branches) treated before and during treatment with 1, 3, 10, 30, 100, 300, and 1,000 µM diamide. *P < 0.05 and **P < 0.01 vs. PE.

View larger version (19K): [in this window] [in a new window]   Fig. 6. Thiol reductant reverses diamide-induced pulmonary vasodilation. A: representative tracing showing tension changes in a PA ring preconstricted by PE (2 µM) before, during, and after application of diamide (100 µM) alone or diamide + DTT (1 mM). B: summarized data (means ± SE) showing the amplitude of PE-induced active tension before (control) and during application of diamide or diamide + DTT (n = 8). **P < 0.01 vs. control and diamide + DTT.

View larger version (22K): [in this window] [in a new window]   Fig. 7. Diamide-mediated pulmonary vasodilation is not dependent of cGMP and protein kinase G (PKG). A: representative tracing showing tension changes when diamide (100 µM) was applied after superfusion with 8-bromoguanosine-3'-5'-cyclic monophosphate (8-BrcGMP; 50 µM) in a PA ring constricted by 2 µM PE. B: summarized data (means ± SE; n = 6) showing the amplitude of PE-induced active tension before (control), during, and after (washout) application of 8-BrcGMP or 8-BrcGMP and diamide (100 µM). C: representative tracing showing PE (2 µM)-mediated tension change before, during, and after application of 100 µM diamide in a PA ring treated with 30 µM HA-1004, an inhibitor of PKG/PKA. D: summarized data (means ± SE; n = 5) showing the amplitude of PE-induced active tension before, during, and after application of diamide in the presence of HA-1004.

Diamide-mediated PA relaxation depends on transmembrane K⁺ gradient. As shown in Fig. 8, elevation of [K⁺]_o from 25 mM (25K) to 80 mM (80K) increased the high-K⁺-induced active tension but markedly inhibited diamide-mediated PA relaxation. For example, diamide caused a 34.4 ± 3.3% reduction of active tension in PA rings preconstricted by 25K (P < 0.001) but only caused a 7.2 ± 1.6% reduction of active tension in PA constricted by 80K (Fig. 8, A and B). The dose-response curves of high K⁺-induced PA contraction (Fig. 8C, open circles) and diamide-mediated PA relaxation (Fig. 8C, solid circles) show that the amplitude of K⁺-induced contraction is inversely proportional to the amplitude of diamide-induced PA relaxation when [K⁺]_o increases from 20 to 80 mM. These results indicate that diamide-mediated pulmonary vasodilation depends on (or is regulated by) the concentration gradient of K⁺ across the plasma membrane. A decrease of the transmembrane K⁺ gradient (or the K⁺ driving force), for example, by raising [K⁺]_o, inhibits diamide-mediated PA contraction.

View larger version (26K): [in this window] [in a new window]   Fig. 8. Diamide-mediated pulmonary vasodilation is significantly inhibited by reducing transmembrane K+ gradient. A: representative tracings showing 100 µM diamide-mediated tension changes in PA rings constricted by 25 mM K+ (25K; left) and 80 mM K+ (80K; right). B: summarized data (means ± SE) showing the amplitude of 25K-mediated (left; n = 4) and 80K-mediated (right; n = 5) active tension before (control), during, and after (washout) extracellular application of 100 µM diamide. **P < 0.01 vs. control and washout. C: summarized data (means ± SE) showing the amplitude of active tension induced by raising extracellular K+ from 4.7 to 20, 25, 30, 35, 40, and 80 mM (; n = 4–8), respectively, and the percent reduction of corresponding active tension induced by 100 µM diamide (; n = 4–8).

View larger version (30K): [in this window] [in a new window]   Fig. 9. Diamide-mediated pulmonary vasodilation is not significantly changed in PA rings constricted with different concentrations of PE. A: representative tracings showing 100 µM diamide-mediated tension changes in PA rings constricted by 0.03 µM PE (left) and 100 µM PE (right). B: summarized data (means ± SE) showing the amplitude of 0.03 µM PE-mediated (left; n = 4) and 100 µM PE-mediated (right; n = 6) active tension before (control), during, and after (washout) extracellular application of 100 µM diamide. **P < 0.01 vs. control and washout. C: summarized data (means ± SE) showing the amplitude of active tension induced by 0.01, 0.03, 0.1, 1, 10, and 100 µM PE (; n = 4–7), respectively, and the percent reduction of corresponding active tension induced by 100 µM diamide (; n = 4–9).

