Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of California School of Medicine, San Diego, California 92103
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ABSTRACT |
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Agonist-induced increases in cytosolic Ca2+ concentration ([Ca2+]cyt) in pulmonary artery (PA) smooth muscle cells (SMCs) consist of a transient Ca2+ release from intracellular stores followed by a sustained Ca2+ influx. Depletion of intracellular Ca2+ stores triggers capacitative Ca2+ entry (CCE), which contributes to the sustained increase in [Ca2+]cyt and the refilling of Ca2+ into the stores. In isolated PAs superfused with Ca2+-free solution, phenylephrine induced a transient contraction, apparently by a rise in [Ca2+]cyt due to Ca2+ release from the intracellular stores. The transient contraction lasted for 3-4 min until the Ca2+ store was depleted. Restoration of extracellular Ca2+ in the presence of phentolamine produced a contraction potentially due to a rise in [Ca2+]cyt via CCE. The store-operated Ca2+ channel blocker Ni2+ reduced the store depletion-activated Ca2+ currents, decreased CCE, and inhibited the CCE-mediated contraction. In single PASMCs, we identified, using RT-PCR, five transient receptor potential gene transcripts. These results suggest that CCE, potentially through transient receptor potential-encoded Ca2+ channels, plays an important role in agonist-mediated PA contraction.
transient receptor potential gene; pulmonary hypertension; pulmonary artery smooth muscle cells
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INTRODUCTION |
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EXCESSIVE PULMONARY VASOCONSTRICTION and elevated circulating vasoactive agonists have been implicated in pulmonary hypertension (7, 12, 28, 40). An increase in the cytosolic free Ca2+ concentration ([Ca2+]cyt) in pulmonary artery (PA) smooth muscle cells (SMCs) is a major trigger for pulmonary vasoconstriction when the vessels are stimulated by vasoactive agonists such as L-phenylephrine (PE) (9) and serotonin (30). The two sources of increased cytosolic Ca2+ on activation of various membrane receptors are 1) intracellular Ca2+ stores, mainly the sarcoplasmic reticulum (SR) in muscle cells, and 2) extracellular Ca2+ that enters the cell through opened Ca2+-permeable channels in the plasma membrane (8, 37). The SR in SMCs has a limited capacity for Ca2+ storage. Therefore, the receptor activation-mediated Ca2+ mobilization depletes the internal stores, which have to be replenished through the entry of Ca2+ from the extracellular milieu. Indeed, depletion of Ca2+ from the SR due to agonist-induced Ca2+ release triggers Ca2+ influx, referred to as capacitative Ca2+ entry (CCE) (31). Store depletion-mediated CCE is therefore a critical mechanism for refilling the empty SR with Ca2+ and maintaining a sustained increase in [Ca2+]cyt (5, 29).
In single vascular SMCs in vitro, agonist-mediated changes in [Ca2+]cyt consist of a transient increase in [Ca2+]cyt due to Ca2+ release from the SR and a sustained increase in [Ca2+]cyt due to Ca2+ influx through sarcolemmal Ca2+ channels. There are at least three classes of Ca2+-permeable channels: 1) store-operated Ca2+ channels (SOCs) (29), 2) receptor-operated Ca2+ channels (ROCs) (11, 37), and 3) voltage-dependent Ca2+ channels (VDCCs) (24). Agonist-induced Ca2+ influx is caused mainly by receptor-mediated activation of ROCs and store depletion-mediated opening of SOCs (5, 29, 37). Membrane depolarization-mediated activation of VDCCs has also been implicated in agonist-induced increases in [Ca2+]cyt (8, 24).
Electrophysiological studies on the store depletion-activated CCE current, also termed SOC current (ISOC), indicate that there are multiple types of SOCs with different single-channel conductances (19, 20). It has recently been demonstrated that a novel gene family, transient receptor potential (TRP), is essential for encoding putative Ca2+-permeable cation channels responsible for agonist-activated CCE (5, 26, 45). Expression of TRP genes in Xenopus oocytes or mammalian cell lines does, indeed, result in the formation of Ca2+ channels that are activated by Ca2+ store depletion. These findings suggest that SOCs may be composed of subunits encoded in TRP genes (5, 20, 45).
