Important role of PLC-γ1 in hypoxic increase in intracellular calcium in pulmonary arterial smooth muscle cells

Vishal R. Yadav, Tengyao Song, Leroy Joseph, Lin Mei, Yun-Min Zheng, Yong-Xiao Wang

Abstract

An increase in intracellular calcium concentration ([Ca2+]i) in pulmonary arterial smooth muscle cells (PASMCs) induces hypoxic cellular responses in the lungs; however, the underlying molecular mechanisms remain incompletely understood. We report, for the first time, that acute hypoxia significantly enhances phospholipase C (PLC) activity in mouse resistance pulmonary arteries (PAs), but not in mesenteric arteries. Western blot analysis and immunofluorescence staining reveal the expression of PLC-γ1 protein in PAs and PASMCs, respectively. The activity of PLC-γ1 is also augmented in PASMCs following hypoxia. Lentiviral shRNA-mediated gene knockdown of mitochondrial complex III Rieske iron-sulfur protein (RISP) to inhibit reactive oxygen species (ROS) production prevents hypoxia from increasing PLC-γ1 activity in PASMCs. Myxothiazol, a mitochondrial complex III inhibitor, reduces the hypoxic response as well. The PLC inhibitor U73122, but not its inactive analog U73433, attenuates the hypoxic vasoconstriction in PAs and hypoxic increase in [Ca2+]i in PASMCs. PLC-γ1 knockdown suppresses its protein expression and the hypoxic increase in [Ca2+]i. Hypoxia remarkably increases inositol 1,4,5-trisphosphate (IP3) production, which is blocked by U73122. The IP3 receptor (IP3R) antagonist 2-aminoethoxydiphenyl borate (2-APB) or xestospongin-C inhibits the hypoxic increase in [Ca2+]i. PLC-γ1 knockdown or U73122 reduces H2O2-induced increase in [Ca2+]i in PASMCs and contraction in PAs. 2-APB and xestospongin-C produce similar inhibitory effects. In conclusion, our findings provide novel evidence that hypoxia activates PLC-γ1 by increasing RISP-dependent mitochondrial ROS production in the complex III, which causes IP3 production, IP3R opening, and Ca2+ release, playing an important role in hypoxic Ca2+ and contractile responses in PASMCs.

  • hypoxia
  • phospholipase c-γ1
  • mitochondria
  • Rieske iron-sulfur protein
  • reactive oxygen species

in response to low alveolar oxygen content, termed hypoxia, pulmonary arteries (PAs) constrict to increase vascular resistance and help direct blood to well-ventilated lung alveoli. This phenomenon is known as hypoxic pulmonary vasoconstriction (HPV), an important physiological response. Persistent HPV can be a critical pathological factor in the development of pulmonary hypertension and right ventricular failure. An increase in intracellular Ca2+ concentration ([Ca2+]i) plays an essential role in producing HPV; however, the underlying signaling mechanisms are not fully understood, and identification of the important molecular players involved in the hypoxic increase in [Ca2+]i in pulmonary arterial smooth muscle cells (PASMCs) and attendant HPV as well as their mechanisms is imperative (6, 22, 30).

Growing evidence suggests that the hypoxic increase in [Ca2+]i in PASMCs is attributed to an increase in intracellular reactive oxygen species (ROS), which are mainly produced by mitochondria and NADPH oxidase (20, 30). The hypoxic generation of mitochondrial ROS is primarily mediated by the mitochondrial electron train chain molecules before the complex III ubisemiquinone site, in which Rieske iron-sulfur protein (RISP) in the complex III is an indispensable element (11, 27). Interestingly, the hypoxic increase in RISP-dependent mitochondrial ROS may cause activation of PKC-ε and subsequently NADPH oxidase, leading to further ROS production; this newly discovered ROS-induced ROS production (RIRP) production plays a significant role in hypoxic Ca2+ and contractile responses in PASMCs (18, 19).

Phospholipase C (PLC) consists of at least 13 divergent isozymes and is important for numerous physiological and pathological cellular responses in the cardiovascular system (4). It has been reported that hypoxia-induced mitogenic factor dose dependently increases [Ca2+]i in cultured human PASMCs; this response is completely abolished by pharmacological inhibition of PLC with U73122 (2), implying the possible role of PLC in the hypoxic Ca2+ response in PASMCs. Intermittent hypoxia, in parallel, increases intracellular ROS generation, augments PLC-γ activity, and increases [Ca2+]i in PC-12 cell line (32). Application of hydrogen peroxide (H2O2), a more diffusible and reactive form of ROS, causes an increase in [Ca2+]i and PLC-γ activity in cultured human venous SMCs, which are blocked by U73122 (5). H2O2 can also enhance the activity of PLC-γ and cell survival in mouse embryonic fibroblasts (28). Moreover, treatment with H2O2 evokes an increase in PLC-γ1 activity and [Ca2+]i in cultured rat astrocytes, and H2O2-evoked increase in [Ca2+]i is blocked by PLC-γ1 gene knockdown (9). These results, together with the facts that mitochondrial ROS are critical for the hypoxic increase in [Ca2+]i in PASMCs (30) and PLC-γ1 is highly expressed in the lungs (8), imply that the activity of PLC-γ1 is augmented by hypoxia as a result of increased mitochondrial ROS production, contributing to the hypoxic Ca2+ response in PASMCs. In this study, thus, we first sought to determine whether hypoxia might activate PLC-γ1 in PASMCs by increasing RISP-dependent mitochondrial ROS production in the complex III.

