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(Circulation Research. 2004;95:496.)
© 2004 American Heart Association, Inc.

Cellular Biology

Chronic Hypoxia–Induced Upregulation of Store-Operated and Receptor-Operated Ca²⁺ Channels in Pulmonary Arterial Smooth Muscle Cells

A Novel Mechanism of Hypoxic Pulmonary Hypertension

Mo-Jun Lin, George P.H. Leung, Wei-Min Zhang, Xiao-Ru Yang, Kay-Pong Yip, Chung-Ming Tse, James S.K. Sham

From the Division of Pulmonary and Critical Care Medicine (M.-J.L., W.-M.Z., X.-R.Y., J.S.K.S.), Division of Gastroenterology (G.P.H.L., C.-M.T.), Johns Hopkins School of Medicine, Baltimore, Md; the Department of Physiology and Biophysics (K.-P.Y.), College of Medicine, University of South Florida, Tampa; and the Department of Physiology and Pathophysiology (M.-J.L.), Fujian Medical University, Fuzhou, Fujian, People’s Republic of China.

Correspondence to James S.K. Sham, PhD, Division of Pulmonary and Critical Care Medicine, Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224. E-mail jsks{at}welchlink.welch.jhu.edu

	Abstract

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Chronic hypoxic pulmonary hypertension is associated with profound vascular remodeling and alterations in Ca²⁺ homeostasis in pulmonary arterial smooth muscle cells (PASMCs). Recent studies show that transient receptor potential (TRPC) genes, which encode store-operated and receptor-operated cation channels, play important roles in Ca²⁺ regulation and cell proliferation. However, the influence of chronic hypoxia on TRPC channels has not been determined. Here we compared TRPC expression, and store- and receptor-operated Ca²⁺ entries in PASMCs of normoxic and chronic hypoxic rats. Reverse-transcription polymerase chain reaction (RT-PCR), Western blot, and immunostaining showed consistently that TRPC1, TRPC3, and TRPC6 were expressed in intralobar pulmonary arteries (PAs) and PASMCs. Application of 1-oleoyl-2-acetyl-sn-glycerol (OAG) to directly activate receptor-operated channels, or thapsigargin to deplete Ca²⁺ stores, caused dramatic increase in cation entry measured by Mn²⁺ quenching of fura-2 and by Ca²⁺ transients. OAG-induced responses were 700-fold more resistant to La³⁺ inhibition than thapsigargin-induced responses. siRNA knockdown of TRPC1 and TRPC6 specifically attenuated thapsigargin- and OAG-induced cation entries, respectively, indicating that TRPC1 mediates store-operated entry and TRPC6 mediates receptor-operated entry. In hypoxic PAs, there were 2- to 3-fold increases in TRPC1 and TRPC6 expression. They were accompanied by significant increases in basal, OAG-induced, and thapsigargin-induced cation entries in hypoxic PASMCs. Moreover, removal of Ca²⁺ or inhibition of store-operated Ca²⁺ entry with La³⁺ and SK&F-96365 reversed the elevated basal [Ca²⁺]_i in PASMCs and vascular tone in PAs of chronic hypoxic animals, but nifedipine had minimal effects. Our results for the first time to our knowledge show that both store- and receptor-operated channels of PASMCs are upregulated by chronic hypoxia and contribute to the enhanced vascular tone in hypoxic pulmonary hypertension.

Key Words: pulmonary hypertension • transient receptor potential channels • store-operated Ca²⁺ channels • receptor-operated Ca²⁺ channels

	Introduction

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Prolonged exposure to alveolar hypoxia causes pulmonary hypertension with profound vascular remodeling and increase in vasomotor tone. The increase in vascular tone is in part attributable to alterations in vasoconstricting and vasorelaxing influences imposed by the endothelially derived and circulating factors.¹ Recent evidence indicates that chronic hypoxia also causes intrinsic changes in ionic balance and Ca²⁺ homeostasis in pulmonary arterial smooth muscle cells (PASMCs), including membrane depolarization, elevation in resting [Ca²⁺]_i, and changes in electrophysiological and Ca²⁺ responses to vasoconstrictors and vasodilators.^2–6