View larger version (29K): [in this window] [in a new window]   Fig. 10. Pulmonary vasodilation induced by the K+ channel opener cromakalim is abolished by reducing transmembrane K+ gradient. A: representative tracings showing 1 µM cromakalim-mediated tension changes in PA rings constricted by 25K (left) and 80K (right). B: summarized data (means ± SE) showing the amplitude of 25K-mediated (left; n = 6) and 80K-mediated (right; n = 6) active tension before (control), during, and after (washout) extracellular application of cromakalim. ***P < 0.001 vs. control and washout.

View larger version (25K): [in this window] [in a new window]   Fig. 11. Thiol oxidants increase whole cell K+ currents. Aa: representative record of outward K+ current elicited by a test potential of +60 mV (holding potential, –70 mV) before (control) and after (diamide) extracellular application of 100 µM diamide in a rat pulmonary artery smooth muscle cells (PASMC). Ab: summarized data (means ± SE; n = 9 cells) showing the amplitudes of the transient (ITr) and steady-state (ISS) components of the currents at +60 mV in PASMC before (control) and after application of diamide. **P < 0.01 vs. control. Ba: representative currents elicited by a series of test potentials ranging from –60 to +80 mV in 20-mV increments (holding potential, –70 mV) before (control) and during extracellular application of hydrogen peroxide (H2O2, 50 µM) in the presence (H2O2+4-AP) or absence (H2O2) of 4-amynopyridine (4-AP; 5 mM) in a H9c2 cell. Bb: summarized data (means ± SE; n = 7 cells) showing the current-voltage (I-V) relationship curves in H9c2 cells before (control; ) and during exposure to H2O2 () or H2O2+4-AP ().

View larger version (20K): [in this window] [in a new window]   Fig. 12. Effect of functional removal of endothelium on diamide-mediated pulmonary vasodilation. A: representative tracings showing active tension induced by 25K in the presence or absence of 10 µM ACh or 100 µM diamide in PA rings with intact (+Endo; top) or denuded (–Endo; bottom) endothelium. Note that ACh-mediated vasodilation is abolished in the endothelium-denuded PA ring. B: time course of the decline phase of diamide-mediated relaxation, corresponding to the shadowed areas in A, in PA rings with (+Endo) or without (–Endo) endothelium. C: summarized data (means ± SE; n = 8) showing amplitude of PE-induced active tension before (control), during, and after (washout) application of 100 µM diamide in PA rings with (+) and without (–) endothelium. ***P < 0.001 vs. control and washout.

In PA rings isolated from the right branches, PE caused a transient contraction in the absence of extracellular Ca²⁺. Approximately 30 min later, restoration of extracellular Ca²⁺ in the presence of the -adrenergic-receptor blocker phentolamine (1 µM) caused a sustained PA contraction due to CCE (Fig. 13Aa). The CCE-induced PA contraction, induced by active store depletion, was significantly inhibited by treatment with 100 µM diamide (Fig. 13Ab). The summarized data show that the PE-mediated transient PA contraction was not significantly changed along time; the active tension in the absence of Ca²⁺ was 372.2 ± 50.1 mg/mg (n = 12) when the vessels were first challenged with PE and 330.9 ± 47.9 (n = 12; P = 0.56) when the vessels were challenged with PE 15 min later (Fig. 13Ba). The CCE-induced active tension associated with the first PE challenge was 147.9 ± 18.3 mg/mg (n = 12, without treatment with diamide), and the CCE-induced active tension associated with the second PE challenge was 23.8 ± 6.8 mg/mg (n = 12; P < 0.001) in the presence of 100 µM diamide (Fig. 13Bb). These results show that diamide significantly inhibited CCE-mediated PA contraction and that the relaxant effect of diamide was not related to changes of PA contractility over time.

