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Am J Physiol Lung Cell Mol Physiol 292: L1598-L1608, 2007. First published March 16, 2007; doi:10.1152/ajplung.00323.2006
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Hydrogen peroxide-induced Ca2+ mobilization in pulmonary arterial smooth muscle cells

Mo-Jun Lin,1,2 Xiao-Ru Yang,1 Yuan-Ning Cao,1 and James S. K. Sham1

1Division of Pulmonary and Critical Care Medicine, Johns Hopkins School of Medicine, Baltimore, Maryland; and 2Department of Physiology and Pathophysiology, Fujian Medicial University, Fuzhou, Fujian, People's Republic of China

Submitted 21 August 2006 ; accepted in final form 12 March 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reactive oxygen species (ROS) generated from NADPH oxidases and mitochondria have been implicated as key messengers for pulmonary vasoconstriction and vascular remodeling induced by agonists and hypoxia. Since Ca2+ mobilization is essential for vasoconstriction and cell proliferation, we sought to characterize the Ca2+ response and to delineate the Ca2+ pathways activated by hydrogen peroxide (H2O2) in rat intralobar pulmonary arterial smooth muscle cells (PASMCs). Exogenous application of 10 µM to 1 mM H2O2 elicited concentration-dependent increase in intracellular Ca2+ concentration in PASMCs, with an initial rise followed by a plateau or slow secondary increase. The initial phase was related to intracellular release. It was attenuated by the inositol trisphosphate (IP3) receptor antagonist 2-aminoethyl diphenylborate, ryanodine, or thapsigargin, but was unaffected by the removal of Ca2+ in external solution. The secondary phase was dependent on extracellular Ca2+ influx. It was unaffected by the voltage-gated Ca2+ channel blocker nifedipine or the nonselective cation channel blockers SKF-96365 and La3+, but inhibited concentration dependently by millimolar Ni2+, and potentiated by the Na+/Ca2+ exchange inhibitor KB-R 7943. H2O2 did not alter the rate of Mn2+ quenching of fura 2, suggesting store- and receptor-operated Ca2+ channels were not involved. By contrast, H2O2 elicited a sustained inward current carried by Na+ at –70 mV, and the current was inhibited by Ni2+. These results suggest that H2O2 mobilizes intracellular Ca2+ through multiple pathways, including the IP3- and ryanodine receptor-gated Ca2+ stores, and Ni2+-sensitive cation channels. Activation of these Ca2+ pathways may play important roles in ROS signaling in PASMCs.

nonselective cation channels; sodium-calcium exchange; ryanodine receptor; inositol trisphosphate receptor; pulmonary arteries

REACTIVE OXYGEN SPECIES (ROS) derived from diverse cellular sources, including NADPH oxidase, mitochondrial electron transport chain (ETC), xanthine oxidase, cytochrome P-450, cyclooxygenase, and nitric oxide synthase, are known to play important roles in the regulation of pulmonary and systemic circulation (15). It has been established that vasoconstrictors/mitogens, such as angiotensin II, serotonin, endothelin-1, platelet-derived growth factor, and tumor necrosis factor-{alpha}, stimulate the production of superoxide and hydrogen peroxide (H2O2) to enhance vasoconstriction and activate redox-sensitive gene transcription to promote proliferation, hypertrophy, migration, and survival of vascular smooth muscle cell (14, 34, 36, 58). Recent studies showed that ROS overproduction is associated with pulmonary remodeling and alterations in vascular reactivity observed in chronic hypoxia, and targeted deletion of gp91phox subunit prevented these changes in knockout mice, suggesting that ROS generated from NADPH oxidase contribute significantly to the pathogenesis of hypoxic pulmonary hypertension (23, 37).

More interestingly, accumulated evidence suggests that ROS are critically involved in O2 sensing for initiating acute hypoxic pulmonary vasoconstriction (HPV) (42, 67, 68). However, the details of the mechanism are still controversial and remain the subjects of intense debates. Several mechanisms involving microsomal/cytosolic NAD(P)H oxidases and mitochondrial ETC have been proposed. Among them, two models have presently received the most attention. The first model is based on the redox regulation of voltage-gated K+ (KV) channels (3, 4, 39). It proposes that ROS generated by mitochondrial ETC (complexes I and III) under normoxia are rapidly converted into diffusible H2O2, which causes pulmonary vasorelaxation through activation of the redox-sensitive KV (KV 1.5 and KV 2.1) channels. Reduction in PO2 during hypoxia decreases mitochondrial electron transport and ROS production, leading to the inhibition of KV channels, membrane depolarization, activation of voltage-gated Ca2+ channels, increase in cytosolic Ca2+ concentration ([Ca2+]), and vasoconstriction. The contending model also proposes mitochondria-derived ROS as the mediator of HPV (65, 66). However, hypoxia causes a paradoxical increase in superoxide production, presumably by facilitating the transfer of unpaired electron from ubisemiquinone to O2 (67). H2O2 liberated by superoxide dismutation in mitochondria and cytosol then triggers Ca2+ release from intracellular Ca2+ stores to activate store-operated Ca2+ entry (44, 51, 63) and to inhibit KV channels to further enhance Ca2+ influx through membrane depolarization and voltage-gated Ca2+ channel activation (48). Despite repeated efforts using different probes and techniques of ROS detection in organ, tissue, and cell preparations, the consent for ROS being up or down during HPV has not been reached (55).