The mechanism for alteration in Ca²⁺ homeostasis in hypoxic PASMCs is controversial. Previous studies found significant suppression of voltage-gated K⁺ (K_V) currents and K_V channel expression in PASMCs isolated from chronic hypoxic animals or cultured under hypoxic conditions.^2–4,6,7 Because K_V channel is a major conductance controlling resting membrane potential, it has been postulated that chronic hypoxia inhibits K_V channels, causing membrane depolarization, activation of L-type Ca²⁺ channels, and increase in [Ca²⁺]_i.^2–4 The involvement of L-type Ca²⁺ channel activation has gained support from some studies showing that Ca²⁺ channel antagonists, nifedipine and verapamil, attenuated hypoxia-induced pulmonary hypertension.^8,9 However, other studies showed that the effects of Ca²⁺ channel blockers were often partial, temporary, and sometimes complicated by changes in cardiac output.^10,11 In chronic hypoxic rats, nifedipine was ineffective in reducing pulmonary hypertension, despite another vasodilator, NIP-121, caused significant reduction in pulmonary arterial pressure and vascular resistance.¹² More dramatically, the elevated resting [Ca²⁺]_i in PASMCs of chronic hypoxic rats was unaffected by nifedipine but was reduced instantaneously to the level of control PASMCs by removal of extracellular Ca²⁺.⁵ These results suggest that other Ca²⁺ influx pathway(s) in additional to L-type Ca²⁺ channel are involved in chronic hypoxic pulmonary hypertension.

Nonselective cation channels, encoded by the canonical transient receptor potential (TRPC) gene family, constitute the alternative pathways of Ca²⁺ entry in vascular myocytes. Recent evidence suggests that TRPC1 channel is related to store-operated Ca²⁺ entry, which can be activated by depletion of Ca²⁺ stores using cyclopiazonic acid (CPA) or thapsigargin;^13,14 TRPC3 and TRPC6 channels are involved in receptor-operated entry, which can be activated directly by diacylglycerol (DAG) via a protein kinase C (PKC)-independent mechanism.^15–17 Multiple TRPC subtypes have been identified in canine pulmonary arteries (PAs), fresh or cultured rat main PASMCs, and cultured human PASMCs.^14,18–21 They have been implicated to mediate store-operated Ca²⁺ influx.^19,20 However, receptor-operated Ca²⁺ entry has not been demonstrated in PASMCs, and the effects of chronic hypoxia on TRPC-dependent Ca²⁺ entries have not been determined. Because hypoxic pulmonary hypertension is associated with PASMC proliferation^14,22 and enhanced reactivity to vasoconstrictors,^1,23,24 both processes are related to store- and receptor-operated Ca²⁺ channels, it is possible that the activity/expression of these channels are altered by chronic hypoxia. In the present study, we sought to identify the TRPC channels expressed in rat intralobar PASMCs, characterize the store and receptor-operated cation entries, determine changes in TRPC expression, store-operated and receptor-operated cation entries, and their involvement in the elevated basal [Ca²⁺]_i and vasomotor tone induced by chronic hypoxia.

	Materials and Methods

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Chronic Hypoxic Exposure
Male Wistar rats (150 to 250 grams) were placed in a hypoxic chamber and exposed to either normoxia or normobaric hypoxia 10% O₂ for 3 to 4 weeks to induce hypoxic pulmonary hypertension as described previously.⁵

Isolation and Culture of PASMCs
PASMCs were enzymatically isolated from de-endothelialized intralobar PAs.⁵ PASMCs from chronic hypoxic and normoxic animals were cultured transiently (16 to 24 hours) inside a modular incubator chamber (Billups-Rothenberg, Inc) under 4% O₂/5% CO₂ and 21% O₂/5% CO₂, respectively, before used.

Measurement of Intracellular [Ca²⁺] and Mn²⁺ Quenching of Fura-2
[Ca²⁺]_i was monitored using fluo-3 AM as previously described.²⁵ Ca²⁺ entry through TRPCs was quantified by quenching of fura-2 with Mn²⁺. PASMCs were loaded with fura-2 AM and bathed in a Ca²⁺ free (with 0.1 mmol/L EGTA) nifedipine (10 µmol/L) containing Tyrode solution. Fura-2 was excited at 360 nm, and emission light was recorded at >510 nm. After a stable baseline fluorescence was attained, 500 µmol/L Mn²⁺ was applied through a multibarrel pipette positioned <50 µm from PASMCs. Maximum rate of quenching of fura-2 fluorescence was determined.

Reverse-Transcription Polymerase Chain Reaction
Total RNA was isolated from intralobar PAs and PASMCs, and reverse-transcription polymerase chain reaction (RT-PCR) was performed using standard methods. Specific sense and antisense primers for RT-PCR were designed according to the strategies described in the online data supplement available at http://circres.ahajournals.org. PCR products of TRPC were quantified using ß-actin as an internal standard.