View larger version (31K): [in this window] [in a new window]   Fig. 13. Diamide inhibits CCE-mediated PA contraction induced by active store depletion. A: representative tracings showing active tension induced by PE-mediated Ca2+ release (in the absence of extracellular Ca2+) and by store depletion-mediated Ca2+ influx or CCE (in the presence of Ca2+ and 1 µM phentolamine) under control conditions (a) and during the treatment with 100 µM diamide (which was applied to the vessels with phentolamine; b). B: summarized data (means ± SE; n = 12) showing PE-mediated transient PA contraction in the absence of extracellular Ca2+ (a) when the vessels were first challenged with PE (1st) and challenged with PE after 15 min (2nd) and CCE-induced PA contraction in vessels treated with or without (control) 100 µM diamide (b). ***P < 0.001 vs. control.

View larger version (22K): [in this window] [in a new window]   Fig. 14. CCE-induced PA contraction is similarly preserved with successive challenges. A: representative tracings showing active tension induced by store depletion-mediated Ca2+ influx or CCE (see Fig. 13 legend for details). Store depletion was induced by 2 successive PE challenges in the presence of 1 µM phentolamine; the 2nd challenge was given 90 min after the first challenge. B: summarized data (means ± SE; n = 8) showing the amplitude of CCE-induced PA contraction when the vessels were first challenged with PE (1st) and challenged with PE after 90 min (2nd). *P < 0.05 vs. 1st.

View larger version (23K): [in this window] [in a new window]   Fig. 15. Diamide inhibits CCE-mediated PA contraction induced by passive store depletion. A: representative tracings showing active tension induced by 40K (left) and by store depletion-mediated Ca2+ influx or CCE as a result of CPA (50 µM; right)-mediated passive depletion of intracellular stores in control PA rings (top) and 100 µM diamide-treated PA rings (bottom). B: summarized data (means ± SE; n = 8) showing amplitude of 40K-mediated active tension [a, when the vessels were first challenged (1st) and challenged 15 min later (2nd)] in PA rings without treatment of diamide and CPA-induced PA contraction due to CCE (b) in control and diamide-treated PA rings. CCE-induced active tension was normalized to the 40K-mediated active tension and expressed as percentage of corresponding 40K-mediated contraction. **P < 0.01 vs. control.

Diamide negligibly affects 80K-induced PA constriction. In coronary artery, Iesaki and Wolin (26) reported that diamide-mediated vasodilation is attenuated by L-type Ca²⁺ channel blockers (nifedipine and diltiazem). Therefore, diamide may function through activating a thiol oxidation mechanism that inhibits L-type VDCC and causes coronary arterial relaxation. To examine whether diamide causes pulmonary vasodilation by inhibiting L-type Ca²⁺ channels, we examined the effect of a high dose of diamide (300 µM) on 80K-induced active tension.

In PA rings preconstricted by 80K, the E_K is shifted to –14 mV, which is beyond the threshold (approximately –30 mV) for opening VDCC in PASMC (59). Therefore, the 80K-induced PA constriction is mainly caused by Ca²⁺ influx through L-type VDCC. Under these conditions (i.e., in PA rings constricted by 80K), although opening of K⁺ channels shifts the E_m close to the E_K, it would not be sufficient to close VDCC because the E_K (–14 mV) is less negative than the voltage threshold (–30 mV) for opening VDCC. Furthermore, in PA rings preconstricted by 80K, SOC is not activated because intracellular stores are not depleted and Ca²⁺ entry through voltage-independent cation channels is actually inhibited because of reduced driving force (or transmembrane electrochemical gradient) for Ca²⁺.