Since HPV, pulmonary vasoconstriction, pulmonary arterial smooth muscle cell (PASMC) proliferation induced by agonists/mitogens, as well as vascular remodeling in pulmonary hypertension are all intricately linked to intracellular Ca2+ signaling, it appears to be crucial to understand the Ca2+ processes underlying ROS signal transduction. However, ROS-induced Ca2+ signals have not been characterized in PASMCs, and deductions of ROS-mediated Ca2+ processes in PASMCs have been based mainly on observations made in systemic vessels and other tissues, which are physiologically different from pulmonary vasculatures. In the present study, we sought to characterize in detail the Ca2+ response and to delineate the Ca2+ pathways activated by H2O2 in rat intralobar PASMCs. H2O2 was chosen among various ROS because it is stable, has a long diffusion distance, and is generally accepted as a key messenger for HPV and agonist-induced responses. Even though the actions of exogenous H2O2 might not simulate exactly those of endogenous H2O2 due to the presence of subcellular local concentration gradients and spatial compartmentations, the present study identified functionally the essential H2O2-susceptible Ca2+ pathways and provided basic information on ROS Ca2+ signaling in native PASMCs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Isolation and culture of PASMCs. PASMCs were enzymatically isolated and transiently cultured as previously described (35). The protocols involved were reviewed and approved by the Johns Hopkins University Animal Care and Use Committee. Briefly, male Wistar rats (150–250 g) were injected with heparin and anesthetized with pentobarbital sodium (130 mg/kg ip). They were exsanguinated, and lungs were removed and transferred to a petri dish filled with HEPES-buffered salt solution (HBSS) containing (in mM) 130 NaCl, 5 KCl, 1.2 MgCl2, 1.5 CaCl2, 10 HEPES, and 10 glucose, pH 7.4 (adjusted with NaOH). Second- and third-generation intrapulmonary arteries (~300–800 µm) were isolated and cleaned free of connective tissue. The endothelium was removed by gently rubbing the luminal surface with a cotton swab. Arteries were then allowed to recover for 30 min in cold (4°C) HBSS, followed by 20 min in reduced Ca2+ (20 µM) containing HBSS at room temperature. The tissue was digested at 37°C for 20 min in 20 µM Ca2+ HBSS containing collagenase (type I, 1,750 U/ml), papain (9.5 U/ml), bovine serum albumin (2 mg/ml), and dithiothreitol (1 mM), and then removed and washed with Ca2+-free HBSS to stop digestion. Single smooth muscle cells were gently dispersed by trituration with a small-bore pipette in Ca2+-free HBSS at room temperature. The cell suspension was then placed on 25-mm glass coverslips and transiently (16–24 h) cultured in Ham's F-12 medium (with L-glutamine) supplemented with 0.5% fetal calf serum, 100 U/ml of streptomycin, and 0.1 mg/ml of penicillin.

Intracellular [Ca2+] measurement. Intracellular [Ca2+] ([Ca2+]i) in single PASMCs was measured with the membrane-permeant acetoxymethyl ester (AM) form of the Ca2+-sensitive fluorescent dye fluo 3 (fluo 3-AM). PASMCs were loaded with 5–10 µM fluo 3-AM, which was first dissolved in dimethyl sulfoxide with 20% pluronic acid, for 30–45 min at room temperature (~22°C) in normal Tyrode solution containing (in mM) 137 NaCl, 5.4 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose, pH 7.4 (adjusted with NaOH). Cells were then washed thoroughly with Tyrode solution to remove extracellular fluo 3-AM and rested for 15–30 min in a cell chamber to allow for complete deesterification of cytosolic dye. Fluorescence measurement was performed on a Nikon Diaphot inverted microscope. The collimated light beam from a 75-W xenon arc lamp was filtered by an interference filter at 480/30 nm and focused onto the PASMCs under examination via a x40 fluorescence oil-immersion objective (Fluor 40x, Nikon). Light emitted from the cell was returned through the objective and split with a dichroic mirror, and fluorescence was detected at 535/40 nm by a photomultiplier tube. The emission signals were amplified with a dual-emission fluorometer (Biomedical Instrumentation Group, University of Pennsylvania, Philadelphia, PA). Photobleaching of fluo 3 dye was minimized using a neutral density filter (ND-3, Omega Optics) and an electronic shutter (Vincent Associates). The shutter was opened for 35 ms every second, and the fluorescence signals during the open period were integrated with a sample-and-hold circuit. The protocols were executed, and the data were collected on-line with a Digidata 1200 analog-to-digital interface (Axon Instruments, Union City, CA) and the pClamp software package. [Ca2+]i was calibrated by the equation [Ca2+]i = KD·(F – Fbg)/(Fmax – F), where F is fluorescence, Fbg is background fluorescence, and Fmax is the maximum fluorescence determined in situ in cell superfused with 10 µM 4-bromo A-23187 and 20 mM Ca2+; or by a pseudoratio method, using the following equation: [Ca2+]i = (KD·R)/[{(KD/[Ca2+]rest) + 1} – R], where R is F/F0, and KD of fluo 3 is 1.1 µM. The value of resting Ca2+ ([Ca2+]rest) of PASMCs was assumed to be 176 nM (35).

