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(Circulation Research. 2006;98:1528.)
© 2006 American Heart Association, Inc.

Cellular Biology

Hypoxia Inducible Factor 1 Mediates Hypoxia-Induced TRPC Expression and Elevated Intracellular Ca²⁺ in Pulmonary Arterial Smooth Muscle Cells

Jian Wang, Letitia Weigand, Wenju Lu, J.T. Sylvester, Gregg L. Semenza, Larissa A. Shimoda

From the Division of Pulmonary and Critical Care Medicine (J.W., L.W., W.L., J.T.S., L.A.S.); Vascular Biology Program (G.L.S.), Institute for Cell Engineering; Departments of Medicine (J.W., L.W., W.L., J.T.S., G.L.S., L.A.S.), Pediatrics (G.L.S.), Oncology (G.L.S.), and Radiation Oncology (G.L.S.); and McKusick–Nathans Institute of Genetic Medicine (G.L.S.), The Johns Hopkins University School of Medicine, Baltimore, Md.

Correspondence to Larissa A. Shimoda, PhD, Division of Pulmonary and Critical Care Medicine, Johns Hopkins University, 5501 Hopkins Bayview Circle, JHAAC 4B.82B, Baltimore, MD 21224. E-mail shimodal{at}welch.jhu.edu

	Abstract

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Chronic hypoxia (CH) causes pulmonary vasoconstriction because of increased pulmonary arterial smooth muscle cell (PASMC) contraction and proliferation. We previously demonstrated that intracellular Ca²⁺ concentration ([Ca²⁺]_i) was elevated in PASMCs from chronically hypoxic rats because of Ca²⁺ influx through pathways other than L-type Ca²⁺ channels and that development of hypoxic pulmonary hypertension required full expression of the transcription factor hypoxia inducible factor 1 (HIF-1). In this study, we examined the effect of CH on the activity and expression of store-operated Ca²⁺ channels (SOCCs) and the regulation of these channels by HIF-1. Capacitative Ca²⁺ entry (CCE) was enhanced in PASMCs from intrapulmonary arteries of rats exposed to CH (10% O₂; 21 days), and exposure to Ca²⁺-free extracellular solution or SOCC antagonists (SKF96365 or NiCl₂) decreased resting [Ca²⁺]_i in these cells. Expression of TRPC1 and TRPC6, but not TRPC4, mRNA and protein was increased in PASMCs from rats and wild-type mice exposed to CH, in PASMCs from normoxic animals cultured under hypoxic conditions (4% O₂; 60 hours), and in PASMCs in which HIF-1 was overexpressed under nonhypoxic conditions. Hypoxia-induced increases in basal [Ca²⁺]_i and TRPC expression were absent in mice partially deficient for HIF-1. These results suggest that increased TRPC expression, leading to enhanced CCE through SOCCs, may contribute to hypoxic pulmonary hypertension by facilitating Ca²⁺ influx and increasing basal [Ca²⁺]_i in PASMCs and that this response is mediated by HIF-1.

Key Words: Ca²⁺ channels • hypoxia • hypoxia-inducible factor 1 • hypoxic pulmonary vasoconstriction • vascular smooth muscle

	Introduction

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Prolonged exposure to alveolar hypoxia is associated with changes in the pulmonary vasculature including structural remodeling^1,2 and active contraction of vascular smooth muscle.^3,4 Recent evidence suggests that the latter may play a more prominent role in the elevation of pulmonary vascular resistance because the thickened smooth muscle cell layer caused by chronic hypoxia (CH) in small pulmonary arteries has been found to have no significant impact on luminal diameter.⁵ Moreover, inhibition of Rho kinase, a mediator of myofilament contractility, has been shown to acutely reverse the increase in pulmonary arterial pressure in chronically hypoxic rats.⁶ Based on these findings, it appears that contraction of smooth muscle during CH is the major factor contributing to the pathogenesis of hypoxic pulmonary hypertension.