In PASMCs, production of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and diacylglycerol is a common mechanism by which vasoactive agonists raise [Ca2+]cyt (5, 8). The Ins(1,4,5)P3-sensitive SR serves as an important source for agonist-mediated Ca2+ release into the cytoplasm (36), whereas the diacylglycerol-mediated opening of ROCs and VDCCs (8, 37) is an important pathway for Ca2+ entry from the extracellular milieu. The contribution of CCE to the agonist-mediated vasomotor tone has been elucidated only recently (13, 27, 42). The role of CCE and SOCs in agonist-mediated pulmonary vasoconstriction and the molecular identity of SOCs in PASMCs, however, are still incompletely understood. This study was designed to test the hypothesis that agonist-mediated increases in [Ca2+]cyt due to CCE through TRP-encoded SOCs participate in producing vasoconstriction in isolated rat PAs.
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MATERIALS AND METHODS |
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Isolation of PA rings. The right branch of the main PA was isolated from male Sprague-Dawley rat (100-125 g) lung tissues. The adipose and connective tissues were carefully stripped off with fine forceps, and the remaining PAs were then cut into 2-mm-long rings. In some of the experiments, the endothelium of the PA rings was removed by gently rubbing the inner lumen of the vessels with a rough wooden stick. This procedure appeared not to damage the vessels because high K+-mediated contractions did not differ significantly between PA rings with and without endothelium.
Tension measurement. Two stainless steel hooks (0.1-mm diameter) were inserted through the lumen of the rings. One hook was mounted in a perfusion chamber (0.75-ml volume), and the other hook was connected to an isometric force transducer (Harvard Apparatus). Isometric tension was continuously monitored and recorded on an IBM-compatible PC with DATAQ data acquisition software (DATAQ Instruments). Resting passive tension was maintained throughout the experiments at 600-625 mg, which offered the maximal tension when the rings were exposed to 40 mM K+. The rings were equilibrated for ~60 min at resting tension and then challenged two to three times with 40 mM K+-containing perfusate to obtain a stable contractile response.
Isolated PA rings were superfused with modified Krebs solution (MKS; at 37°C) that contained (in mM) 138 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 NaH2PO4, 5 HEPES, 1.8 CaCl2, and 10 glucose (pH 7.4). In the Ca2+-free solution, CaCl2 was replaced by equimolar MgCl2 and 1 mM EGTA was added to chelate residual Ca2+. In the high-K+ solution, NaCl was replaced by equimolar KCl to maintain osmolarity. PE, phentolamine, nifedipine, verapamil (Sigma), NiCl2, and LaCl3 were dissolved directly in the perfusate on the day of use. The pH values of all solutions were measured after addition of the drugs and readjusted to 7.4.Cell preparation. To isolate PASMCs, the right and left branches of the main PA were isolated and incubated for 20 min in Hanks' solution containing 2 mg/ml of collagenase. The adventitia and endothelium were removed after the incubation. The remaining smooth muscle was digested with 0.5 mg/ml of elastase and 2.0 mg/ml of collagenase at 37°C. Single PASMCs were plated onto 25-mm coverslips (for patch-clamp and fluorescence experiments) and 10-cm petri dishes (for molecular biological experiments). The cells were incubated in a humidified atmosphere of 5% CO2 in air at 37°C and cultured in 10% fetal bovine serum (FBS)-DMEM for 5-6 days before the experiment.
PA endothelial cells (PAECs) were obtained by gently scraping the intima of the PAs with a plastic cell lifter and seeding into a 10-cm petri dish containing 10% FBS-DMEM-F-12 medium. After incubation for 1 wk (5% CO2 in air at 37°C), SMC contaminants were removed by pipette aspiration. The cultures were verified as endothelial cells by positive factor VIII staining and uptake of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-labeled acetylated low-density lipoprotein. PAECs were used for experimentation at passages 6-10 (23).Electrophysiological measurements.
Whole cell ISOC was recorded with an Axopatch-1D
amplifier and a DigiData 1200 interface with patch-clamp techniques
(16, 43). Patch pipettes (2-4 M) were made on a
Sutter electrode puller with borosilicate glass tubes and fire polished
on a microforge. Voltage stimuli lasting 300 ms were delivered from a
holding potential of 0 mV in voltage steps from
120 to +20 mV (in
20-mV increments). Traces recorded before the activation of
SOCs were used as a template to subtract leak currents. All
experiments were performed at room temperature (22-24°C). In the
patch-clamp experiments, SOCs were activated by passive depletion of
the SR with 10 µM cyclopiazonic acid (CPA) dissolved in the
Ca2+-free solution.