Mn quenching of fura 2. Ca2+ entry through nonselective cation channels was quantified by quenching of fura 2 with Mn2+. PASMCs were loaded with fura 2-AM as described above. Fura 2 was excited at the Ca2+-insensitive isobestic point of 360 nm, and emission light was recorded at 535/40 nm. PASMCs were then bathed in a Ca2+-free (with 0.1 mM EGTA) Tyrode solution containing 1 µM nifedipine. After a stable baseline fluorescent measurement was attained, 500 µM Mn2+ were applied through a concentration-clamp system with the multibarrel pipette positioned <50 µm from PASMCs. The rates of quenching of fura 2 fluorescence in PASMCs with and without drug treatments were determined and compared.

Whole cell recording of H2O2-induced current. PASMCs were superfused with external solution containing (in mM) 137 NaCl, 5.4 CsCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose (pH 7.4). Patch pipettes (tip resistance 3–5 M{Omega}) containing (in mM) 118 Cs methane-sulfonate, 20 CsCl, 1 Ca2+-gluconate, 2 EGTA, and 4 Na2ATP (pH 7.2) will be used. The ionic conditions were designed to eliminate K+, and 1 µM nifedipine was added to the external solution to block L-type Ca2+ channel. Membrane current was recorded under whole cell voltage-clamp mode and filtered at 5 kHz with an Axopatch-200B amplifier (Axon Instruments, Union City, CA). Junction potential, cell capacitance, and access resistance were compensated electronically. Voltage-clamp protocols were executed and analyzed with the pClamp software.

Data analysis. Data are expressed as means ± SE. The number of cells is specified in the text. Statistical significance (P < 0.05) was assessed by paired or unpaired Student's t-tests, or ANOVA with Newman-Keuls post hoc analyses, wherever applicable.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
H2O2-induced Ca2+ transient. The effects of exogenous H2O2 on global [Ca2+]i was examined in transiently cultured PASMCs. H2O2 elicited concentration-dependent increases in [Ca2+]i (Fig. 1). At the low concentrations (10–100 µM), H2O2 elicited an increase in [Ca2+]i immediately after application that reached a small, sustained plateau above the resting [Ca2+]i level, and receded after washout of H2O2. By contrast, H2O2 at the higher concentrations (0.3 or 1 mM) caused a biphasic Ca2+ response, with an initial increase (75 ± 6 nM for 0.3 mM and 175 ± 11 nM for 1 mM) followed by a slow, continuous secondary rise in [Ca2+]i, which progressed throughout the period of H2O2 exposure (186 ± 19 nM for 0.3 mM and 1,180 ± 123 nM for 1 mM after 15-min H2O2 treatment, P < 0.001 for both cases). Ca2+ response induced by 1 mM H2O2 was completely abolished by catalytic elimination of H2O2 with catalase (500 U/ml), indicating that the Ca2+ response was specific to H2O2 and unrelated to contamination by impurities.


Figure 1
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  		Fig. 1. Concentration-dependent effects of hydrogen peroxide (H2O2) on intracellular Ca2+ concentration ([Ca2+]i) in pulmonary arterial smooth muscle cells (PASMCs). A–E: ensemble-averaged Ca2+ transients (n > 20 each) elicited by H2O2 at concentration of 10 µM to 1 mM, respectively. E: Ca2+ response induced by 1 mM H2O2 in the absence or presence of catalase (500 U/ml). F: bar graph showing the average changes in [Ca2+]i at 5 and 15 min elicited by H2O2 at various concentrations. {Delta}, Change.

 
To delineate the Ca2+ pathways activated by H2O2, the effects of 300 µM H2O2 in Ca2+ (2 mM)-containing and Ca2+-free (0 Ca2+ plus 1 mM EGTA) Tyrode solution were compared (Fig. 2). Elimination of extracellular Ca2+ had only a minor effect on the initial rise of Ca2+ transients induced by H2O2 measured at 5 min after H2O2 application (2 Ca2+: 49.3 ± 8.9 nM, n = 15; 0 Ca2+: 30.8 ± 11.0 nM, n = 15). However, the secondary rise in [Ca2+]i was significantly inhibited or reversed in the absence of external Ca2+ (0 Ca2+: 16.1 ± 7.3 nM, n = 15; 2 Ca2+: 99.6 ± 18.8 nM, n = 15 at 15 min after H2O2, P < 0.001). Reintroduction of extracellular Ca2+ caused an instantaneous recovery of Ca2+ transient to a magnitude comparable to that elicited in Ca2+-containing Tyrode solution (0 Ca2+: 102.9 ± 19.6 nM; 2 Ca2+: 90.4 ± 18.7 nM at 20 min after H2O2, P = 0.337). These results suggested that the Ca2+ response elicited by H2O2 consisted of an intracellular Ca2+ release component, which contributed to the initial rise, and a Ca2+ influx component, which sustained the secondary elevation of [Ca2+]i.