As shown in Fig. 16, diamide (300 µM) had no relaxant effect on PA rings preconstricted by 80K, whereas nifedipine (0.1 µM) almost abolished the 80K-induced PA contraction. These data suggest that diamide-mediated thiol oxidation is unlikely involved in inhibiting L-type VDCC in rat pulmonary vascular smooth muscle. The relaxing effect of diamide on PA rings preconstricted by 20–40K (in which E_K is from –49 to –31 mV) was thus mainly due to opening of K⁺ channels (which would shift the E_m close to the E_K and close VDCC) and to other intracellular mechanisms (e.g., inhibition of myosin light-chain kinases and contractile proteins). The relaxing effect of diamide on PA rings preconstricted by PE was thus mainly due to inhibition of ROC and/or SOC.

View larger version (29K): [in this window] [in a new window]   Fig. 16. Increase of extracellular K+ to 80 mM abolished diamide-mediated pulmonary vasodilation. A: representative tracings showing active tension induced by 80K before, during, and after application of 300 µM diamide (left), as well as before and during application of 0.1 µM nifedipine (right). B: summarized data (means ± SE; n = 4) showing tension before (basal) and during application of 80K in the presence (80K+diamide) or absence (80K) of diamide (300 µM). C: summarized data (means ± SE; n = 4) showing tension before (basal) and during application of 80K in the presence (80K+nif) or absence (80K) of nifedipine (0.1 µM). ***P < 0.001 vs. 80K.

	DISCUSSION

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Using isolated PA rings, we showed that membrane depolarization by raising [K⁺]_o (from 4.7 to 20–80 mM) causes sustained vasoconstriction because of Ca²⁺ influx through nifedipine-sensitive L-type VDCC. Furthermore, activation of the -adrenergic receptor by PE increases [Ca²⁺]_cyt in PASMC and causes pulmonary vasoconstriction by at least three mechanisms: 1) Ca²⁺ release from the SR, 2) store depletion-mediated Ca²⁺ influx through SOC or CCE, and 3) Ca²⁺ influx through ROC. The CCE-mediated pulmonary vasoconstriction can also be induced by passive depletion of intracellular Ca²⁺ stores with the SERCA inhibitor CPA. Finally, the CCE-mediated active tension appears to be predominant in the right branches of PA, whereas high-K⁺-induced active tension is comparable between right and left PA branches. In other words, CCE-induced PA contraction due to passive vs. active store depletion differs between left and right branches. Although the passive store depletion-mediated PA contraction (by CPA) is comparable, the active store depletion-induced contraction is significantly greater in right PA branches than in left PA branches.

Furthermore, our results demonstrated that 1) acute treatment with the thiol oxidant diamide causes pulmonary vasodilation, 2) the relaxing effect of diamide is not dependent on intact endothelium but modulated by endothelium-derived factors, 3) pretreatment of the vessels with the membrane-permeable PKG/PKA inhibitor HA-1004 negligibly affects diamide-mediated pulmonary vasodilation, 4) the thiol oxidant-mediated pulmonary vasodilation is markedly inhibited when [K⁺]_o is raised (e.g., from 20 to 80 mM) and thus the transmembrane K⁺ driving force is reduced, and 5) the diamide-induced pulmonary vasodilation is abolished in vessels constricted with 80K. These results indicate that thiol oxidation by diamide induces pulmonary vasodilation by opening K⁺ channels (and subsequent membrane hyperpolarization) and blocking SOC that are opened by active and passive store depletion. Although diamide indirectly inhibits VDCC activity by causing membrane hyperpolarization or repolarization (53, 73), thiol oxidation appears not to have a direct inhibitory effect on VDCC because diamide negligibly affected active tension in PA rings contracted by 80K.

Diamide is an oxidant that crosses the plasma membrane rapidly by diffusion and promotes thiol oxidation of membrane and intracellular proteins (31). Diamide decreases the GSH-to-GSSG ratio by oxidizing intracellular GSH to glutathione disulfide (GSSG) (30) and decreases the NADPH-to-NADP⁺ ratio by inhibiting NADPH generation (42). The thiol oxidant diamide has been demonstrated to stimulate soluble guanylate cyclase (sGC) in platelets at low concentrations and to inhibit the sGC activity at higher concentrations (74). In bovine PA, Mingone et al. (42) provide compelling evidence that the thiol oxidant diamide (1 mM) inhibits the ability of NO to activate sGC and attenuates NO-mediated pulmonary vasodilation (42). Our results, however, indicate that treatment of rat PA rings with 8-BrcGMP or with HA-1004, a PKG/PKA inhibitor, failed to abolish diamide (100 µM)-mediated PA relaxation. These results suggest that, in addition to inhibiting sGC activation, the thiol oxidant may have a direct oxidizing effect on membrane ion channels (53). Indeed, our results imply that thiol oxidation by diamide causes pulmonary vasodilation by opening K⁺ channels and by inhibiting SOC in the plasma membrane of PASMC. It remains unclear how the thiol oxidant-mediated cellular redox changes in PASMC cause opposite effects on K⁺ channels and store depletion-activated Ca²⁺ channels.