Western Blotting and Immunostaining of TRPCs
Proteins were extracted from PAs and PASMCs, and TRPC subtypes were detected using a standard Western blot protocol. Polyclonal rabbit anti-TRP1, anti-TRP3, anti-TRP4, or anti-TRP6 antibodies (Alomone Labs, Jerusalem, Israel) were used as the primary antibodies. Rat brain membrane proteins were used as positive controls. Immunostaining of TRPC in PASMCs was performed as described previously.²⁵ Coverslips without exposing to primary antibodies were used as negative control.

siRNA Knockdown of TRPC
PASMCs were isolated and cultured for 24 hours and then transiently transfected with siRNA specific for TRPC1, TRPC6, or a random nonsilencing sequence for 10 to 12 hours with RNAifect Transfection Reagent (Qiagen), according to the manufacturer’s instructions. PASMCs were then further cultured for 24 to 48 hours before used for mRNA and protein determinations and Mn²⁺ quenching experiments (see the online data supplement for all detail methods and primer sequences).

	Results

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Identification of TRPC Subtypes
Figure 1A shows RT-PCR amplified products of TRPCs in normoxic PASMCs after 30 cycles. TRPC1 and TRPC6 were the major, and TRPC3 was the minor, TRPC mRNA expressed in rat intralobar PASMCs. Expression of TRPC4 mRNA was very low and only detected after 35 cycles. TRPC2 and TRPC5 mRNA were practically undetectable. All RT-PCR amplified products from PASMCs had sizes corresponding to the predicted sequences and matched with positive controls generated from brain or heart. Same results were obtained when mRNA were extracted from freshly isolated intralobar PAs (Figure 4A and 4B).

View larger version (26K): [in this window] [in a new window]   Figure 1. A, RT-PCR analysis of TRPC expressions in PASMCs. Brain and heart mRNAs were used as standard. Predicted lengths of PCR products are 402, 496, 519, 423, 831, and 415 base pairs for TRPC1, TRPC2, TRPC3, TRPC4, TRPC5, and TRPC6, respectively. B, Western blot analysis of TRPC1, TRPC3, TRPC4, and TRPC6 proteins in PASMCs. Brain proteins were used as positive control. C, Immunostaining of TRPC channels in PASMCs.

Expressions of TRPC proteins in normoxic PASMCs were examined using Western blot analysis (Figure 1B). TRPC1, TRPC3, and TRPC6 proteins of 140, 150, and 115 kDa, respectively, were clearly detected along with positive controls from rat brain, whereas TRPC4 proteins were undetectable using specific antibodies from 2 different companies. Immunostaining confirmed that the TRPC subtypes identified by RT-PCR and Western blot analysis were indeed expressed in PASMCs and were not attributable to contaminations of endothelial cells. Strong immunofluorescent signals were detected in PASMCs using specific anti-TRPC1, anti-TRPC3, and anti-TRPC6 antibodies, but not with the anti-TRPC4 antibody (Figure 1C).

Thapsigargin-Induced and Diacylglycerol-Induced Ca²⁺/Cation Entry
The functional activities of store-operated and receptor-operated channels were quantified by quenching fura-2 fluorescence with Mn²⁺. In the absence of extracellular Ca²⁺ (0.1 mmol/L EGTA) and in the presence of 10 µmol/L nifedipine, application of 500 µmol/L Mn²⁺ caused a slow quenching of fura-2 fluorescence (0.04±0.004%/second). Depleting SR Ca²⁺ stores after 15 minutes of exposure of PASMCs to 10 µmol/L thapsigargin caused a dramatic 25-fold increase in the maximal rate of Mn²⁺-induced quenching (1.01±0.19%/second; n=8, P<0.001) (Figure 2A). Activation of receptor-operated cation entry by 10 minutes pretreatment with 100 µmol/L 1-oleoyl-2-acetyl-sn-glycerol (OAG) also accelerated significantly the rate of quenching from 0.05±0.01 to 0.43±0.09%/second (n=9, P<0.001). OAG-induced cation entry was not impeded by PKC inhibition using staurosporine. Hence, it was not mediated via PKC-dependent phosphorylation but by direct activation of TRPCs, a hallmark of receptor-operated cation entry.¹⁵ Thapsigargin-induced store-operated cation entry was highly sensitive to La³⁺, which inhibited Mn²⁺ quenching at submicromolar concentrations with an IC₅₀ of 0.27±0.08 µmol/L (Figure 2B and 2D). By contrast, the OAG-induced cation entry was rather insensitive to La³⁺. Significant inhibition by La³⁺ was observed only at concentrations 100 µmol/L, with an IC₅₀ of 183±54 µmol/L.