Figure 2
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  		Fig. 2. Involvement of extracellular Ca2+ in H2O2-induced Ca2+ response in PASMCs. A: ensemble-averaged Ca2+ transients elicited by 300 µM H2O2 in PASMCs superfused with normal Ca2+-containing (2 mM) or Ca2+-free (0 Ca2+ plus 1 mM EGTA) Tyrode solutions. Extracellular Ca2+ was removed from 0 to 15 min during the application of H2O2; extracellular Ca2+ was reintroduced after 15 min. B: bar graph showing the average change in [Ca2+]i at 5- and 15-min exposure to H2O2 and 10 min after readmission of extracellular Ca2+. n = 15 for each group of experiments.

 
H2O2-induced intracellular Ca2+ release. To determine the intracellular Ca2+ stores responsible for the Ca2+ release induced by H2O2, PASMCs were pretreated with 50 µM ryanodine for 30 min, and the complete inhibition of ryanodine receptor (RyR)-gated Ca2+ store was verified by a brief application (10 s) of 10 mM caffeine. In the absence of extracellular Ca2+, the initial Ca2+ release elicited by H2O2 was attenuated by ~38% in the ryanodine-treated PASMCs (Fig. 3). In a separate set of experiments, PASMCs were exposed to 50 µM 2-APB for 30 min to inhibit the inositol trisphosphate (IP3) receptors (IP3R), and extracellular Ca2+ was then removed at 5 min before H2O2 application (Fig. 4). The initial Ca2+ release elicited by H2O2 in the absence of Ca2+ was attenuated significantly by 2-APB (control: change in [Ca2+]i = 28.339 ± 3.874 nM, n = 12 ; 2-APB treated: change in [Ca2+]i = 15.071 ± 3.12 nM, n = 8, P < 0.05). Depletion of both IP3R- and RyR-gated Ca2+ stores by inhibiting the sarcoplasmic Ca2+-ATPase with 10 µM thapsigargin for 30 min significantly reduced the resting [Ca2+]i in Ca2+-free external solution and completely abolished the initial Ca2+ release transient. These results suggest that the H2O2-induced Ca2+ release was composed of Ca2+ released from both IP3R- and RyR-gated stores. In addition, there was a small, residual, slow rise in [Ca2+]i in the presence of thapsigargin (Fig. 4C) that could be related to incomplete block of Ca2+-ATPase or minor Ca2+ release from other thapsigargin-insensitive Ca2+ stores, such as mitochondria and lysosomes.


Figure 3
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  		Fig. 3. Effects of ryanodine on H2O2-induced Ca2+ release. H2O2-induced Ca2+ transients are shown in control (n = 9; A) and ryanodine-treated (n = 6; B) PASMCs under Ca2+-free condition. PASMCs were pretreated with 50 µM ryanodine for 30 min, and depletion of ryanodine receptor (RyR)-gated Ca2+ store was confirmed by applications (10 s) of 10 mM caffeine. C: bar graph showing the average change in [Ca2+]i at 5 min after H2O2 exposure. There was a 38% decrease in the Ca2+ release response in the ryanodine-treated cells measured after 5 min of H2O2 exposure.

 

Figure 4
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  		Fig. 4. Role of inositol trisphosphate (IP3)-gated Ca2+ stores in H2O2-induced Ca2+ release. H2O2-induced Ca2+ transients in control (n = 12; A), the IP3-receptor inhibitor 2-APB (n = 8; B), and the sarcoplasmic reticulum (SR) Ca2+-ATPase inhibitor thapsigargin (TG)-treated (n = 8; C) PASMCs are shown in the absence of extracellular Ca2+. PASMCs were pretreated with 50 µM 2-APB or 10 µM TG for 30 min before H2O2 exposure. D: bar graph showing the average change in [Ca2+]i at 5 min after H2O2 exposure. There were 50 and 80% decrease in the Ca2+ release in the 2-APB and TG-pretreated cells, respectively. *Significant difference from control; **significant difference from 2-APB.

 
H2O2-induced extracellular Ca2+ influx. H2O2-induced Ca2+ influx could be mediated via voltage-gated Ca2+ channels and/or nonselective cation channels. The possible involvement of voltage-gated L-type Ca2+ channels was examined by pretreatment of PASMCs with nifedipine (1 µM) before the application of 300 µM H2O2 (Fig. 5, A and B). Neither the initial nor the secondary rise of [Ca2+]i in the nifedipine-treated PASMCs differed significantly from that of the time-matched controls. Removal of extracellular Ca2+ in the presence of nifedipine caused a reduction of the plateau phase/secondary rise in the H2O2-induced Ca2+ transient, and the recovery of Ca2+ transient upon readmission of extracellular Ca2+ was not blocked by nifedipine (Fig. 5, C and D). These results suggested that L-type Ca2+ channels did not participate in the Ca2+ entry elicited by H2O2.