On activation, PLC hydrolyzes membrane-bound phosphatidylinositol bisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3), which activates IP3 receptors (IP3Rs) on the sarcoplasmic reticulum (SR), inducing Ca2+ release from the SR. Presumably, the increased activity PLC-γ1 during hypoxic exposure may augment IP3 production to induce Ca2+ release through IP3Rs, mediating the hypoxic Ca2+ response in PASMCs. In support of this assumption, application of hypoxia-induced mitogenic factor results in U73122-sensitive increase in [Ca2+]i and IP3 generation in cultured human PASMCs (2). In addition, hypoxia increases IP3 generation as well in pulmonary artery fibroblasts (31). Considering all these facts, in the present study, we also intended to test whether the hypoxic augmentation in the activity of PLC-γ1 could increase IP3 generation and accordingly induce Ca2+ release from the SR, playing an important role in the hypoxic increase in [Ca2+]i in PASMCs.

MATERIALS AND METHODS

Reagents.

Fura-2 AM was purchased from Molecular Probes (Eugene, OR); phospho-PLC-γ1 (Tyr783) antibody from Cell Signaling (Beverly, MA); PLC-γ1 antibody, smooth muscle actin antibody, and protein G-coated beads from Santa Cruz Biotechnology (Santa Cruz, CA); U73122, U73433, 2-aminoethoxydiphenyl borate (2-APB), xestospongin-C, myxothiazol, and hydrogen peroxide from Sigma Aldrich (St. Louis, MO); IP-One HTRF assay kit from Cisbio Bioassays (Bedford, MA); and a set of mouse pLKO.1 lentiviral PLC-γ1 shRNAs (TRC-Mm1.0; catalog no. RMM4534) and lentiviral scrambled (non-silencing) shRNAs (catalog no. RHS4346) from Open Biosystems (Lafayette, CO).

Preparation of isolated blood vessels and vascular SMCs.

All animal experiments were approved by the Institutional Animal Care and Use Committee of Albany Medical College. PAs and PASMCs were prepared from Swiss-Webster mice (Taconic, Germantown, NY), as we described previously (11, 12, 18, 19, 27, 33, 34). Briefly, the heart and lungs were carefully removed from mice and placed in ice-cold physiological saline solution (PSS) containing (in mM): 130 NaCl, 1 MgSO4, 1.8 CaCl2, 5.4 KCl, 10 HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid], and 10 glucose (pH 7.4). Resistance PAs and mesenteric arteries (MAs) were then carefully dissected. To obtain PASMCs, isolated PAs were enzymatically digested using the two-step digestion method, in which the arteries were first incubated in 2-ml PSS containing (mg): 2.4 papain, 0.4 dithioerythritol, and 2 bovine serum albumin (BSA) for 20 min at 37°C (prewarmed for 20 min at 37°C), and then in 1.5-ml PSS (prewarmed at 37°C for 7–8 min) containing (mg): 1.5 collagenase II, 1.2 collagenase F, 1.2 dithiothreitol, and 1.5 BSA for 15 min at 37°C. The digested arteries were incubated in ice-cold PSS containing 1% BSA on ice for 15 min and washed every 5 min. Single cells were harvested by gentle trituration using a Pasteur pipette. To examine the effects of pharmacological reagents, control experiments were carried out in the same mouse cells.

Hypoxic exposure.

Hypoxic responses were obtained by exposing vascular issues and cells to hypoxia for 5 min (11, 12, 18, 19, 27, 33, 34). To test the effect of hypoxia on the activity of PLC, tissues or cells were first incubated in a normoxic PSS (that was continuously aerated with a normoxic gas) for 10 min and then a hypoxic PSS (continuously bubbled with a hypoxic gas) for 5 min. In control experiments, vascular tissues or cells were incubated in the normoxic PSS for 15 min. The composition of PSS was (in mM): 110 NaCl, 3.4 KCl, 2.4 CaCl2, 0.8 MgSO4, 25.8 NaHCO3, 1.2 KH2PO4, and 5.6 glucose (pH 7.4). The normoxic PSS was made by bubbling 20% O2, 5% CO2, 75% N2, whereas the hypoxic PSS was achieved by gassing 1% O2, 5% CO2, 94% N2.

In determination of the effect of hypoxia on [Ca2+]i in PASMCs, single cells were first perfused with the normoxic PSS for 10 min and then with the hypoxic PSS for 5 min. As control, cells were treated identically, but only perfused with the normoxic PSS.

To assess HPV, PAs were incubated in normoxic solution for 90 min. After that, PGF-2α (0.33 μM) was added to cause pretone. When the pretone reached at its plateau, a hypoxic gas mixture (1% O2, 5% CO2, and 94% N2) was applied for 45 min to induce HPV.

Total PLC and PLC-γ1 activity assay.

Total PLC activity was determined using PIP2 assay (25). Twenty microliters of total lysates from isolated vascular tissues or cells were mixed and incubated with 20 μl PLC activity assay buffer containing (in mM) 100 HEPES, 6 EGTA, 160 KCL, and 1 CaCl2 (pH 7.2) with [3H]-PIP2 (10,000 CPM count/reaction) for 30 min at 37°C. The reaction was stopped using 200 μl 10% TCA and 100 μl of 1% BSA. The reaction content was vortexed and centrifuged at 8,600 g for 5 min. Three hundred microliters of the supernatant were mixed with 5 ml Formular-989 liquid scintillation cocktail (Perkin-Elmer, Foster City, CA), and then subjected to scintillation counting to read [3H]-IP3 production from [3H]-PIP2 to reflect PLC activity.