Although the cellular mechanisms underlying the development of pulmonary hypertension remain poorly understood, both growth and contraction of pulmonary arterial smooth muscle cells (PASMCs) are associated with alterations in Ca²⁺ homeostasis. We previously demonstrated that basal intracellular Ca²⁺ concentration ([Ca²⁺]_i) was increased in PASMCs from chronically hypoxic rats,⁷ indicating profound changes in Ca²⁺ regulation. Because PASMCs from chronically hypoxic animals are depolarized, presumably secondary to decreased voltage-gated K⁺ channel activity,^8–10 it was hypothesized that an increase in [Ca²⁺]_i attributable to activation of voltage-dependent Ca²⁺ channels (VDCCs) is the mechanism underlying hypoxic pulmonary hypertension.^9,10 This possibility was contradicted, however, by data indicating that VDCC antagonists did not prevent development of hypertension secondary to CH^4,11,12 and that acute administration of vasodilators,³ but not VDCC antagonists,^11,13 reduced pulmonary artery pressure in patients with hypoxic pulmonary hypertension. Furthermore, we demonstrated that removal of extracellular Ca²⁺, but not VDCC blockers, decreased [Ca²⁺]_i during CH,⁷ indicating a role for Ca²⁺ influx through pathways other than VDCCs. Similarly, removal of extracellular Ca²⁺ relaxed arteries from chronically hypoxic rats, whereas blockade of VDCCs had no effect.⁷ These data confirmed that the CH-induced increase in resting [Ca²⁺]_i was responsible for maintaining pulmonary vasoconstriction but eliminated a role for VDCCs.

In addition to VDCCs, Ca²⁺ influx in PASMCs can also occur via nonselective cation channels, which include both receptor-operated Ca²⁺ channels and store-operated Ca²⁺ channels (SOCCs). In contrast to VDCCs, these channels are not activated by depolarization. Instead, receptor-operated channels are activated by ligand binding to membrane receptors, whereas SOCCs are activated by depletion of intracellular stores. In the case of SOCCs, the Ca²⁺ influx induced by store depletion is believed to replenish stores and is termed capacitative Ca²⁺ entry (CCE). CCE through SOCCs is present in PASMCs^14–16 and studies using inhibitors of SOCCs revealed a role for these channels in PASMC contraction and growth.^14,17–20 SOCCs are believed to be composed of mammalian homologs of transient receptor potential (TRP) proteins. The exact molecular identity of the proteins encoding SOCCs remains unclear, although isoforms in the canonical TRP (TRPC) subfamily are primary candidates.^18,21,22 Recently, we²³ and others²⁴ have demonstrated that exposure to CH enhanced the expression of TRPC proteins and activation of SOCCs, contributing to the maintenance of elevated basal [Ca²⁺]_i in PASMCs. However, the mechanism by which hypoxia induces the expression of these channels remains unknown.

Numerous adaptive responses to hypoxia are mediated by the transcription factor, hypoxia inducible factor 1 (HIF-1). HIF-1 exists as a heterodimer, consisting of HIF-1 and HIF-1ß subunits.²⁵ HIF-1ß is ubiquitously overexpressed, whereas HIF-1 is found in low levels under normoxic conditions because of ubiquitination and proteasomal degradation.^26–28 During hypoxia, decreased ubiquitination correlates with rapid stabilization of HIF-1 protein,²⁸ followed by accumulation in the nucleus,²⁶ where HIF-1dimerizes with HIF-1ß and binds to the core DNA sequence 5'-RCGTG-3',²⁹ resulting in the transactivation of numerous target genes.³⁰ Thus, HIF-1 confers sensitivity and specificity for hypoxic induction.

In studies using transgenic mice heterozygous for a null allele at the Hif1a locus encoding HIF-1 (Hif1a^+/–), we found that the development of pulmonary hypertension, polycythemia, and vascular remodeling was blunted and the electrophysiologic changes in PASMCs induced by hypoxia were markedly attenuated in Hif1a^+/– mice exposed to CH.^31,32 The transcriptional regulation of TRPC proteins is just beginning to be explored and the role of HIF-1 in the oxygen-dependent regulation of TRPCs has not been studied. Therefore, we tested the hypothesis that HIF-1 regulates the maintenance of elevated resting [Ca²⁺]_i in pulmonary vascular smooth muscle during CH via induction of TRPCs proteins and enhanced Ca²⁺ influx through SOCCs.

	Materials and Methods

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An expanded Materials and Methods section can be found in the online data supplement available at http://circres.ahajournals.org.

Chronic Hypoxic Exposure
All procedures were approved by the Animal Care and Use Committee of The Johns Hopkins University School of Medicine. Adult male Wistar rats (200 to 350 g) or mice (8 weeks) were exposed to normoxia or hypoxia (10% O₂) for 21 days as previously described.^7,31

Cell Isolation and Culture
The method for obtaining single PASMCs has been described previously.^7,31 Rat PASMCs were transiently cultured in Ham’s F-12 medium with L-glutamine (Mediatech) supplemented with 0.5% FCS, 1% streptomycin, and 1% penicillin for 24 to 48 hours. Murine PASMCs were cultured in SmBM (Cambrex) complete media supplemented with 10% FCS 24 to 48 hours and placed in serum-free media 24 hours before experiments.