Figure 5
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  		Fig. 5. Effects of the L-type Ca2+ channel antagonist nifedipine on H2O2-induced Ca2+ influx. A: averaged Ca2+ transients elicited by H2O2 in control (n = 8) and nifedipine-treated (n = 9) PASMCs. B: bar graph showing the average changes in [Ca2+]i at 5 and 15 min of H2O2 exposure in the absence and presence of 1 µM nifedipine. C: H2O2-induced Ca2+ influx during extracellular Ca2+ readmission in the presence of nifedipine (n = 19 for 100 µM H2O2 and n = 15 for 300 µM H2O2). D: average changes in [Ca2+]i in the initial Ca2+ release response at 5 min and the Ca2+ influx during Ca2+ readmission measured at 20 min after exposure to H2O2.

 
To explore the role of nonselective cation channels in H2O2-induced Ca2+ entry, the effects of the several nonselective cation channel antagonists were examined. In the presence of nifedipine (1 µM), application of 100 or 300 µM of La3+ or 5 µM SKF-96365 had no apparent effect on the secondary phase of Ca2+ transients elicited by H2O2 (Fig. 6, A and C). In contrast, Ni2+ at concentrations of 300 µM to 3 mM caused concentration-dependent inhibition of the H2O2-induced Ca2+ transient. The reduction of Ca2+ transients ranged from 21.0 ± 4.1 to 76.5 ± 7.2% (Fig. 6, B and D). In a separate set of experiments, Mn2+ quenching technique was employed to further evaluate the nonselective cation entry during H2O2 exposure. In fura 2-AM loaded PASMCs, application of 500 µM Mn2+ caused a slow quenching of fluorescence signal in the presence of 1 µM of nifedipine (Fig. 7). Depletion of sarcoplasmic reticulum (SR) Ca2+ stores with 10 µM thapsigargin caused a dramatic increase in the rate of Mn2+-induced quenching of fura 2 fluorescence, indicative of store-operated nonselective cation entry. However, pretreatment of PASMCs with H2O2 at 1 mM did not elicit nonselective cation entry in the absence or presence of extracellular Ca2+ (Fig. 7). Similar results were observed when 300 µM H2O2 was used. The inability of La3+ and SKF-96365 to block the H2O2-induced Ca2+ influx and the failure of H2O2 to enhance Mn2+-induced quenching of fura 2 signal suggested that the Ca2+ entry activated by H2O2 was unrelated to the store- and receptor-operated cation channels reported in PASMCs (35).


Figure 6
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  		Fig. 6. Effects of nonselective cation channel antagonists on H2O2-induced Ca2+ response. A: effect of La3+ (100 µM, n = 8; 300 µM, n = 6) and SKF-96365 (n = 9) on Ca2+ transients induced by 300 µM H2O2. B: concentration-dependent inhibition of H2O2-induced Ca2+ transients by 0.3, 1, and 3 mM Ni2+; number of experiments were 7, 11, and 8, respectively. Inhibitors were applied at the plateau phase 10 min after H2O2 application. C: bar graph showing the average percent inhibition of Ca2+ response by La3+ and SKF-96365 at 2 min after application of the blocker. D: bar graph showing the average percent inhibition of Ca2+ response at 2 min after the addition of various concentrations of Ni2+.

 

Figure 7
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  		Fig. 7. Effects of H2O2 on nonselective divalent cation entry measured by Mn2+ quenching of fura 2 fluorescence. Mn2+ quenching in PASMCs pretreated for 15 min with 10 µM TG (n = 8; A), and 1 mM H2O2 in the absence (n = 7; B) and in the presence of extracellular Ca2+ (n = 6; C) are shown. Top: averaged traces from all the experiments in each group. Bottom: average maximum rate of quenching.

 
H2O2-activated cation current and Na+/Ca2+ exchange. The nature of H2O2-induced Ca2+ entry was further examined electrophysiologically using whole cell patch-clamp technique. External and internal solutions were designed to eliminate both the voltage-gated K+ and Ca2+ currents. At a holding potential of –70 mV, 1 mM H2O2 elicited an inward current (IH2O2) of 13.0 ± 4.9 pA/pF 1–3 min after application (Fig. 8). IH2O2 was dependent on extracellular Na+. Replacement of Na+ with N-methyl-D-glucamine (NMDG+) almost completely abolished the current (2.6 ± 0.6 pA/pF, n = 5, P < 0.05). It was also sensitive to 3 mM Ni2+, consistent with the Ca2+ transient experiments. The averaged amplitude of IH2O2 at –70 mV was reduced from –14.7 ± 3.4 to –5.6 ± 1.2 pA/pF (n = 10, P < 0.05) in the presence of Ni2+. Current-voltage (I-V) relations of IH2O2, generated by a ramp protocol with voltages ranging from –100 to +80 mV, exhibited a voltage dependence of slight outward rectification (Fig. 9A). Application of 3 mM Ni2+ inhibited the IH2O2 in both inward and outward direction (Fig. 9B).