PLC-γ1 activity was assessed by examining phosphorylation of PLC-γ1 at tyrosine 783 using Western blot analysis, as described by Wahl et al. (25) and our group (11, 19, 34). Fifteen micrograms of proteins of cell lysates were loaded onto 8% SDS-PAGE and subsequently transferred to PVDF membranes. The membranes were incubated with primary antibodies for PLC-γ1 or PLC-γ1 phosphorylated on tyrosine 783 at a 1:250 dilution and then secondary horseradish peroxidase-conjugated antibodies at a 1:1,500 dilution. Blots were developed using an enhanced chemiluminescence detection kit (Santa Cruz Biotechnology). The intensity of bands was quantified using Multi Gauge software (Fujifilm, Tokyo, Japan).

Western blotting and immunofluorescence staining.

Western blotting was performed as described above (11, 19, 25, 34). Immunofluorescence staining was conducted as reported previously (27, 33, 34). Primary antibodies against PLC-γ1 at a 1:250 dilution and secondary Alexa488-labeled anti-rabbit fluorescent antibodies at a 1:750 dilution were used. Images were taken using a LSM510 laser scanning confocal microscope (Carl Zeiss, Jena, Germany).

Measurement of [Ca2+]i.

Measurement of [Ca2+]i was made using a fluorescence imaging system (TILL Photonics) (11, 12, 18, 19, 27, 33, 34). PASMCs were incubated with the fluorescent dye Fura-2-AM (10 μM) for 30 min. Fura-2 was excited at 340 and 380 nm, respectively. The emitted fluorescence detected at 510 nm was used to determine [Ca2+]i.

Muscle tension measurement.

Muscle tension measurement was performed using a PowerLab data acquisition system (AD Instruments, Colorado Springs, CO), as described earlier (11, 12, 34). Endothelium-denuded-resistant PA rings were placed in organ bath chambers that contained PSS aerated with a normoxic gas mixture (20% O2, 5% CO2 and 75% N2), given a preload of 800 mg, and equilibrated for 90 min. During an equilibration period of time, the α-adrenergic receptor agonist phenylephrine (10 μM) was applied to induce contraction for the verification of the responsiveness of PAs. Following phenylephrine-induced contraction, acetylcholine (10 μM) was added to confirm no relaxation (i.e., the complete removal of the endothelium). A difference between the basal tension and maximal tension following hypoxic and H2O2 exposure was calculated and normalized to tissue weight (mg/mg), which was presented as hypoxic and H2O2-evoked muscle tension.

Lentiviral shRNA preparation and infection.

Lentiviral particles containing mouse pLKO.1 shRNAs specific for PLC-γ1 and scrambled shRNA were produced using a classic calcium phosphate transfection protocol modified from Icardi et al. (10). Lentiviral packaging pCMV-dR8.2 dvpr and pVSV-G constructs were kindly provided by Dr. Peter A. Vincent (Center for Cardiovascular Sciences, Albany Medical College, Albany, NY). In brief, 11.1 μg of the plasmid shRNAmir vectors were cotransfected with 8.33 μg of pCMV-dR8.2 dvpr and 5.55 μg of pVSV-G in Hek293T cells seeded in a 10-cm dish. Supernatant containing the lentiviral particles was harvested 48 and 72 h after transfection and concentrated by ultracentrifugation for 2 h at 22,000 g in an SW28 rotor at 4°C. Primary cultured PASMCs in DMEM medium were infected with lentiviral particles for 24 h, followed by incubation with the medium in the absence of lentiviral particles for 48 h. Cells were then incubated with puromycin (4 μM) for 48 h to kill the noninfected cells and also ensure near 100% infection of cells. The infected cells were incubated in 0.3% fetal bovine serum medium for 48 h. The knockdown efficiency of each clone was determined using Western blotting. The clones showing maximum knockdown efficiency were selected for further experiments.

IP3 production assay.

Production of IP3 was measured by analyzing production of inositol monophosphate (IP1, a stable downstream metabolite of IP3 in the presence of LiCl) using IP-One HTRF assay (24) according to the manufacturer's protocol. Primary cultured PASMCs were incubated in assay buffer containing LiCl to inhibit IP1 degradation for 1.5 h at 37°C. Eu3+-labeled anti-IP1 antibody (acceptor) was added, followed by IP1-d2 conjugate (donor). After incubation for 1 h at room temperature, the reaction plate wells were excited at 343 nm on a FlexStation-III Microplate Reader (Molecular Devices, Sunnyvale, CA), and emitted light was measured at 620 and 665 nm. The time-resolved fluorescence resonance energy transfer (ratio of 665/620 nm), which is inversely proportional to IP1 amount, was used to determine IP3 production.

Statistical analysis.

All the data were presented as means ± SE. The comparison between two groups was made using Student's t-test. Experiments comprising three or more groups were compared using one-way ANOVA. Values at P < 0.05 were considered statistical significance.

RESULTS

Hypoxia causes an increase in the activity of PLC-γ1 in PASMCs.