Measurement of Intracellular Ca²⁺
[Ca²⁺]_i was measured in single PASMCs using FURA-2 dye and fluorescent microscopy as previously described.¹⁶

Isometric Tension Recording
The methods for measurement of isometric tension recording in pulmonary arteries have been previously described.⁷

Reverse-Transcription PCR
Total RNA was prepared from endothelium-denuded intralobar pulmonary arteries by TRIzol extraction. Two arteries each from 3 rats or mice were combined per sample. Reverse transcription and PCR were performed as described previously¹⁶ and in the online data supplement.

Immunoblot Assays
For each sample, cells from 1 to 3 animals were isolated grown in 60-mm Petri dishes for 3 to 5 days and then serum starved for 24 hours before harvest. Aliquots of cell lysates were subjected to immunoblot assay as described previously¹⁶ and in the online data supplement.

Adenovirus Infection
PASMCs were cultured to 80% confluence in Ham’s media. After 24 hours in serum-free media, cells were infected with replication-defective recombinant adenoviruses encoding either ß-galactosidase (AdLacZ) or a constitutively active form of HIF-1 (AdCA5), which contains mutations that inhibit degradation of the protein under nonhypoxic conditions.^33,34 PASMCs were inoculated with 50 plaque-forming units (pfu) per cell and incubated for 48 hours at 37°C under nonhypoxic conditions.

	Results

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Role of Ca²⁺ Influx Through SOCCs in Maintenance of Elevated Baseline [Ca²⁺]_i in PASMCs From Chronically Hypoxic Rats
Basal [Ca²⁺]_i was significantly greater in PASMCs from chronically hypoxic rats, consistent with our previous observations.⁷ In PASMCs isolated from normoxic animals and perfused for 10 minutes with modified Krebs solution containing the SOCC inhibitor SKF96362 (SKF) (50 µmol/L) or NiCl₂ (500 µmol/L) at concentrations that inhibited CCE by >80%,¹⁶ neither inhibitor altered resting [Ca²⁺]_i (149.3±20.5 to 163.7±22.6 nmol/L, n=8 experiments in 76 cells for SKF; and 144.8±20.1 to 146.2±19.9 nmol/L, n=8 experiments in 75 cells for NiCl₂). In contrast, SKF decreased baseline [Ca²⁺]_i from 234±11.5 to 186.5±16.2 nmol/L (Figure 1A; n=9 experiment in 87 cells) in PASMCs from chronically hypoxic rats. Similarly, NiCl₂ decreased resting [Ca²⁺]_i from 233±9.6 to 156.1±14.8 nmol/L in PASMCs following exposure to CH (Figure 1B; n=5 experiments in 52 cells).

View larger version (25K): [in this window] [in a new window]   Figure 1. A and B, Role of Ca2+ influx through SOCCs in maintenance of basal [Ca2+]i in PASMCs from normoxic (N) and chronically hypoxic (CH) rats. Left, PASMCs were perfused with solution containing the SOCC inhibitors SKF96365 (A) or NiCl2 (B). Right, Bar graphs representing mean change in [Ca2+]i ( [Ca2+]i) in response to SKF96365 or NiCl2 in PASMCs from normoxic and chronically hypoxic rats. *P<0.01 between normoxia and chronic hypoxia values. C and D, Role of Ca2+ influx through SOCCs in maintenance of active tone in pulmonary arteries from normoxic and chronically hypoxic rats. Left, Time course of change in basal tension (% of the initial tension) following addition of SKF96356 (C) or NiCl2 (D). Right, Bar graphs representing the mean±SEM percent change in tension (% Tension) measured 20 minutes after treatment.

Effect of CCE Inhibitors on Baseline Tension in Rat Intrapulmonary Arteries
To determine whether Ca²⁺ entry through SOCCs contributes to the maintenance of tone, the effects of SKF and NiCl₂ on baseline tension were examined in both normoxic and chronically hypoxic intrapulmonary arteries. In arterial rings from normoxic rats, treatment for 20 minutes with SKF (50 µmol/L) and NiCl₂ (500 µmol/L) had little effect on tension, causing decreases of 1.9±0.7% (n=5) and 1.5±0.9% (n=5), respectively (Figure 1C and 1D). In contrast, chronically hypoxic intrapulmonary arteries treated with SKF exhibited a significant decrease in tension of 11.8±4.3% (n=5). Similarly, NiCl₂ caused a 10.1±5.1% decrease in baseline tension in intrapulmonary arteries from chronically hypoxic rats (n=4).