Figure 8
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  		Fig. 8. H2O2 activates an inward current carried by Na+. A: representative traces of H2O2-induced inward current (IH2O2) at a holding potential (HP) of –70 mV. The current was partially inhibited by 3 mM Ni2+ and completely abolished by the replacement of extracellular Na+ with N-methyl-glucamine (NMG+). B: bar graph showing the mean IH2O2 before and after replacement of external Na+ with NMG+ (n = 5) or in the absence or presence of 3 mM Ni2+ (n = 10). *Significant difference from currents before Na+ replacement or Ni2+ application.

 

Figure 9
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  		Fig. 9. Current-voltage (I-V) relationship of membrane current induced by H2O2 in the absence or presence of Ni2+ and KB-R 7943. Membrane currents were activated by a voltage ramp from –100 mV to 80 mV from a holding potential of –70 mV. A, top: representative traces of ramp currents activated before and after application of 1 mM H2O2. Bottom: I-V relation of H2O2-activated current generated by subtracting the control ramp current from the H2O2 ramp current shown in the top panel. B, top: representative traces of the ramp currents recorded in the presence of H2O2 before and after application of 3 mM Ni2+. Bottom: I-V relation of H2O2-induced Ni2+-sensitive current generated by subtracting the ramp current recorded after Ni2+ application from the ramp current recorded before Ni2+ application shown in the top panel. C, top: representative traces of the ramp currents recorded in the presence of H2O2 before and after application 10 µM of KB-R 7943. Bottom: I-V relation of H2O2-induced KB-R 7943-sensitive current generated by subtracting the ramp current recorded after KB-R 7943 application from the ramp current recorded before KB-R 7943 application shown in the top panel. Vm, membrane potential.

 
IH2O2 might be mediated via nonselective cation channels, but it could be related alternatively to Na+/Ca2+ exchange, which is also Na+ dependent and sensitive to millimolar Ni2+. The latter possibility was examined by using the specific Na+/Ca2+ exchange antagonist, KB-R 7943. Ten micromolar KB-R 7943 caused a significant inhibition of the outward, but only a minor reduction of the inward, IH2O2. The I-V relation of the KB-R 7943-sensitive current was outward rectifying with a reverse potential of about –20 mV (Fig. 9C). This apparent reverse potential was close to the estimated equilibrium potential of Na+/Ca2+ exchange (ENa/Ca = 3ENa – 2ECa, {approx} –25 mV), assuming an exchange stoichiometry of three Na+ to one Ca2+ ion. These results suggested that Na+/Ca2+ exchange was a significant component of the outward IH2O2.

Since Na+/Ca2+ exchange is bidirectional, activation of Na+/Ca2+ exchange by H2O2 may either enhance cytosolic Ca2+ removal via forward mode or facilitate Ca2+ influx through reverse exchange. To distinguish between these two possibilities, the effect of Na+/Ca2+ exchange inhibition on the H2O2-induced Ca2+ transient was examined. Application of 10 µM KB-R 7943 during the secondary phase of Ca2+ transient elicited by H2O2 caused a significant increase in the Ca2+ transients from 222 ± 8 to 320.7 ± 39.6 nM (n = 8, P < 0.001) (Fig. 10), indicating that Na+/Ca2+ exchange operated predominantly in the forward direction to stabilize the increase in cytosolic [Ca2+] activated by H2O2 through other Ca2+ entry and release pathways.


Figure 10
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  		Fig. 10. Effects of the Na+/Ca2+ exchange antagonists KB-R 7943 on H2O2-induced Ca2+ response. A: ensemble-averaged Ca2+ transients elicited by 300 µM H2O2 in PASMCs superfused with or without application of 10 µM KB-R 7943 between 10 and 19 min. B: bar graph showing the average change in [Ca2+]i at 10- and 19-min exposure to H2O2. n = 8 for each group of experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
Previous studies showed that exogenous application of H2O2 caused either pulmonary vasoconstriction or vasodilatation, depending on the concentration and on the vascular tone (27, 28). H2O2 at 1–100 µM elicited concentration-dependent relaxation in isolated bovine pulmonary arteries preconstricted with 5-hydroxytryptamine and in isolated perfused rabbit lungs primed with U-46619 (8, 9). The vasodilatation was attributed to a H2O2/catalase-dependent activation of guanylate cyclase and cGMP production in pulmonary smooth muscle (10). Another study showed that H2O2 at low concentration caused vasorelaxation, but, at millimolar concentration, it activated vasoconstriction in hypoxia-contracted guinea pig pulmonary arteries (1). In contrast, H2O2-induced pulmonary vasoconstriction has been reported in many other studies in the absence of agonist-induced preconstriction (26, 46, 50, 56). Depending on the preparations, H2O2-induced pulmonary vasoconstriction has been attributed to the activation of different signaling pathways, including cyclooxygenase, lipoxygenase, calmodulin, protein kinase C, phospholipase C, serine esterase, and tyrosine kinase. Our observation of Ca2+ response elicited by H2O2, at a concentration as low as 10 µM, corroborates with the latter studies that suggest that the primary response of PASMCs to a physiological concentration of H2O2 is vasoconstriction. It is also consistent with a previous report in PASMCs (66) and other studies in systemic vascular myocytes (32, 52, 70) that H2O2 primarily increases [Ca2+]i in quiescence cells.