It is known that PLC-γ1, which is highly expressed in the lungs (8), can be activated by ROS in several types of cells (5, 9, 28), and ROS serve as essential molecules for hypoxic Ca2+ signaling in PASMCs (30); thus we hypothesized that hypoxia might activate PLC-γ1 in PASMCs. To test this interesting hypothesis, we first investigated whether acute hypoxia could activate PLC in PAs using [3H]-PIP2 assay as described previously (25). The results are shown in Fig. 1A, in which total PLC activity was significantly increased in PAs following acute hypoxic exposure for 5 min compared with control PAs after normoxic exposure for 5 min. The hypoxic increase in the activity of total PLC was completely blocked by pretreatment with the specific PLC inhibitor U73122 (1 μM) for 30 min. On the other hand, U73122 (1 μM) had no effect on the basal activity of PLC in isolated PASMCs (Fig. 1B).

Fig. 1.

Hypoxia causes a significant increase in the activity of total phospholipase C (PLC) in pulmonary arteries (PAs), but not in mesenteric arteries (MAs). A: effect of hypoxia on the activity of total PLC in PAs and MAs. Isolated arteries were first exposed to normoxia for 10 min and then hypoxia or normoxia for 5 min. In experiments testing the effect of PLC inhibition, PAs was treated with U73122 (1 μM) for 30 min before hypoxic exposure. Data in each group were obtained from 4 independent experiments. *P < 0.05 compared with control. B: effect of U73122 on the basal activity of total PLC in PASMCs. PASMCs were pretreated with and without U73122 (1 μM) for 30 min and then subjected to PLC activity assay. Data were obtained from 4 separate experiments.

Considering that hypoxia causes a large increase in [Ca2+]i in PASMCs, but not in mesenteric and other systemic artery myocytes (29), we examined whether hypoxia was unable to augment the activity of total PLC in MAs. As expected, different from PAs, MAs had no change in the activity of total PLC in response to acute hypoxia (Fig. 1A).

To extend the above-described findings, we looked at PLC-γ1 protein expression in PAs. Western blot analysis showed that PLC-γ1 protein was expressed (Fig. 2A). The expression of PLC-γ1 protein in PASMCs was further confirmed by immunofluorescence staining (Fig. 2B).

Fig. 2.

Hypoxia leads to an increase in the activity of PLC-γ1 in pulmonary artery smooth muscle cells (PASMCs), and the hypoxic increase in the activity of PLC-γ1 is blocked by Rieske iron-sulfur protein (RISP) gene knockdown or the mitochondrial complex III inhibitor myxothiazol. A: representative image of Western blot from 3 different experiments illustrates expression of PLC-γ1 and smooth muscle actin protein in PAs. B: immunofluorescence staining of PLC-γ1 protein expression in isolated PASMCs. C: Western blot shows protein expression of PLC-γ1 phosphorylated at tyrosine 783 (pTyr-783) and total PLC-γ1 in PASMCs uninfected (control), infected with lentiviral particles containing shRNAs specific for RISP, and treated with myxothiazol (10 μM) for 15 min. D: bar graph summarizes the ratio of pTyr-783 expression level relative to total PLC-γ1 protein expression level from 3 separate experiments. *P < 0.05 compared with normoxia; #P < 0.05 compared with hypoxia in control (uninfected or untreated cells).

Consistent with the increased activity of total PLC and expression of PLC-γ1, acute hypoxia largely increased the activity of PLC-γ1, assessed by determining phosphorylation of PLC-γ1 at tyrosine 783, as described previously (5, 9, 28), in PASMCs (Fig. 2, C and D). Thus the activity of PLC-γ1 is augmented in PASMCs upon hypoxic stimulation.

Hypoxic increase in the activity of PLC-γ1 in PASMCs is mediated by augmented RISP-dependent mitochondrial ROS production in the complex III.

Our previous studies have demonstrated that RISP-dependent mitochondrial ROS production in complex III plays a vital role in the hypoxic Ca2+ response in PASMCs (11); accordingly, we asked whether RISP-dependent mitochondrial ROS production in the complex III might also be crucial for the hypoxic increase in the activity of PLC-γ1 in PASMCs. To address this interesting question, we used lentiviral shRNAs in PASMCs to specifically knockdown RISP expression and prevent mitochondrial ROS production, as reported in our recent publication (11). Unsurprisingly, we have found that RISP knockdown mostly diminished the hypoxic increase in the activity of PLC-γ1 in PASMCs (Fig. 2, C and D).

In support of the effect of RISP knockdown, treatment with the mitochondrial complex III inhibitor myxothiazol (10 μM) for 15 min to impede mitochondrial ROS production in complex III (30) also inhibited the hypoxia-induced increase in activity of PLC-γ1 in PASMCs (Fig. 2, C and D). Taken together, our present data reveal that the hypoxic increase in the activity of PLC-γ1 in PASMCs is secondary to the increased, RISP-reliant mitochondrial ROS production in the complex III.

PLC-γ1 plays an important role in hypoxia-induced vasoconstriction in PAs and increase in [Ca2+]i in PASMCs.

Because the activity of PLC in PASMCs is significantly augmented by hypoxia (Fig. 2) and the augmented activity of PLC-γ1 is involved in H2O2-evoked increase in [Ca2+]i in astrocytes (9), we assumed that PLC might play an important role in HPV. In agreement with this assumption, we observed that specific inhibition of PLC by treatment with U73122 (1 μM) for 30 min significantly reduced hypoxia-evoked contraction in isolated PAs (Fig. 3A).

Fig. 3.