Assessment of CCE in PASMCs From Normoxic and Chronically Hypoxic Rats
CCE was assessed by restoration of extracellular calcium to PASMCs perfused for 10 minutes with Ca²⁺-free modified Krebs solution containing 10 µmol/L cyclopiazonic acid (CPA) to deplete intracellular calcium stores, 5 µmol/L nifedipine to prevent calcium entry through VDCCs, and 0.5 mmol/L EGTA to chelate any residual Ca²⁺. [Ca²⁺]_i was measured at 1-minute intervals before and after restoration of extracellular [Ca²⁺] to 2.5 mmol/L (by perfusion with normal modified Krebs solution containing CPA and nifedipine) and CCE was evaluated by the increase in [Ca²⁺]_i caused by restoration of extracellular [Ca²⁺]_i. As shown in Figure 2A, CPA given in the absence of extracellular Ca²⁺ and the presence of nifedipine caused a small, transient increase in [Ca²⁺]_i. The CPA-induced increase in [Ca²⁺]_i was similar in PASMCs from normoxic and chronically hypoxic rats ([Ca²⁺]_i=92.3±42.3 nmol/L, n=8 experiments in 125 cells for normoxic; and 133.9±43.3 nmol/L, n=5 experiments in 73 cells for chronically hypoxic cells). Subsequent restoration of extracellular Ca²⁺ induced a second increase in [Ca²⁺]_i, which rose quickly to a peak ([Ca²⁺]_i=385.2±47.9 nmol/L; n=8 experiments in 93 cells) in PASMCs from hypoxic rats. This peak change in [Ca²⁺]_i was significantly greater than that measured in PASMCs from normoxic rats ([Ca²⁺]_i= 189.2±10.6 nmol/L; n=5 experiments in 53 cells).

View larger version (26K): [in this window] [in a new window]   Figure 2. Effect of exposure to chronic hypoxia on CCE. A, Change in [Ca2+]i in PASMCs from normoxic (N) or chronically hypoxic (CH) rats and subjected to restoration of extracellular Ca2+ following store depletion with CPA. Bar graph illustrates mean±SEM change in [Ca2+]i ([Ca2+]i) in response to Ca2+ restoration. All experiments were performed in the presence of nifedipine. B and C, Comparison of Mn2+ quenching of Fura-2 fluorescence in PASMCs from normoxic and chronically hypoxic rats. Composite traces (left) and bar graphs (right) illustrating the time course and magnitude of Mn2+ quenching. For each experiment, the fluorescence as a function of time (F) was normalized to the fluorescence value measured immediately before addition of Mn2+ (F0). D, Effect of SKF96365 and NiCl2 on Mn2+ quenching of Fura-2 fluorescence in unstimulated cells (absence of CPA) from chronically hypoxic rats. *P<0.01 compared with normoxia (A through C) or control (D).

To confirm that the response to restoration of extracellular Ca²⁺ reflected Ca²⁺ influx, we measured the effect of extracellular MnCl₂ (Mn²⁺; 200 µmol/L) on Fura-2 fluorescence excited at 360 nm (F₃₆₀) at 30-second intervals in PASMCs perfused with Ca²⁺-free modified Krebs solution containing nifedipine. EGTA was omitted in these experiments as it chelates Mn²⁺. CCE was evaluated by the rate at which F₃₆₀ was quenched by Mn²⁺, which enters the cell as a Ca²⁺ surrogate and reduces fluorescence on binding to the dye. F₃₆₀ is the same for Ca²⁺-bound and Ca²⁺-free Fura-2; therefore, changes in fluorescence can be assumed to be caused by Mn²⁺ alone. In the absence of Mn²⁺, F₃₆₀ decreased slowly in cells from normoxic (n=5 experiments in 67 cells) and chronically hypoxic (n=4 experiments in 55 cells) rats, suggesting minimal photobleaching of the dye (data not shown). The ability of Mn²⁺ to quench F₃₆₀ was assessed both in the absence and presence of CPA, to allow measurement of influx under resting conditions and when SOCCs were maximally activated, respectively. In untreated cells, F₃₆₀ decreased in PASMCs from both normoxic and chronically hypoxic rats (Figure 2B), although the rate of quenching was significantly greater in cells from hypoxic animals at 23.3±3.2% (n=7 experiments in 95 cells) compared with 15.5±1.0% (n=10 experiments in 138 cells) in normoxic cells. As shown in Figure 2C, quenching of F₃₆₀ by Mn²⁺ in the presence of CPA was 61.0±2.9% in PASMCs from chronically hypoxic rats (n=5 experiments in 61 cells), significantly greater than that observed in the absence of CPA. With CPA, F₃₆₀ decreased by only 35.9±3.3% in PASMCs from normoxic rats (n=9 experiments in 112 cells), suggesting that Ca²⁺ entry through SOCCs was greater in PASMCs from chronically hypoxic rats both at baseline and when stores are depleted.