H2O2-induced Ca2+ response in systemic vascular smooth muscle involves activation of voltage-gated Ca2+ channel (52, 59, 70, 71), caffeine-sensitive/RyR-gated Ca2+ stores (57, 72), IP3-induced Ca2+ release (52), and inhibition of SR Ca2+-ATPase (16, 17). However, the sources of Ca2+ mobilized by H2O2 have not been explored in isolated PASMCs. The present study confirmed that H2O2 stimulates intracellular Ca2+ release from both ryanodine and IP3-sensitive Ca2+ stores in PASMCs. Mechanistic studies on RyRs showed that H2O2 causes direct modification of cysteine residues to form intersubunit cross-linkages within the release channel tetramer (2, 18, 19) and causes S-glutathionylation in combination with glutathione (12, 20). These sulfhydryl modifications enhance RyR activity by altering subunit-subunit interactions and by preventing the inhibitory binding of calmodulin and Mg2+. It is also accepted that H2O2 activates IP3R (5, 74) through sulfhydryl modifications, which lead to the increase in IP3R's sensitivity/affinity to IP3 (21, 24). Moreover, Ca2+ release via IP3-sensitive Ca2+ stores could be further enhanced by H2O2-induced activation of PLC (22, 56).

In addition to ryanodine- and IP3-sensitive Ca2+ stores, mitochondria and acidic lysosomal organelles may act as effective intracellular Ca2+ stores in PASMCs. Previous studies showed that H2O2 at 1 mM causes a slow and sustained Ca2+ release from mitochondria of bovine pulmonary smooth muscle and mouse pancreatic acinar cells (45, 53). We and others have found a thapsigargin-insensitive lysosomal Ca2+ store, which contributes to endothelin-1 and integrin-ligand-induced Ca2+ mobilization in PASMCs (7, 30, 60). The small residual increase in Ca2+ induced by H2O2 in thapsigargin-pretreated cells in Ca2+-free solution could be related to Ca2+ mobilization from these intracellular Ca2+ stores.

In contrast to the findings in aortic, mesenteric, and cerebral arterial smooth muscle cells (52, 59, 70, 71), the enhancement of Ca2+ influx in rat PASMCs by H2O2 is completely independent of the voltage-gated Ca2+ channels. This is attested by the insensitivity of the Ca2+ response to the L-type Ca2+ channel antagonist nifedipine or to a low concentration of Ni2+ (e.g., <100 µM), which blocks T-type Ca2+ channels. H2O2-induced Ca2+ influx also appears unrelated to the major Ca2+ influx pathways, such as the transient receptor potential (TRPC) encoded store- and receptor-operated Ca2+ channels (35, 38, 43, 63), because the commonly used TRPC blockers, La3+ and SKF-96365, were unable to block Ca2+ responses stimulated by H2O2. Moreover, H2O2 failed to enhance Mn2+-induced quenching of fura 2 fluorescence, a hallmark of nonselective cation entry via store- and receptor-operated TRPC channels (35).

However, H2O2 may facilitate Ca2+ influx via other, less recognized Ca2+ entry pathways in PASMCs. Our laboratory has recently identified multiple subtypes of transient receptor potential melastatin- (TRPM) and vanilloid-related (TRPV) channels in rat PASMCs (69), including TRPM2, TRPM3, TRPM4, TRPM7, and TRPM8 of the TRPM family, and TRPV1, TRPV2, TRPV3, and TRPV4 of the TRPV family. Among them, TRPM2 is the most relevant candidate for mediating H2O2-induced Ca2+ influx in PASMCs. TRPM2 is redox sensitive and can be activated by nicotinamide adenine dinucleotide, ADP-ribose, and H2O2 in neurons, immune cells, and other heterologous expression systems (47). It has been proposed as an endogenous redox sensor for mediating oxidative stress/ROS-induced Ca2+ entry (33). It is interesting to note that TRPM2 channel is La3+ insensitive, and even though it is nonselective to monovalent and divalent cations, its inward current is carried primarily by Na+ ions due to high extracellular Na+ concentration (31). These properties coincide with our observations in PASMCs that the H2O2-induced Ca2+ influx was not affected by 300 µM La3+, and the sustained inward current activated by H2O2 was almost completely abolished by the replacement of Na+ with NMG+. However, the functional properties and physiological roles of TRPM2 channels in vascular smooth muscle have not been characterized. It is unclear whether the native TRPM2 channels are permeable to Mn2+. This information is particular important because H2O2 failed to enhance Mn2+ quenching of fura 2 in PASMCs. Nevertheless, future experiments using siRNA will be essential for providing a more definitive answer to the possible involvement of TRPM2 in Ca2+ signaling in PASMCs.