Pharmacological inhibition of PLC diminishes the hypoxia-induced vasoconstriction in PAs and increase in [Ca2+]i in PASMCs. A: original recording exhibits hypoxia-induced tension (hypoxic pulmonary vasoconstriction, HPV) in an isolated PA. Bar graph summarizes HPV in PAs untreated and treated with the PLC inhibitor U73122 (1 μM) for 30 min. B: quantification of the hypoxic increase in [Ca2+]i in PASMCs untreated and treated with U73122 or U73433 (1 μM) for 30 min.

Comparable to the effect on HPV, treatment with U73122 (1 μM) for 30 min also greatly reduced the hypoxia-induced increase in [Ca2+]i in PASMCs (Fig. 3B). In contrast, its inactive analog U73433 had no effect. Moreover, we found that the basal [Ca2+]i was not altered following treatment with U73122. It is evident that hypoxia increases the activity of PLC, contributing to the hypoxic increase in [Ca2+]i in PASMCs and attendant HPV in PAs.

To provide direct evidence for the functional importance of PLC-γ1 in PASMCs, we wanted to study the effect of PLC-γ1 gene knockdown on hypoxia-induced increase in [Ca2+]i in PASMCs. PLC-γ1 gene knockdown was achieved by infecting mouse pLKO.1 lentiviral particles containing shRNAs specific for PLC-γ1 in PASMCs. As control, cells were infected with lentiviral particles containing scrambled shRNAs. The knockdown efficiency was examined using Western blot analysis. Relative to control (infection of scrambled shRNAs), infection of lentiviral particles comprising Construct 74 shRNAs specific for PLC-γ1 (Reference no. TRCN0000024974) remarkably knocked down PLC-γ1 protein expression (Fig. 4, A and B). In contrast, infection of lentiviral particles containing Construct 75 shRNAs targeted to PLC-γ1 (TRCN0000024975) had no effect. Moreover, infection of lentiviral particles containing Construct 77 and 78 shRNAs targeted to PLC-γ1 (TRCN0000024977 and TRCN0000024978) suppressed PLC-γ1 protein expression as well.

In close agreement with the effect on its protein expression, infection of lentiviral particles consisting of scrambled shRNAs had no effect on the hypoxic increase in [Ca2+]i in PASMCs, infection of lentiviral particles containing Construct 74 shRNAs targeted to PLC-γ1 significantly reduced the hypoxic Ca2+ response, and infection of lentiviral particles comprising construct 75 shRNAs specific for PLC-γ1 did not produce an effect (Fig. 4C). These data prove that PLC-γ1 plays an important role in the hypoxia-induced increase in [Ca2+]i in PASMCs.

Fig. 4.

PLC-γ1 gene knockdown inhibits the hypoxic increase in [Ca2+]i in PASMCs. A: representative Western blot of PLC-γ1 expression in PASMCs infected with mouse pLKO.1 lentiviral particles containing scrambled shRNAs and Construct 74, 75, 77, and 78 shRNAs specific for PLC-γ1. B: quantification of PLC-γ1 protein expression in PASMCs infected with lentiviral particles containing scrambled and specific PLC-γ1 shRNAs. Data were summarized from 3 experiments. *P < 0.05 compared with cells infected with scrambled shRNAs. C: effect of scrambled and PLC-γ1 shRNAs on the hypoxia-induced increase in [Ca2+]i in PASMCs. *P < 0.05 compared with uninfected cells (control).

IP3Rs are involved in hypoxia-induced increase in [Ca2+]i in PASMCs.

It is widely accepted that activation of PLC produces IP3, which opens IP3Rs to induce Ca2+ release from the SR in a variety of cells. As such, we speculated that hypoxia might increase IP3 production in PASMCs. IP3 production was determined by analyzing production of IP1 (a stable downstream metabolite of IP3) using IP-One HTRF assay (24). After acute hypoxia exposure, IP3 production was significantly increased in PASMCs (Fig. 5A). The increased IP3 production was fully abolished by pretreatment with U73122 (1 μM) for 30 min. Noticeably, U73122 did not affect the basal IP3 level.

Fig. 5.

PLC inhibition abolishes the hypoxic increase in inositol 1,4,5-trisphosphate (IP3) production in PAs, and IP3 receptor (IP3R) blockade curbs the hypoxia-induced increase in [Ca2+]i in PASMCs. A: effect of hypoxia on IP3 production in PASMCs untreated and treated with U73122 (1 μM) for 30 min. Data were obtained from 4 separate experiments. *P < 0.05 compared with normoxia (control). B: hypoxic increase in [Ca2+]i in PASMCs untreated and treated with the IP3R inhibitor 2-aminoethoxydiphenyl borate (2-APB) (50 μM) for 30 min. Numbers in parentheses indicate the number of cells tested from at least 3 animals. *P < 0.05 compared with control (cells untreated with 2-APB). C: effect of xestospongin-C (Xest-C, 7.5 μM, 30 min) on the hypoxia-induced increase in [Ca2+]i. *P < 0.05 compared with control (cells untreated with xestospongin-C).

In a match for the hypoxic increase in IP3 production, inhibition of IP3Rs following treatment with its antagonist 2-APB (50 μM) for 30 min significantly decreased the hypoxia-induced increase in [Ca2+]i in PASMCs (Fig. 5B). In addition to its effect on IP3Rs, 2-APB has been shown to block other ion channels as well. Considering this fact, we also examined the effect of xestospongin-C, a more specific IP3R blocker. The results are summarized in Fig. 5C. Similar to 2-APB, application of the IP3R antagonist xestospongin-C (7.5 μM) for 30 min blocked the hypoxic Ca2+ responses as well. The inhibitory effect of these two IP3R antagonists are at one with that of the PLC inhibitor U73122, providing evidence that hypoxia causes activation of PLC and production of IP3, opening of IP3Rs, and Ca2+ release from the SR in PASMCs.