View larger version (36K): [in this window] [in a new window]   Figure 2. Characterization of thapsigargin- and OAG-induced cation entry by Mn2+ quenching of fura-2 in PASMCs. A, Representative tracings recorded in control, thapsigargin, OAG, and OAG+staurosporine (stau)-treated PASMCs. B and C, Effects of La3+ on thapsigargin- and OAG-induced cation entries. D, Concentration-dependent inhibition of thapsigargin- and OAG-induced Mn2+ quenching by La3+. Lines are the least-square fit of the data using the Hill equation; 7 to 12 experiments from at least 3 different animals were performed for each concentration of La3+.

Store-operated and receptor-operated Ca²⁺ entries were further evaluated by recording the Ca²⁺ transients (Figure 3). In the presence of nifedipine (10 µmol/L), removal of extracellular Ca²⁺ for 10 minutes caused a slight decrease in resting [Ca²⁺]_i. Re-introduction of Ca²⁺ resulted in a minimal increase of [Ca²⁺]_i (57.9±25.7 nmol/L) to the basal level. When SR Ca²⁺ was depleted by thapsigargin, a large overshoot of Ca²⁺ transient (772.51±120 nmol/L, n=18) was elicited immediately on switching back to Ca²⁺ containing solution. This store-operated Ca²⁺ entry was abolished by 30 µmol/L La³⁺ (87.37±68 nmol/L, n=13, P<0.005). Pretreatment of PASMCs with OAG also activated a Ca²⁺ entry transient (293±51 nmol/L, n=9), which was unaffected by 100 µmol/L La³⁺ (343±54 nmol/L, n=9). These results suggest that PASMCs possess two functionally distinctive thapsigargin-activated and OAG-activated Ca²⁺ pathways, which have different sensitivity to the inorganic blocker La³⁺.

View larger version (47K): [in this window] [in a new window]   Figure 3. Thapsigargin- and OAG-induced Ca2+ entry transients in PASMCs in the presence of nifedipine. External Ca2+ was removed for 10 minutes and then reintroduced to allow store- or receptor-operated Ca2+ entry. A, Representative Ca2+ transients generated in control (upper panel), and thapsigargin-pretreated (middle and lower panels) PASMCs. B, The averaged change in peak Ca2+ transients elicited by thapsigargin in the absence or presence of La3+; n indicates the number of experiments; **significant differences. C, Representative Ca2+ transients generated in OAG-pretreated PASMCs. D, The averaged change in [Ca2+]i elicited by OAG in the absence and presence of 100 µmol/L La3+.

Chronic Hypoxia–Altered TRPC Expression and Cation Entry
TRPC expression in intralobar PAs of normoxic and chronic hypoxic rats was quantified by RT-PCR, using ß-actin as the internal standard. TRPC1 and TRPC6 mRNA levels were approximately tripled in hypoxic, compared with normoxic PAs, but TRPC3 levels were the same (Figure 4A and 4B). Western blot analysis showed that TRPC1 and TRPC6 protein levels were increased by 150%, whereas TRPC3 protein expression was unaltered, in chronic hypoxic PAs. The increase in the expression of TRPC mRNA and protein in hypoxic PA was accompanied by enhanced cation entry. The rate of thapsigargin-induced Mn²⁺ quenching was increased from 1.01±0.19%/second (n=8) in normoxic to 2.23±0.37%/second (n=8, P<0.01) in hypoxic PASMCs, whereas OAG-induced cation entry was accelerated from 0.31±0.05%/second (n=9) in normoxic to 0.98±0.31%/second (n=9) in hypoxic cells. Moreover, the basal rate of cation entry was slightly, but significantly, higher in hypoxic PASMCs (normoxia: 0.041±0.004%/second, n=23; hypoxia: 0.053±0.003%/second, n=24, P<0.05). Ca²⁺ entry transients also showed that both thapsigargin- and OAG-induced Ca²⁺ entries were significantly enhanced in hypoxic PASMCs (Figure 6). After pretreatment with thapsigargin in nifedipine containing Ca²⁺ free solution, reintroduction of Ca²⁺ to hypoxic PASMCs elicited a large Ca²⁺ transient (1.71±0.39 µmol/L, n=6) of twice the magnitude of Ca²⁺ entry transients elicited in normoxic PASMCs (772.51±120 nmol/L, n=18). OAG-induced Ca²⁺ entry transients were also increased by >200% (normoxia: 0.29±0.05 µmol/L, n=9; hypoxia: 0.99±0.26 µmol/L, n=8) in hypoxic PASMCs. These results clearly indicate that the expression of putative store-operated and receptor-operated Ca²⁺ channels and their functional activities are proportionately augmented in PASMCs after chronic exposure to hypoxia.

