Hypoxia Inducible Factor 1 Mediates Hypoxia-Induced TRPC Expression and Elevated Intracellular Ca2+ in Pulmonary Arterial Smooth M
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Jian Wang, Letitia Weigand, Wenju Lu, J.
参见附件。
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.)
Radiation Oncology (G.L.S.)
McKusick–Nathans Institute of Genetic Medicine (G.L.S.), The Johns Hopkins University School of Medicine, Baltimore, Md.
Abstract
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
Prolonged exposure to alveolar hypoxia is associated with changes in the pulmonary vasculature including structural remodeling1,2 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 CH4,11,12 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 PASMCs14–16 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-1 subunits.25 HIF-1 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-1 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
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 -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
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).
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).
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.
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).
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).
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.
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 -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.
Discussion
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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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.)
Radiation Oncology (G.L.S.)
McKusick–Nathans Institute of Genetic Medicine (G.L.S.), The Johns Hopkins University School of Medicine, Baltimore, Md.
Abstract
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
Prolonged exposure to alveolar hypoxia is associated with changes in the pulmonary vasculature including structural remodeling1,2 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 CH4,11,12 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 PASMCs14–16 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-1 subunits.25 HIF-1 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-1 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
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 -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
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).
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).
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.
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).
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).
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.
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 -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.
Discussion
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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