We verified that increased Mn²⁺ quenching of dye in PASMCs from chronically hypoxic rats was attributable to Ca²⁺ influx through SOCCs by measuring the effects of SKF and NiCl₂ on the response (Figure 2D). In the presence of either SKF or NiCl₂ the Mn²⁺-induced decrease in F₃₆₀ was significantly reduced to 11.4±1.3% (n=5 experiments in 76 cells) and 14.2±0.9% (n=5 experiments in 86 cells), respectively, levels similar to that observed in PASMCs from normoxic animals.

TRPC Expression in Pulmonary Vascular Smooth Muscle From Chronically Hypoxic and Normoxic Rats
SOCCs are likely composed of TRPC proteins.^21,22 We previously demonstrated the expression of 3 TRPC isoforms in cultured myocytes from rat distal pulmonary arteries: TRPC1, TRPC4, and TRPC6.¹⁶ In the present study, we used RT-PCR and immunoblot assays to determine whether the increase in CCE observed in PASMCs from chronically hypoxic rats was associated with increased expression of these channel proteins. We found that exposure to CH increased both TRPC1 and TRPC6 mRNA (Figure 3A) and protein (Figure 3B) levels in endothelium-denuded intralobar pulmonary arteries. In contrast, hypoxia had no effect on TRPC4 mRNA or protein levels.

View larger version (36K): [in this window] [in a new window]   Figure 3. Effect of chronic hypoxia on TRPC expression. TRPC1, TRPC4, and TRPC6 mRNA (A) and protein (B) expression were analyzed in pulmonary arterial tissue from chronically hypoxic (CH) and normoxic (N) rats. Bar graphs show mean±SEM data for TRPC mRNA expression normalized to ß-actin mRNA and TRPC protein expression normalized to -actin protein (n=3 to 4 separate experiments on samples pooled from 3 to 4 animals; *P<0.05).

TRPC Expression in PASMCs Cultured Under Hypoxic Conditions
To distinguish between a direct effect of hypoxia on TRPC expression and effects of circulating factors or increased pulmonary arterial pressure that may occur during pulmonary hypertension, we isolated PASMCs from normoxic rats and cultured these cells under hypoxic conditions (4% O₂; 60 hours). Hypoxia induced a significant increase in TRPC1 and TRPC6 mRNA and protein levels (Figure 4A and 4B) but had no effect on TRPC4 expression. As shown in Figure 4C, the increase in TRPC expression was associated with an increase in basal [Ca²⁺]_i, from 120.3±5.6 nmol/L in PASMCs cultured under nonhypoxic conditions (n=9 experiments in 190 cells) to 191.0±9.5 nmol/L in PASMCs cultured under hypoxic conditions (n=6 experiments in 141 cells).

View larger version (45K): [in this window] [in a new window]   Figure 4. Exposure of cultured PASMCs to hypoxia increased TRPC expression and [Ca2+]i. TRPC1, TRPC4, and TRPC6 mRNA (A) and protein (B) expression in PASMCs from normoxic rats cultured under nonhypoxic (N) and hypoxic (H) conditions (4% O2; 60 hours). Bar graphs show TRPC expression normalized to - or ß-actin (mean±SEM; n=3 independent experiments). C, Analysis of [Ca2+]i in PASMCs cultured under nonhypoxic and hypoxic conditions. *P<0.05.