View larger version (46K): [in this window] [in a new window]   Figure 4. Expression of TRPC mRNA and protein in PAs of normoxic (N) and chronic hypoxic (CH) rats. A, Semi-quantitative RT-PCR analysis of TRPC mRNA expression in PAs using ß-actin as internal standard. B, The normalized amount of TRPC products generated from PAs of 7 normoxic and chronic animals. C, Western blot analysis of TRPC proteins in PAs of normoxic and chronic hypoxic rats. D, The normalized amount of TRPC proteins from 4 normoxic and 4 hypoxic animals. *Significant difference between normoxic and chronic hypoxic PAs.

View larger version (22K): [in this window] [in a new window]   Figure 5. Comparison of basal, thapsigargin-induced, and OAG-induced cation entry by Mn2+ quenching in PASMCs. A, Averaged time-course of Mn2+ quenching in control (left panel), thapsigargin (middle panel), and OAG-treated (right panel) PASMCs. C, The averaged maximum rate of quenching in control (left panel), thapsigargin (middle panel), and OAG-treated (right panel) PASMCs. *Significant difference between normoxic and hypoxic PASMCs.

View larger version (46K): [in this window] [in a new window]   Figure 6. Comparison of thapsigargin- and OAG-induced Ca2+ transients in normoxic and chronic hypoxic PASMCs. A, Representative traces of thapsigargin-induced Ca2+ transients generated in normoxic (upper panel) and hypoxic (lower panel) PASMCs. B, The averaged change in peak Ca2+ transients elicited by thapsigargin in 18 normoxic and 6 hypoxic cell experiments. C, Representative traces of OAG-induced Ca2+ transients generated in normoxic and hypoxic PASMCs. D, The averaged change in peak Ca2+ transients elicited by OAG in 9 normoxic and 8 hypoxic cell experiments. *Significant difference between normoxic and hypoxic PASMCs.

siRNA Knockdown of TRPC1 and TRPC6
To verify whether TRPC1 and TRPC6 expressions indeed dictated the store- and receptor-operated cation entries in PASMCs, the channels were knockdown specifically using siRNA. Transfection of normoxic PASMCs with siRNA against TRPC1 resulted in 70% reduction in mRNA and protein of TRPC1 compared with PASMCs transfected with a nonsilencing random sequence (Figure 7). There was no significant change in the expression of TRPC6 mRNA and protein in TRPC1 siRNA transfected cells. The maximum rate of thapsigargin-induced Mn²⁺-quenching was significantly slower in TRPC1 siRNA transfected (0.28±0.07%/second, n=15) than in control sequence transfected PASMCs (0.75±0.14%/second, n=14, P<0.01). OAG-induced Mn²⁺-quenching was similar in the two groups of myocytes. Transfection of PASMCs with TRPC6 siRNA caused a 50% reduction of mRNA and protein of TRPC6, without affecting the expression of TRPC1. The reduction of TRPC6 expression was associated with a comparable reduction in the rate of OAG-induced Mn²⁺ quenching (control: 0.50±0.08%/second; TRPC6 siRNA: 0.19±0.05%/second, n= 9, P<0.05), whereas the store-operated cation entry was unaffected. These results indicate that TRPC1 and TRPC6 are the major determinants of the store- and receptor-operated Ca²⁺ entries, respectively, in rat PASMCs.

View larger version (59K): [in this window] [in a new window]   Figure 7. A, RT-PCR analysis of TRPC mRNA expressions in PASMCs transfected with siRNA against TRPC1, TRPC6, and control nonsilencing sequence. B, Averaged normalized amount of TRPC PCR products (n=4 to 5). C, Western blot of TRPC proteins in PASMCs transfected with siRNA. D, Bar graph showing averaged normalized amount of TRPC protein (n=4 to 7). E, Averaged time-course of thapsigargin- and OAG-induced Mn2+ quenching in PASMCs transfected with TRPC1 and TRPC6 siRNA and a control RNA sequence.

Roles of Store-Operated and Receptor-Operated Ca²⁺ Entry on Baseline [Ca²⁺] and Vasomotor Tone
As reported previously,⁵ basal [Ca²⁺]_i was significantly elevated in chronic hypoxic PASMCs (normoxia: 176±9 nmol/L, n=23; hypoxia: 267±30 nmol/L, n=9, P<0.05) (Figure 8). It was quickly reversed by the removal of extracellular Ca²⁺. Application of 10 µmol/L La³⁺ to block store-operated Ca²⁺ channels caused a similar reduction in [Ca²⁺]_i to 159±17 nmol/L (n=12, P<0.05), whereas complete inhibition of L-type Ca²⁺ channels with 1 µmol/L nifedipine⁵ only caused a 30% to 40% reduction, as induced by Ca²⁺ removal or La³⁺. In the presence of nifedipine, 10 µmol/L La³⁺ caused a further decrease in basal [Ca²⁺]_i; increase [La³⁺] to 300 µmol/L to inhibit receptor-operated Ca²⁺ entry failed to cause additional decrease in [Ca²⁺]_i (n=4). In contrast, removal of Ca²⁺, application of La³⁺, or nifedipine caused only minor reduction in basal [Ca²⁺]_i in normoxic PASMCs. These results suggest that store-operated Ca²⁺ entry is the major Ca²⁺ entry pathway responsible for the elevated [Ca²⁺]_i in chronic hypoxic PASMCs.