Exogenous ROS, similar to hypoxia, induce an increase in [Ca2+]i in PASMCs and vasoconstriction in PAs via PLC-reliant activation of IP3Rs.

Various studies demonstrate that ROS are the major molecular determinants for the hypoxic increase in [Ca2+]i in PASMCs and associated HPV (30). Our present data unveil that the hypoxic increase in the activity of PLC results from an increase in mitochondrial ROS production, thereby leading to IP3 production, IP3R opening, and Ca2+ release in PASMCs. In light of these facts, we investigated the effect of PLC-γ1 gene knockdown on H2O2-induced increase in [Ca2+]i in PASMCs. Infection of lentiviral particles enclosing construct 77 shRNAs for PLC-γ1, which repressed PLC-γ1 protein expression (Fig. 4, A and B), significantly reduced H2O2-induced increase in [Ca2+]i in PASMCs (Fig. 6A). As control, infection of lentiviral particles composing scrambled shRNAs had no effect.

Fig. 6.

PLC-γ1 reduces H2O2-induced increase in [Ca2+]i in PASMCs and vasoconstriction in PAs. A: effect of PLC-γ1 gene knockdown on H2O2 (500 μM)-induced increase in [Ca2+]i in PASMCs. Cells were uninfected (control) or infected with lentiviral particles containing scrambled shRNAs or Construct 77 shRNAs for PLC-γ1. *P < 0.05 compared with control (uninfected cells). B: summary of H2O2-evoked increase in [Ca2+]i in PASMCs untreated (control) or treated with U73122 or U73433 (1 μM) for 30 min. *P < 0.05 with control. C: H2O2 (500 μM)-induced vasoconstriction in PAs untreated or treated with U73122 or U73433 (1 μM) for 30 min. *P < 0.05 with control.

Likewise, pharmacological inhibition of PLC following application of U73122 (1 μM) for 30 min diminished H2O2-evoked Ca2+ response in PASMCs (Fig. 6B), whereas application of U73433 (1 μM) for 30 min did not inhibit H2O2-induced increase in [Ca2+]i. Moreover, treatment with U73122, but not U73433 (1 μM), for 30 min decreased H2O2-elicited vasoconstriction in PAs (Fig. 6C).

In accordance with PLC inhibition, IP3R inhibition by treatment with 2-APB (50 μM) for 30 min also markedly reduced H2O2-induced increase in [Ca2+]i in PASMCs (Fig. 7A). Besides, treatment with 2-APB (50 μM) for 30 min inhibited H2O2-evoked vasoconstriction in isolated PAs as well (Fig. 7B).

Fig. 7.

IP3R inhibition diminishes H2O2-induced Ca2+ and contractile responses in PASMCs. A: summary of H2O2 (500 μM)-elicited increase in [Ca2+]i in PASMCs pretreated without (control) and with 2-APB (50 μM) for 30 min. B: effect of treatment of 2-APB (50 μM) for 30 min on H2O2 (500 μM)-evoked vasoconstriction in isolated PAs. C: effect of pretreatment with xestospongin-C (7.5 μM) for 30 min on H2O2 (500 μM)-induced increase in [Ca2+]i in PASMCs. D: effect of pretreatment with xestospongin-C (7.5 μM) for 30 min on H2O2 (500 μM)-caused vasoconstriction in PAs. *P < 0.05 compared with control (untreated with either 2-APB or Xest-C).

Consistent with the effect of 2-APB, treatment with the more specific IP3R blocker xestospongin-C (7.5 μM) for 30 min could also significantly reduce H2O2-induced increase in [Ca2+]i in PASMCs (Fig. 7C) and vasoconstriction in isolated PAs (Fig. 7D).

DISCUSSION

It is well documented that hypoxia causes an increase in [Ca2+]i in PASMCs, playing an essential role in hypoxic physiological and pathological cellular responses in the lungs. As recapitulated in our recent review article and others (17, 20, 30), a series of pharmacological and genetic studies from Schumacker's, Perez-Vizcaino's, Wang's and other groups have shown that the hypoxic increase in [Ca2+]i in PASMCs is closely linked to mitochondrial ROS production, particularly in complex III. Our recent investigations have demonstrated that hypoxia can result in ROS production in isolated mitochondria and complex III from PASMCs, and the hypoxic ROS production in mitochondria and complex III are primarily attributed to the RISP-dependent ROS generation system (11). The primary role of RISP in ROS production has been also verified in human 143b and HEK293 cell lines (1, 7). However, the signaling interplay between the hypoxic, RISP-dependent mitochondrial ROS generation and hypoxic Ca2+ response remains incompletely understood. PLC is a family of enzymes that consist of at least 13 different members and function as crucial intracellular signaling molecules in a variety of cells. PLC-γ1 has been shown to be expressed and central for many cellular responses in numerous types of cells. This important PLC member is expressed in the lungs (8); however, its functional importance is largely unknown in PASMCs. In this study, we have discovered that acute hypoxia results in a significant increase in the activity of total PLC in PAs (Fig. 1). The hypoxic increase in the activity of total PLC is completely blocked by pretreatment with PLC inhibitor U73122. Unlike in PAs, hypoxia fails to alter the activity of total PLC in mesenteric arteries. These data are consistent with the widely accepted view that hypoxia causes a large increase in [Ca2+]i in PASMCs, but not in mesenteric and other systemic artery myocytes (29). Using Western blot analysis and immunofluorescence staining, we have found expression of PLC-γ1 protein in PAs and PASMCs (Fig. 2, A and B). Phosphorylation of PLC-γ1 has been commonly recognized to be essential for its activation (16). Our present study has shown that acute hypoxia causes a large increase in phosphorylation of PLC-γ1 in PASMCs (Fig. 2, C and D), thus indicating the hypoxic activation (increased activity) of PLC-γ1. Presumably, PLC-γ1 activation is likely to be involved in the hypoxia-induced increase in [Ca2+]i in PASMCs.