Effect of Partial Deficiency of HIF-1 on Basal [Ca²⁺]_i and TRPC Expression in Chronically Hypoxic Mice
CH induced a significant increase in basal [Ca²⁺]_i in murine PASMCs, which could be reversed with removal of extracellular Ca²⁺ (n=4 experiments in 103 cells) or application of SKF (n=3 experiments in 68 cells) or NiCl₂ (n=3 experiments in 70 cells) (Figure 5A). CCE, measured by Ca²⁺ restoration following store depletion, was also significantly enhanced in PASMCs from chronically hypoxic mice (Figure 5B). Basal [Ca²⁺]_i was similar in PASMCS from normoxic Hif1a^+/+ (121.2±3.8 nmol/L; n=10 experiments in 10 cells) and Hif1a^+/– (117.1±12.6 nmol/L; n=13 experiments in 13 cells) littermate mice (Figure 5C). Exposure to CH resulted in a significant increase in [Ca²⁺]_i in PASMCs from Hif1a^+/+ mice, to 182.7±27.4 nmol/L (n=16 experiments in 16 cells), an increase similar in magnitude to that observed in the chronically hypoxic rats. In contrast, basal [Ca²⁺]_i in PASMCs from chronically hypoxic Hif1a^+/– mice (128.3±8.9 nmol/L; n=17 experiments in 17 cells) was similar to that in PASMCs from normoxic Hif1a^+/– mice, indicating a loss of hypoxia-induced increase in [Ca²⁺]_i in mice partially deficient for HIF-1. Immunoblot analysis confirmed that PASMCs from Hif1a^+/+ mice exhibited significantly greater HIF-1 protein during hypoxia (1% O₂; 6 hours) compared with PASMCs from Hif1a^+/– mice, although -actin expression was similar in both groups (Figure 5D).

View larger version (28K): [in this window] [in a new window]   Figure 5. A, Composite traces showing removal of extracellular Ca2+ and application of SKF96365 or NiCl2 decreased basal [Ca2+]i in PASMCs from chronically hypoxic but not normoxic mice. B, Change in [Ca2+]i in PASMCs from normoxic (N) or chronically hypoxic (CH) mice and subjected to restoration of extracellular Ca2+ following store depletion with CPA. Bar graph illustrates mean±SEM change in [Ca2+]i ([Ca2+]i) in response to Ca2+ restoration. All experiments were performed in the presence of nifedipine. C, Resting [Ca2+]i is increased in PASMCs from hypoxic Hif1a+/+ mice but not Hif1a+/– mice. Mean±SEM data for each group derived from analysis of 10 to 20 cells from 3 animals. D, PASMCs from Hif1a+/+ mice exhibited greater HIF-1 protein during hypoxia compared with PASMCs from Hif1a+/– mice. Similar results were observed in two other experiments. *P<0.05.

Consistent with our results in rats, exposure to CH increased TRPC1 and TRPC6 mRNA (Figure 6A) and protein expression (Figure 6B) but had no effect on TRPC4 expression (data not shown) in endothelium-denuded pulmonary arteries of Hif1a^+/+ mice. The hypoxia-induced increase in TRPC1 and TRPC6 mRNA and protein was absent in pulmonary arteries from Hif1a^+/– mice.

View larger version (40K): [in this window] [in a new window]   Figure 6. Effect of hypoxia on TRPC expression in Hif1a+/+ and Hif1a+/– mice. TRPC1 and TRPC6 mRNA (A) and protein (B) expression in pulmonary arterial tissue from hypoxic (CH) and normoxic (N) Hif1a+/+ mice. Bar graphs represent mean±SEM values from 3 independent experiments on samples pooled from 3 mice each. *P<0.05.

Effect of HIF-1 Overexpression on TRPC Expression
The analysis of Hif1a^+/– mice indicated that partial HIF-1 loss of function impaired hypoxia-induced TRPC expression. To further evaluate the role of HIF-1 in the regulation of TRPC expression, we used a gain-of-function model using AdCA5, an adenovirus that encodes a constitutively active form of HIF-1, which on transfection, activates HIF-1–dependent gene transcription under nonhypoxic conditions.^33–35 In PASMCs isolated from normoxic rats and cultured under nonhypoxic conditions, transfection with AdCA5 resulted in an increase in both TRPC1 and TRPC6 RNA (Figure 7A) and protein (Figure 7B) expression compared with PASMCs transfected with AdLacZ, a control adenovirus encoding Escherichia coli ß-galactosidase. In contrast, AdCa5 had no effect on TRPC4 mRNA and protein expression. Thus, AdCA5 induced changes in TRPC expression that were identical to those induced by hypoxia.

View larger version (37K): [in this window] [in a new window]   Figure 7. Effect of HIF-1 gain-of-function on TRPC expression. Left, Analysis of TRPC mRNA (A) and protein (B) expression in PASMCs transfected with an adenovirus encoding a constitutively active form of HIF-1 (AdCA5) or a control adenovirus encoding ß-galactosidase (AdLacZ). Right, TRPC mRNA and protein expression normalized to - or ß-actin (mean±SEM; n=3 experiments for each group; *P<0.05).