View larger version (49K): [in this window] [in a new window]   Figure 8. A, Ca2+ transients showing the effects of 0 Ca2+, La3+, nifedipine, and La3+ in the presence of nifedipine on the resting [Ca2+]i in normoxic and hypoxic PASMCs. Each trace is the ensemble averaged Ca2+ transient from the whole group with number indicated in Results. B, Averaged data from experiments in (A). *Significant difference between before and after blocker application. #Significant difference between normoxic (N) and chronic hypoxic (CH) PASMCs. C, The effects of 0 Ca2+, SK&F-96365, nifedipine, and SK&F-96365 in the presence of nifedipine on resting tension of normoxic and hypoxic PA rings. N=7 for each conditions. D, Averaged data from experiments in C. *Significant difference between normoxic and hypoxic PAs. #Significant difference from 0 Ca2+, SK&F-96365, and SK&F-96365+nifedipine.

Parallel experiments were performed to evaluate basal vascular tone in PA rings. SK&F-96365 was used to inhibit nonselectively the store- and receptor-operated cation entry because La³⁺ precipitates in Krebs bicarbonate solution. Removal of extracellular Ca²⁺ or 30 µmol/L SK&F-96365 caused a 10% to 12% reduction in resting tension in hypoxic PAs but had minimal effects on normoxic PAs. Nifedipine, which has no effect on resting tension in normoxic PAs, relaxed the chronic hypoxic PAs by 3.8±1.4%; and subsequent application of SK&F-96365 further relaxed the hypoxic PA to 9.1±1.5% (n=7, P<0.05), a reduction comparable to Ca²⁺ removal.

	Discussion

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The present study demonstrated the coexistence of functionally distinctive receptor-operated and store-operated cation entry pathways in PASMCs. The evidence includes: (1) the putative store-operated Ca²⁺ channel TRPC1 and receptor-operated Ca²⁺ channel TRPC6 are coexpressed in rat PASMCs and PAs; (2) the cation/Ca²⁺ entry can be elicited by depleting SR Ca²⁺ stores using thapsigargin and by direct activation of receptor-operated channel using OAG; (3) thapsigargin- and OAG-induced cation/Ca²⁺ entries are distinguishable pharmacologically by a large difference in the sensitivity to La³⁺; and (4) siRNA knockdown of TRPC1 and TRPC6 inhibited specifically the thapsigargin and OAG-induced cation entry, respectively. As far as we are aware, these are the first direct evidence of receptor-operated cation entry in PASMCs.

The expression of TRPC subtypes has been reported in several types of vascular smooth muscle cells including PASMCs, but their specific expression vary among species and cell preparations. RT-PCR detected TRPC4, TRPC6, and TRPC7 mRNAs in canine main PAs, with TRPC4 being the major expressed isoform.²¹ In cultured rat main PASMCs, TRPC1, TRPC2, TRPC4, TRPC5, and TRPC6 mRNAs were found highly expressed, whereas TRPC3 and TRPC7 were undetectable.¹⁹ In contrast, a similar study using freshly isolated PASMCs from rat main PAs found prominent expression of TRPC1 and TRPC6, less expression of TRPC3, and exceedingly low levels of expression of TRPC4 and TRPC5 mRNA.²⁰ The expression profile of TRPC in our freshly isolated intralobar PAs and transient cultured PASMCs (<1 day) is similar to those of freshly isolated rat main PASMCs, suggesting that TRPC1, TRPC3, and TRPC6 are the major TRPCs expressed in rat pulmonary vasculature. The expression of other TRPC subtypes (eg, TRPC2, TRPC4, TRPC5) in cultured rat main PASMCs could be related to prolonged cell culture and/or contamination by endothelial cells, because TRPC expression changes during PASMC proliferation^14,18,22 and TRPC4 is prominently expressed in pulmonary endothelium.