In addition to nonselective cation entry, H2O2 also activated an outward rectifying current IH2O2, which was blocked significantly by 3 mM Ni2+ and partially blocked by the specific Na+/Ca2+ exchange antagonist KB-R 7943 (25). The KB-R 7943-sensitive component of IH2O2 reversed at a potential similar to the predicted ENa/Ca, suggesting that ENa/Ca current is a component of IH2O2. Previous studies have identified transcripts and proteins of Na+/Ca2+ exchangers in PASMCs (54, 64, 73), and studies in cardiac vesicles and myocytes have reported a stimulatory effect of H2O2 on Na+/Ca2+ exchange (13, 29, 49). Despite a few contrasting reports suggesting that H2O2 and oxidative stress may impair the exchanger activity (61), our present results support the notion that H2O2 stimulates the exchanger in intact PASMCs.

Na+/Ca2+ exchanger is a reversible transporter, and its direction of exchange is determined by the electrochemical gradients of Na+ and Ca2+, according to a stoichiometry of three Na+ ions exchange for one Ca2+ ion (6). Under normal physiological conditions, the exchanger operates mainly in the forward mode to remove excessive Ca2+, which enters the cytoplasm via various Ca2+ entry pathways. However, under conditions of strong depolarization and/or increased intracellular [Na+], Na+/Ca2+ exchange can operate in reverse mode to support Ca2+ influx. A recent study suggests that Na+ entry via store-operated cation channels contributes to the increase in [Ca2+]i through reverse Na+/Ca2+ exchange in PASMCs (73). Our results show that the enhanced Na+/Ca2+ exchange induced by H2O2 operates in the forward direction for the removal of cytosolic Ca2+, because inhibition of the exchanger potentiated significantly the magnitude of the H2O2-induced Ca2+ response. It, therefore, acts as a negative feedback mechanism for stabilizing the elevation of cytosolic [Ca2+] caused by Ca2+ release and influx through pathways activated by H2O2.

Our present results provide some interesting insights into the roles of H2O2 in Ca2+ signaling in PASMCs. A current debate in pulmonary circulation is on whether a decrease or an increase in ROS, in particular H2O2, in PASMCs is responsible for triggering HPV (42, 55, 67, 68). The proponents for a decrease in ROS during acute hypoxia propose a paradigm that H2O2 at the basal level provides continuous stimulation to KV channels to maintain membrane hyperpolarization; a reduction in H2O2 during hypoxia causes inhibition of KV channels, membrane depolarization, and activation of voltage-gated Ca2+ channels (40). In contrast, others suggest that a reduction in PO2 causes a paradoxical increase in H2O2 that activates Ca2+ release from SR Ca2+ stores, leading to store-operated Ca2+ entry, membrane depolarization, and activation of voltage-gated Ca2+ channels (67). Since both sides have provided substantial evidence of ROS measurements in support of their own hypotheses, the answer appears to depend decisively on whether H2O2 operates as a positive or a negative signal for Ca2+ mobilization in PASMCs. Our observation of an increase in [Ca2+] in response to H2O2 at physiologically relevant concentrations of 10 to 100 µM suggests that H2O2 is a Ca2+ activator in PASMCs and therefore lends support for the hypothesis of ROS increase as a mechanism of HPV. Recruitment of ryanodine-sensitive Ca2+ release by H2O2 is also consistent with previous studies that RyRs play a major role in hypoxic vasoconstriction (11, 41, 62). However, the Ca2+ responses elicited by H2O2 and hypoxia do not completely resemble each other. For example, Ca2+ responses elicited by H2O2 at low concentrations are small and independent of L-type Ca2+ channel and store-operated Ca2+ entry, in contrast to the hypoxia-induced Ca2+ response in PASMCs (44, 63). Moreover, it has been reported that hypoxia inhibits Na+/Ca2+ exchange in PASMCs (64). Hence, it is possible that hypoxia activates other mechanisms in addition to H2O2 for the generation of Ca2+ response. It is important, however, to note that the H2O2 concentration used in the study could be significantly higher than the level within PASMCs during hypoxia, and H2O2 applied exogenously may not mimic exactly the actions of endogenously generated H2O2.

In conclusion, we have characterized in detail the Ca2+ signals elicited by H2O2 in PASMCs. We found that H2O2 at a wide range of concentrations causes significant increase in [Ca2+]i through coordinated activation of multiple Ca2+ pathways of intracellular Ca2+ release from IP3- and RyRs and extracellular Ca2+ influx via Ni2+-sensitive cation channels. These H2O2-triggered Ca2+ signals in PASMCs may play important roles in physiological processes, such as agonist/hypoxia-induced pulmonary vasoconstriction and vascular remodeling.


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This work is supported in part by National Heart, Lung, and Blood Institute Grants HL-071835 and HL-075134 to J. S. K. Sham, and M. J. Lin is supported by National Science Foundation (Fujian, China) Grant C0620002.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. S. K. Sham, Division of Pulmonary and Critical Care Medicine, Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224 (e-mail: jsks{at}welchlink.welch.jhu.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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