	Discussion

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Development of chronic hypoxic pulmonary hypertension is associated with elevated resting [Ca²⁺]_i in PASMCs and contraction of pulmonary vascular smooth muscle. The maintenance of increased PASMC [Ca²⁺]_i and tone during CH requires Ca²⁺ influx through pathways other than VDCCs.⁷ In this study, we found that exposure to CH caused enhanced activity of SOCCs, which was required for maintenance of elevated resting [Ca²⁺]_i and active tone and correlated with elevated expression of TRPC proteins. In addition, we established a critical role for HIF-1 in regulating both hypoxic induction of TRPC proteins and elevated basal [Ca²⁺]_i.

We have previously demonstrated the presence of CCE and SOCCs in rat PASMCs.¹⁶ We found that CCE, measured via Ca²⁺ restoration following store depletion, was greater in PASMCs from chronically hypoxic rats compared with that measured in PASMCs from normoxic rats. Because the increase in [Ca²⁺]_i following readdition of Ca²⁺ can be influenced by both Ca²⁺ influx through SOCCs and Ca²⁺ efflux through plasmalemmal Ca²⁺-ATPases and Na⁺/Ca²⁺ exchange, we also used Mn²⁺ quenching of F₃₆₀ as a more direct evaluation of Ca²⁺ entry. In the absence of Mn²⁺, no significant decrease in F₃₆₀ was observed in PASMCs from either normoxic or chronically hypoxic rats, verifying that photobleaching did not contribute significantly to any decrease in F₃₆₀ over the time of our measurements. Following depletion of intracellular Ca²⁺ stores with CPA, a maneuver that maximally activates CCE, addition of Mn²⁺ caused significantly greater quenching of F₃₆₀ in PASMCs from chronically hypoxic rats. The increased rise in [Ca²⁺]_i following restoration of extracellular Ca²⁺ and faster rate of Mn²⁺ quenching of F₃₆₀ in PASMCs from chronically hypoxic animals indicate enhanced Ca²⁺ influx through SOCCs.

Given the apparent increase in SOCC activity following exposure to CH, we next evaluated whether enhanced CCE might contribute to the elevated basal [Ca²⁺]_i observed in these cells. In PASMCs from normoxic rats, inhibitors of CCE had no effect on basal [Ca²⁺]_i, whereas in PASMCs from chronically hypoxic rats, both inhibitors caused immediate decreases in [Ca²⁺]_i to levels similar to those measured in PASMCs from normoxic rats. Although SKF and NiCl₂ may inhibit VDCCs in some cells, we do not believe that this action contributed to the decrease in resting [Ca²⁺]_i measured in PASMCs because nifedipine, given at a concentration that blocked KCl-induced increases in [Ca²⁺]_i, had no effect on resting [Ca²⁺]_i.⁷ Moreover, whereas SKF and NiCl blocked CCE in our cells, neither drug affected the increase in [Ca²⁺]_i caused by KCl.¹⁶ These results rule out a nonspecific effect of SKF and NiCl₂ on VDCCs in our preparation.

Our observation that SOCC inhibitors reduced resting [Ca²⁺]_i following exposure to CH suggested that SOCCs were active under resting (unstimulated) conditions. To test this possibility, we measured Mn²⁺ quenching of F₃₆₀ without prior depletion of intracellular stores. Under these conditions, Mn²⁺ quenching still occurred in cells from normoxic and chronically hypoxic rats, albeit at a substantially slower rate than that observed in cells in which stores had been depleted. Moreover, the decrease in F₃₆₀ was significantly greater in PASMCs from chronically hypoxic rats and could be blocked by pretreatment with SOCC inhibitors. These results suggest that Ca²⁺ entry through SOCCs is enhanced following exposure to CH, both at rest and following store depletion with CPA.

One possible explanation for enhanced CCE following exposure to CH is a greater degree of store depletion. However, the increase in [Ca²⁺]_i in response to CPA was not significantly different in normoxic and chronically hypoxic PASMCs, suggesting equal depletion of stored Ca²⁺. Moreover, we previously tested the ability of caffeine to induce Ca²⁺ release and found similar increases in [Ca²⁺]_i in response to caffeine in PASMCs from normoxic and chronically hypoxic rats, again indicating intact intracellular Ca²⁺ stores following exposure to CH.⁷ Thus, differences in store depletion do not explain enhanced CCE under conditions of CH.