Detection of the putative receptor-operated channel TRPC6,^15–17 in addition to TRPC1, provides the hint that receptor-operated Ca²⁺ entry pathway is present in PASMCs. Previous studies using vasoactive agonists to activate receptor-operated Ca²⁺ entry in native tissues were complicated by the fact that they activate both store- and receptor-operated channels, and common blockers of nonselective cation channel inhibit both types of Ca²⁺ entries. To circumvent these complications, OAG was used as the stimulant to activate directly the receptor-operated TRPC channels,¹⁵ without causing SR Ca²⁺ depletion or IP₃R and RyR activation in PASMCs.²⁵ Indeed, OAG elicited a dramatic cation entry, which was unaffected by PKC inhibition with staurosporine and was highly resistant to La³⁺ (Kd 200 µmol/L), in contrast to the La³⁺-sensitive thapsigargin-induced cation entry (Kd 0.3 µmol/L) in the same PASMCs. The several hundred-fold difference in La³⁺ sensitivity is consistent with reports that lanthanides, La³⁺ and Gd³⁺, have a higher affinity to store-operated than to receptor-operated cation channels²⁶ and can be used as a "yardstick" for distinguishing the two Ca²⁺ entry pathways in PASMCs.

The thapsigargin-induced cation entry in rat intralobar PASMCs is mediated by TRPC1. Using TRPC1 siRNA in this study and using TRPC1 specific antisense oligonucleotides or specific antibody in other studies have demonstrated unequivocally that inhibition of TRPC1 expression/function selectively blocks store-operated Ca²⁺ entry.^13,14,27 Pharmacological evidence also shows that a low concentration of lanthanides (<1 µmol/L), which effectively blocks store-operated cation entry in rat PASMCs, PAs, and systemic myocytes,^26,28 also blocks TRPC1 channels in heterologous expression systems.²⁹ More importantly, the present study demonstrated that TRPC6 is a major constituent for receptor-operated Ca²⁺ entry in PASMCs, because TRPC6 knockdown in siRNA transfected myocytes caused a proportional reduction, and TRPC6 upregulation in chronic hypoxic PASMCs resulted in a comparable increase in OAG-induced cation/Ca²⁺ entry. This is consistent with findings that TRPC6 mediates agonist induced receptor-operated Ca²⁺ entry in systemic myocytes.^16,17 Moreover, the observations that TRPC1 siRNA had no effect on OAG-induced cation entry and TRPC6 siRNA did not alter the thapsigargin-induced response provided the molecular evidence that the store- and receptor-operated pathways are mutually independent in rat PASMCs. This is coherent with functional studies that DAG/OAG activates directly TRPC3/6/7 channels but not TRPC1,¹⁵ and with biochemical studies that TRPCs are divided into two subfamilies of TRPC1/4/5 and TRPC3/6/7, in which members within a subfamily, but not between different subfamilies, can form heterotetrameric channels.^30,31

It is noteworthy, however, that a study shows that TRPC6 expression is closely related to CPA-induced Ca²⁺ entry in proliferating PASMCs.²² It is unclear if TRPC6 exhibits a different phenotype in proliferating PASMCs, eg, by heteromerization with proteins such as TRPC3, which has been implicated as a receptor- and store-operated Ca²⁺ channel,^15,32 or if CPA activates TRPC6 through mechanism(s) other than SR Ca²⁺ depletion. These interesting possibilities require future investigations.

The major finding of the present study is that chronic hypoxia upregulates TRPC expression and enhances both store-and receptor-operated Ca²⁺ entries in PASMCs. TRPC upregulation by hypoxia is subtype-specific, such that TRPC1 and TRPC6 expressions are doubled or tripled, whereas other TRPCs are unaltered. Hypoxia may directly promote TRPC expression through the activation of O₂-regulated transcription factors, including hypoxia inducible factor, which upregulates dozens of target genes in hypoxic cells, including pulmonary cells.³³ Previous studies showed that partial deficiency of hypoxia inducible factor-1 in mice annihilated the hypoxia-induced structural and physiological changes in pulmonary vasculature and PASMCs.³⁴ However, evidence for hypoxia inducible factor-1 directly regulates TRPC transcription is unavailable. Alternatively, hypoxia may regulate TRPC expression indirectly through mitogenic/growth factors. It has been reported that hypoxia stimulates the release of PDGF, fibroblast growth factor, vascular endothelial growth factor, and endothelin-1 (ET-1). In vitro studies show that TRPC1 mRNA is upregulated, resting [Ca²⁺]_i is elevated, and store-operated Ca²⁺ entry is enhanced during serum-induced proliferation in cultured PASMCs,¹⁸ and TRPC6 expression is increased by PDGF-induced PASMC proliferation.²² Moreover, PASMC proliferation can be blocked by antisense oligonucleotides against TRPCs,^14,22 suggesting that TRPC upregulation is a required step for the growth processes. Even though these observations may not applied equivalently to in vivo situations, nevertheless the release of mitogens and the massive medial thickening in chronic hypoxic pulmonary hypertension raise the likely possibility that TRPC upregulation is related at least in part to PASMC proliferation.