H2O2, a more diffusible and reactive radical, has been reported to cause phosphorylation of PLC-γ1, which results in an increase in cell survival in mouse embryonic fibroblasts (28) and in [Ca2+]i in cultured rat astrocytes (9). We have unveiled that specific knockdown of RISP in the mitochondrial complex III blocks the hypoxic ROS production in isolated PASMCs, mitochondria, and complex III (11). In addition, we have disclosed that RISP gene knockdown eliminates the hypoxia-induced activation (increase in phosphorylation) of PLC-γ1 in PASMCs (Fig. 2, C and D). Furthermore, treatment with myxothiazol, a mitochondrial complex III inhibitor, to inhibit ROS generation blocks the hypoxic response as well. Taken together, hypoxia may give rise to an increase in the activity of PLC-γ1 in PASMCs due to the augmented mitochondrial ROS production via RISP-based ROS production system in the complex III.

Consistent with our conjecture that activation of PLC-γ1 may contribute to hypoxic cellular responses in PASMCs, we have discovered that treatment with the specific PLC inhibitor U73122 markedly inhibits the hypoxia-evoked vasoconstriction in endothelium-denuded isolated PAs (Fig. 3A). The successful denudation of the endothelium was confirmed by the absence of acetylcholine-induced relaxation. The full HPV is held to require an intact endothelium; however, this unique response occurs primarily in PASMCs (6, 22). Thus our data suggest that PLC-γ1 in PASMCs is important for the development of HPV. In support, a recent study has also revealed that U73122 dose-dependently inhibits pulmonary vasoconstriction in isolated lungs following alveolar hypoxia (3).

Treatment with U73122 also diminishes the hypoxic increase in [Ca2+]i in PASMCs (Fig. 3B). In contrast, U73443, an inactive analog of U73122, does not produce an inhibitory effect. To extend these pharmacological findings, we have further examined the effect of PLC-γ1 gene knockdown on the hypoxic response in PASMCs. The results indicate that lentiviral shRNA-mediated PLC-γ1 gene knockdown greatly suppresses its protein expression in PASMCs (Fig. 4, A and B). More importantly, we have found that PLC-γ1 knockdown significantly inhibits the hypoxic increase in [Ca2+]i (Fig. 4C). Thus PLC-γ1 plays a significant role in the hypoxic Ca2+ signaling in PASMCs. In agreement with our findings, U73122 has been shown to be able to block HPV in isolated lungs (3). Furthermore, the increase in [Ca2+]i induced by hypoxia-induced mitogenic factor in cultured human PASMCs is eliminated by U73122 (2). In contrast, Tang et al. (23) have reported that U73122 only produces a slight (∼20%), although statistically significant, inhibition of the hypoxic increase in [Ca2+]i in cultured human PASMCs (23). Noticeably, the “hypoxic gas” in their report did not contain O2, whereas 1% O2 was included in our hypoxic gas. This dissimilar hypoxic gas may account for the different extent of U73122-mediated inhibition of the hypoxic response in PASMCs observed in these two studies. In fact, 1% O2 is very often applied to induce hypoxic responses in isolated PASMCs, PAs, and lungs although no O2 is also used in many studies (6, 22, 30). As for the normoxic gas, there appears to be no exception using ∼20% O2 (the O2 content in the air). This is probably based on the fact that PASMCs may encounter the air in the lungs.

The functional importance of PLC can be often implemented by its product IP3, which opens IP3Rs on the SR and subsequently induces Ca2+ release, mediating numerous cellular responses in many types of cells. Because hypoxia gives rise to a large increase in the activity of PLC-γ1 and inhibition of PLC-γ1 brings about a large reduction in the hypoxic increase in [Ca2+]i in PASMCs, we anticipated that hypoxia may also cause a significant increase in IP3 production, IP3R opening and SR Ca2+ release. In accord with our expectation, we have observed that hypoxia results in a significant increase in production of IP3 in PASMCs (Fig. 5A). The hypoxic increase in IP3 production is fully abolished by pretreatment with the specific PLC inhibitor U73122. Along with these results, treatment with 2-APB to inhibit IP3Rs remarkably inhibits the hypoxic increase in [Ca2+]i in PASMCs (Fig. 5B). It is known that 2-APB inhibits multiple ion channels such as store-operated Ca2+ channels that are involved in hypoxic responses in PASMCs (6, 22, 30). In consolidation of the role of 2-APB as a result of the inhibition of IP3Rs, application of the more specific IP3R blocker xestospongin-C can reduce the hypoxic response as well. It is interesting to point out that a recent report has also shown that xestospongin-C produces an inhibitory effect on the hypoxic Ca2+ release in rat PASMCs (26). In concert, the role of PLC-γ1 in mediating the hypoxic increase in [Ca2+]i in PASMCs is due to IP3R-mediated Ca2+ release from the SR as a result of the increased IP3 production.