Another possibility is that increased CCE caused by CH results from increased expression of SOCCs, which are believed to be composed of TRPC proteins.^18,21,22 TRPC expression in the pulmonary vasculature has been studied by several laboratories, with somewhat varying results. In distal pulmonary vascular smooth muscle, most studies have identified TRPC1, TRPC4, and TRPC6,^14,16,19 although others have failed to find TRPC4 and instead found expression of TRPC3.²⁴ Consistent with our previous results, we found that both rat and mouse PASMCs express only TRPC1, TRPC4, and TRPC6. Moreover, following exposure to CH, we found that expression of TRPC1 and TRPC6, but not TRPC4, increased in both the rat and murine models, in agreement with other labs.²⁴

We next sought to determine the mechanism by which TRPC expression was increased by CH. During development of hypoxic pulmonary hypertension in the intact animal, pulmonary arteries are subjected to numerous stimuli in addition to decreased oxygen tension, including increased pressure and altered exposure to circulating factors; however, gene and protein expression of both TRPC1 and TRPC6 were increased in PASMCs from normoxic animals cultured under hypoxic conditions for 60 hours. These results suggest that CH might upregulate TPRC expression through a direct effect on gene expression in PASMCs.

Little is known about the regulation of TRPC gene transcription, although the promoter regions of rat and mouse genes encoding TRPC1 and TRPC6 may contain HIF-1 binding sites. Because our previous work demonstrated a crucial role for HIF-1 in the pathogenesis of hypoxic pulmonary hypertension,³² we hypothesized that HIF-1 was involved in the hypoxic induction of TRPC proteins. In mice with partial HIF-1 deficiency, both the elevation of basal [Ca²⁺]_i and the hypoxic induction of TRPC1 and TRPC6 were lost, indicating that full HIF-1 activity was necessary for these responses to hypoxia. Conversely, expression of a constitutively active form of HIF-1 under nonhypoxic conditions increased TRPC1 and TRPC6 expression, indicating that increased HIF-1 activity was sufficient for induction. Loss of HIF-1 function increased basal TRPC expression and impaired hypoxia-induced TRPC expression, whereas gain of HIF-1 function had the opposite effect, indicating that HIF-1 may act as a negative regulator of TRPC expression under nonhypoxic conditions and a positive regulator under hypoxic conditions. In this regard, it is interesting to note that PASMCs, unlike most cultured primary cells, express high levels of HIF-1 protein under nonhypoxic as well as hypoxic conditions.³⁶

The increase in resting [Ca²⁺]_i observed in PASMCs from chronically hypoxic animals had a functional consequence. Removal of extracellular Ca²⁺ caused an immediate decrease in isometric tension in pulmonary arteries from chronically hypoxic rats,⁷ suggesting that Ca²⁺ influx was required not only for the CH-induced elevation of [Ca²⁺]_i but also for maintenance of active vasoconstriction. Similarly, in the current study, we found that SOCC inhibitors also decreased baseline tension in pulmonary arteries from chronically hypoxic rats and that the decrease in tension was similar in magnitude to that observed in response to removal of extracellular Ca²⁺.

In summary, we found that exposure to CH increased TRPC expression in PASMCs, leading to enhanced Ca²⁺ entry through SOCCs and a sustained increase in resting cytosolic [Ca²⁺]_i. The increase in [Ca²⁺]_i was required for maintenance of active tone in pulmonary arteries following exposure to CH. In addition to modulating vasoconstriction, elevated [Ca²⁺]_i also plays an important role in the regulation of PASMC growth,^14,18,19 and may contribute to the development of hypoxic pulmonary hypertension via regulation of both PASMC contraction and proliferation. Our findings that both the elevation of basal [Ca²⁺]_i and increase in TRPC expression induced by hypoxia are regulated by HIF-1 indicates that HIF-1 has a profound effect on pulmonary vascular and, specifically, PASMC, responses to CH and provide compelling evidence that HIF-1 plays a critical role in the pathogenesis of hypoxia-induced pulmonary hypertension.

	Acknowledgments

 
Sources of Funding

This work was supported by the National Heart, Lung, and Blood Institute (grants HL51912 and HL75113 to J.T.S.; HL67919 and HL67191 to L.A.S.; and HL55338 to G.L.S.) and by an American Heart Association Scientist Development Grant (AHA0430037N to J.W.).

Disclosures

None.

	Footnotes

 
This manuscript was sent to Donald D. Heistad, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

Original received August 22, 2005; resubmission received March 21, 2006; revised resubmission received April 27, 2006; accepted May 8, 2006.

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