PASMC functionally express multiple K⁺ channels, including Kv channels, K_Ca channels, K_ATP channels, and inward rectifier K⁺ channels (63). In PA rings constricted by, for example, 20K, opening of any K⁺ channels would shift membrane potential toward the E_K, which is estimated to be approximately –49 mV (given that [K⁺]_o is 140 mM), close to the activation threshold for VDCC approximately –40 to –30 mV; the peak of window currents is approximately –25 to –15 mV) (17, 45, 58), and cause PASMC relaxation. In PA rings constricted by 80K, however, opening of K⁺ channels would only shift the membrane potential toward –14 mV (the calculated E_K in PASMC superfused with 80K-containing solution) and would therefore be unable to close VDCC and cause PASMC relaxation. As shown in Figs. 8 and 16, increasing [K⁺]_o from 20 to 80 mM increased the active tension in PA rings but significantly decreased diamide-mediated PA relaxation. These data strongly indicate that diamide causes pulmonary vasodilation by opening K⁺ channels in PASMC. The oxidation-mediated opening of different types of K⁺ channels (e.g., Kv, K_ATP, and K_Ca channels) is well known (4, 48, 65), and oxidation of thiol or sulfide hydrogen groups to form disulfide bridges may cause conformational changes in the channel protein and open the channel by modulation its gating or inactivation kinetics (53, 57, 80).

In bovine coronary arteries, Iesaki and Wolin (26) reported that diamide-mediated vasodilation was significantly inhibited in arteries constricted by high K⁺ compared with arteries constricted by agonist (e.g., U-46619). These observations further support our contention that diamide at least in part causes vasodilation by opening K⁺ channels in PASMC, which subsequently induces membrane hyperpolarization (or repolarization), closure of voltage-dependent L-type Ca²⁺ channels, and inhibition of Ca²⁺ influx. Furthermore, Iesaki and Wolin demonstrated that blockade of VDCC with nifedipine (1 µM) or diltiazem (10 µM) inhibited diamide-mediated vasodilation in bovine coronary arteries preconstricted by U-46619 (26), suggesting that thiol oxidation-mediated vasodilation may be partially induced by inhibiting L-type Ca²⁺ channels. In rat PA rings, however, diamide (100 or 300 µM) had a negligible effect on 80K-mediated vasoconstriction (Figs. 8 and 16), which is mainly caused by Ca²⁺ influx through L-type VDCC in PASMC. It is possible that thiol oxidation induced by diamide may target different Ca²⁺ channels in different arteries (e.g., pulmonary vs. coronary arteries) or in PA isolated from different species. Another possibility that nifedipine or diltiazem attenuates diamide-induced coronary arterial relaxation (26) is because of the potential nonselective inhibitory effect of high doses of nifedipine and diltiazem on SOCs and ROCs.