As described in our recent review articles and others (6, 22, 30, 35), a number of studies from our laboratory and other groups have shown that IP3Rs are highly colocalized with ryanodine receptors (RyRs) in PASMCs; as such, Ca2+ release via IP3Rs may activate RyRs to cause further Ca2+ release, i.e., IP3R/RyR interaction-mediated local Ca2+-induced Ca2+ release (CICR). This local CICR largely amplifies agonist-induced cellular responses. RyR antagonists, like IP3R inhibitors, also block the hypoxic increase in [Ca2+]i in isolated PASMCs and HPV in isolated PAs. All the three known RyR subtypes (RyR1, RyR2, and RyR3) are expressed in PASMCs and involved in hypoxic Ca2+ and contractile responses in PASMCs. However, RyR2 is the most important player as RyR2 gene knockout completely abolishes, whereas RyR1 and RyR only partially inhibit, the hypoxic increase in [Ca2+]i and contraction in PASMCs. We have further demonstrated that RyR2 amplifies IP3R-mediated Ca2+ release following stimulation of G protein-coupled receptors and intracellular application of exogenous IP3 in airway SMCs (15). Apparently, it would be intriguing to perform additional studies in the future to determine whether RyR2 may mediate PLC-γ1-dependent, IP3R-mediated hypoxic cellular responses in PASMCs.

In addition to IP3R opening, another known downstream consequence of the increased activity PLC-γ1 is the activation of PKC. Our previous studies have shown that activation of PKC, particularly PKC-ε, occurs in response to hypoxia in PASMCs (18). Using pharmacological and genetic approaches, we and others have revealed that PKC-ε plays a significant role in the hypoxic increase in [Ca2+]i and contraction in PASMCs (18) as well as HPV in isolated lungs (14). The hypoxic activation of PKC-ε is secondary to the increase in mitochondrial ROS production (18, 19). It is also interesting to point out that mitochondrial ROS-dependent activation of PKC-ε can subsequently increase the activity of NADPH oxidase, inducing further ROS production. This novel RIRP process makes contributes to hypoxic Ca2+ and contractile responses in PASMCs (19). Thus further studies in the future are worth pursuing to explore whether PLC-γ1 may mediate the hypoxic activation of PKC-ε and its role in hypoxic cellular responses in PASMCs.

Exogenous ROS, mimicking hypoxia, lead to an increase in the activity of PLC-γ1 and [Ca2+]i in cultured astrocytes (9), whereas inhibition of mitochondrial ROS production blocks the hypoxic activation of PLC-γ1 (Fig. 2, C and D) and increase in [Ca2+]i in PASMCs (11, 30). Conceivably, PLC inhibition may also produce an oppressive effect on H2O2-evoked increase in [Ca2+]i in PASMCs, as it does hypoxic response. In support, specific PLC-γ1 gene knockdown reduces H2O2-elicited Ca2+ response in PASMCs (Fig. 6A). Likewise, we have shown that treatment with U73122 to block PLC can block H2O2-induced increase in [Ca2+]i as well (Fig. 6B). In agreement with our data, an earlier publication has reported that H2O2-induced pulmonary vasoconstriction in isolated rabbit PAs is blocked by the PLC inhibitor 2-nitro-4-carboxy-phenyl-N,N-diphenylcarbamate (21). Akin to the PLC inhibition, IP3R blockade following treatment with 2-APB and xestospongin-C can also significantly curb H2O2-induced increase in [Ca2+]i in PASMCs and vasoconstriction in PAs (Fig. 7). A similar inhibitory effect on H2O2-induced increase in [Ca2+]i has also been observed in isolated rat PASMCs (13). All these data further support our concept that ROS are the important signaling molecules to mediate the hypoxic Ca2+ response in PASMCs by causing PLC-γ1-dependent opening of P3Rs.

In conclusion, our present studies utilizing combined biochemical, genetic, pharmacological, and physiological approaches present comprehensible evidence that acute hypoxia causes a significant increase in the activity of PLC-γ1, which plays an important role in the hypoxic increase in [Ca2+]i and attendant contraction in PASMCs. The hypoxic increase in the activity of PLC-γ1 is secondary to the increased mitochondrial ROS production primarily through RISP-based ROS generation system in complex III. The role of PLC-γ1 in mediating hypoxic Ca2+ and contractile responses in PASMCs is a result of the increased PLC-γ1-reliant IP3 production, IP3R opening, and SR Ca2+ release.

GRANTS

This work was supported by AHA Established Investigator Award 0340160N (Y.-X. Wang) and Scientist Development Grant 0630236N (Y.-M. Zheng), and NIH R01HL64043, HL064043-S1, HL075190, and HL108232 (Y.-X. Wang).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

Author contributions: V.R.Y., Y.-M.Z., and Y.-X.W. conception and design of research; V.R.Y., T.S., L.J., L.M., and Y.-M.Z. performed experiments; V.R.Y., L.J., and L.M. analyzed data; V.R.Y., T.S., L.J., L.M., Y.-M.Z., and Y.-X.W. interpreted results of experiments; V.R.Y., L.J., L.M., and Y.-X.W. prepared figures; V.R.Y. drafted manuscript; V.R.Y., T.S., L.J., L.M., Y.-M.Z., and Y.-X.W. edited and revised manuscript; V.R.Y., T.S., L.J., L.M., Y.-M.Z., and Y.-X.W. approved final version of manuscript.

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