The enhanced TRPC expression and activity play a critical role in the increase in vascular tone of hypoxic PAs. It is evident in the present study that the basal Ca²⁺ influx via TRPC channels, as indicated by Mn²⁺ quenching, was significantly augmented in chronic hypoxic PASMCs. More dramatically, La³⁺ and the nonselective cation channel blocker, SK&F-96365, reduced the elevated [Ca²⁺] and vascular tone in hypoxic PAs to a level similar to Ca²⁺ removal. The prominent effects of La³⁺ and SK&F-96365 are not attributable to the inhibition of voltage-gated Ca²⁺ channels, because nifedipine at a concentration that completely abolishes voltage-gated Ca²⁺ entry⁵ only had a small effect, and subsequent application of La³⁺ and SK&F-96365 in the presence of nifedipine caused further reduction in basal [Ca²⁺]_i and vascular tone. In addition, 10 µmol/L of La³⁺ is sufficient to cause the maximum reduction in basal [Ca²⁺]_I, suggesting that the store-operated Ca²⁺ entry is likely the major pathway responsible for the enhanced basal Ca²⁺ entry in chronic hypoxic PASMCs. TRPC upregulation may contribute to vasomotor tone through several mechanisms. Ca²⁺ influx via TRPC increases [Ca²⁺]_i to initiate actin–myosin interactions; Ca²⁺ influx via store-operated channel replenishes SR Ca²⁺ stores to allow further Ca²⁺ release;³⁵ and Na⁺ influx via TRPCs because of their nonselective nature may cause subsarcolemmal increase in [Na⁺] to promote Ca²⁺ influx via reverse Na⁺–Ca²⁺ exchange.³⁶ Moreover, activation of TRPC can also lead to membrane depolarization and further activation of L-type Ca²⁺ channels,³⁷ a process that has previously been attributed solely to the downregulation of K_V channel in chronic hypoxic PASMCs.^4,6,7

Increase in store- and receptor-operated Ca²⁺ entries in chronic hypoxic PASMCs may contribute to alterations in pulmonary vasoreactivity.¹ Previous studies showed that hypoxic PAs exhibit dramatic increase in contractile response to vasoconstrictors, such as endothelin-1, angiotensin II, and serotonin,^1,23,24 presumable because of an increase or a shift in receptor expression.^38,39 However, most of these vasoconstrictors activate G-protein coupled receptors that stimulate phospholipase C to generate IP₃ and DAG, which may work synergistically to promote Ca²⁺ entry through TRPC channels. IP₃ activates Ca²⁺ release via IP₃ receptors, and cross-activates neighboring ryanodine receptors (RyRs) through Ca²⁺-induced Ca²⁺ release,²⁵ leading to local reduction/depletion of SR Ca²⁺ to activate store-operated Ca²⁺ channels, whereas DAG directly activates the receptor-operated Ca²⁺ channels.¹⁵ The coactivation of store- and receptor-operated channels by vasoconstrictors is exemplified by ET-1, which activates multiple channels with pharmacological and functional properties of store- and receptor-operated TRPC channels.^40,41 Hence, the upregulation of TRPC channels, in conjunction with elevated circulating levels of vasoconstrictors and increased agonist receptors in PASMCs,^38,39 may provide a powerful mechanism for increasing vascular reactivity in hypoxic pulmonary hypertension.

In conclusion, we have identified multiple TRPC channels and characterized distinctive store- and receptor-operated cation entry pathways in rat intralobar PASMCs. TRPC1 and TRPC6 are upregulated and both store- and receptor-operated cation entries are potentiated in hypoxic pulmonary hypertension. The enhanced TRPC-dependent pathways are crucial to the elevated basal [Ca²⁺]_i and vascular tone in chronic hypoxic PAs. These results may provide the physiological basis for targeting TRPC channels for future treatments of hypoxia related pulmonary hypertension.

	Acknowledgments

 
This work is supported in part by National Institutes of Health grants HL-075134, HL-071835, and HL-063813 (to J.S.K.S.). X.R.Y. is supported by an American Lung Association postdoctoral fellowship.

	Footnotes

 
This manuscript was sent to Elizabeth Nabel, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

Original received January 29, 2004; revision received July 1, 2004; accepted July 2, 2004.

	References

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