Studies on the molecular identification of SOCs indicate that multiple channel subunits participate in forming functional SOCs in vascular smooth muscle and endothelial cells (14, 15, 29, 36, 60, 69, 76). Transient receptor potential (TRP) genes have been demonstrated to encode the channel subunits that are involved in the formation of functional SOC in human and animal PASMC (10, 77, 78). Similar to the pore-forming Kv channel -subunit, the TRP channel subunit also contains a cytoplasmic NH₂ terminus, six transmembrane domains (S1 to S6), a pore region between S5 and S6 domains, and a cytoplasmic COOH terminus. The functional TRP channels or TRP-encoded SOC are either homotetramers or heterotetramers; thus differences in composition may determine the channel's sensitivity to store depletion or receptor activation. As shown in Figs. 13 and 15, the thiol oxidant diamide not only inhibited CCE-induced PA contraction due to PE-mediated active store depletion (Fig. 13) but also attenuated CCE-induced PA contraction as a result of CPA-mediated passive store depletion (Fig. 15). The greater inhibition on PA contraction induced by active store depletion than that induced by passive store depletion implies that diamide-mediated PA relaxation may involve inhibition of agonist-mediated receptor activation and downstream signaling cascades.

In porcine aortic endothelial cells, Poteser et al. (52) demonstrated that TRPC3 and TRPC4 formed a redox-sensitive heterotetrameric cation channel, indicating that TRPC3 and TRPC4 are involved in forming native cation channels that are regulated by the cellular redox state. The TRPC3 and TRPC4 channels overexpressed in porcine aortic endothelial cells and HEK-293 cells could be activated by incubation with carbachol (200 µM), cholesterol oxidase (0.5 U/ml), or 1-oleoyl-2-acetyl-sn-glycerol (100 µM, an analog of diacylglycerol). Our data in isolated PA rings suggest that thiol oxidation induced by diamide may cause pulmonary vasodilation by closing SOC in PASMC. Further study is thus needed to define 1) whether the TRPC3 and TRPC4 heterotetrameric channels in PASMC are regulated by the cellular redox state in the same way as they are in vascular endothelial cells, 2) whether TRPC3 and TRPC4 homo- and heterotetrameric channels in PASMC are regulated indirectly by a redox-sensitive intermediate, and 3) whether canonical TRP channels functionally expressed in PASMC can be oppositely regulated by different reducing agents.

Acute hypoxia causes pulmonary vasoconstriction, which is believed to be a unique and intrinsic property of PASMC. It has been demonstrated that hypoxia-induced pulmonary vasoconstriction is associated with inhibition of Kv channels (2, 51, 80) and activation of TRP or SOC channels (13, 70). Hypoxia-mediated changes in cellular redox status [determined by the ratios of NAD(P)H/NAD(P) and GSH/GSSG] are likely an important mechanism by which acute hypoxia inhibits Kv channels and activates TRP channels in PASMC (16, 34, 70). It is still controversial whether hypoxia leads to decreased or increased production of ROS and how changes in ROS production affect cellular response. However, studies in vitro demonstrate that whole cell K⁺ currents in rat PASMC were increased by intracellular application of GSSG but decreased by GSH (73, 80). In addition, NADH decreases the single-channel activity of K_Ca channels in PASMC, whereas NAD increases the channel activity (34). The mechanism by which hypoxia or redox status change modulates Kv channel activity appears to involve the cytoplasmic -subunit, which contains an active oxidoreductase domain with a NADPH cofactor pocket and a substrate binding site (18). Therefore, the Kv channel -subunit may be an important target for the thiol oxidant diamide, ROS, and/or redox status changes to affect Kv channel function (4).

In summary, the data from this study present evidence that thiol oxidation of membrane proteins and intracellular enzymes in PASMC play an important role in regulating pulmonary vascular tone. The thiol oxidant diamide causes pulmonary vasodilation by potentially opening K⁺ channels and inhibiting SOC in PASMC; the thiol oxidation-mediated effect appears to be independent of cellular cGMP/PKG and intact endothelium. Our observations also indicate that shifting the redox status in PASMC to a more oxidized level leads to pulmonary arterial relaxation.

	GRANTS

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This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-066012, HL-054043, and HL-064945.

	ACKNOWLEDGMENTS

 
We thank A. Nicholson for technical assistance.

	FOOTNOTES

 

Address for reprint requests and other correspondence: J. X.-J. Yuan, Division of Pulmonary and Critical Care Medicine, Medical Teaching Facility, Rm. 252, Univ. of California, San Diego, 9500 Gilman Drive, MC 0725, La Jolla, CA 92093-0725 (e-mail: xiyuan{at}ucsd.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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