Measurement of [Ca2+]cyt. In single PASMCs, [Ca2+]cyt was measured with the Ca2+-sensitive fluorescent indicator fura 2 and the quantitative fluorescence microscopy system (15, 43). Cells were loaded with the acetoxymethyl ester form of fura 2 (fura 2-AM; 3 µM for 30 min) in the dark at room temperature (22-24°C) under an atmosphere of 5% CO2-95% air. The fura 2-loaded cells on the coverslips were then transferred to a recording cell chamber mounted on a microscope stage and superfused with MKS for 30 min to remove the extracellular fura 2, which may have diffused out of the cell after initial loading, and to allow cytosolic esterases to cleave fura 2-AM into active fura 2. The fluorescence microscopy experiments were carried out at 32°C.
Fura 2 fluorescence (510-nm light emission excited by 340- and 380-nm illumination) from the cell as well as background fluorescence was collected with Nikon UV-Fluor objectives. The fluorescence signals emitted from the cells were monitored continuously with an intracellular imaging fluorescence microscopy system and recorded on an IBM-compatible computer for later analysis. The 340- to 380-nm fluorescence ratio was used to indicate the changes in [Ca2+]cyt in PASMCs treated with the various agonists. The resting [Ca2+]cyt, calculated according to the method previously reported by Grynkiewicz et al. (15), was 70 ± 11 nM in rat PASMCs. CPA (Sigma) was dissolved in DMSO to make a stock solution of 30 mM. Aliquots of the stock solution were then diluted 1:3,000 in the bath solution to make a final concentration of 10 µM CPA (pH 7.4). In [Ca2+]cyt and ISOC measurement experiments, the same amount of DMSO (0.03%) used for dissolving CPA was added to the control solutions. Vehicle (DMSO) alone had negligible effects on [Ca2+]cyt and ISOC in PASMCs.RT-PCR.
Total RNA [3 µg; 260- to 280-nm optical density (OD) ratio > 1.7] was prepared from single cells (PASMCs and PAECs) and brain tissues by the acid guanidinium thiocyanate-phenol-chloroform extraction method and reverse transcribed with random hexamers [pd(N)6 primer] (41). The sense and
antisense primers chosen to amplify the cDNA were specifically designed
from the coding regions of various TRPs (Table
1). The fidelity and specificity of the
sense and antisense oligos were examined with the BLAST program. PCR
was performed by a GeneAmp PCR system with AmpliTaq DNA
polymerase and accompanying buffers. The first-strand cDNA reaction
mixture (3 µl) was used in a 50-µl PCR consisting of 0.2 nmol of
each primer, 10 mM Tris · HCl, pH 8.3, 50 mM KCl, 2 mM
MgCl2, 200 µM each deoxynucleotide triphosphate, and 2 U of Taq DNA polymerase. The cDNA samples were amplified in a
DNA thermal cycler under the following conditions: the mixture was annealed at 52-61°C (1 min), extended at 72°C (2.0 min), and
denatured at 94°C (1 min) for 20-30 cycles. This was followed by
a final extension at 72°C (10 min) to ensure complete product
extension. The PCR products were electrophoresed through a 2% agarose
gel, and the amplified cDNA bands were visualized by ethidium bromide staining. To quantify the PCR products of TRPs, an invariant mRNA of
smooth muscle -actin was used as an internal control. Immediately after each experiment, the OD value for each band on the gel was measured by a gel documentation system. The OD values in TRP signals were normalized to the OD values in the
-actin signals. The
normalized values are expressed as arbitrary units for quantitative
comparison.
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Statistical analysis. The composite data are expressed as means ± SE. Statistical analyses were performed with unpaired Student's t-test or ANOVA and post hoc tests (Student-Newman-Keuls) where appropriate. Differences were considered to be significant when P < 0.05.
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RESULTS |
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Requirement of extracellular Ca2+ for
pulmonary vasoconstriction.
Cytosolic Ca2+ stemming from extracellular fluids is
important in triggering and maintaining agonist-mediated
vasoconstriction. In isolated rat PA rings, removal of extracellular
Ca2+ almost abolished the active tension induced by 40 mM
K+ or 2 µM PE, an -receptor agonist (Fig.
1A). Chelation of
extracellular Ca2+ with 2 mM EGTA, which decreases free
Ca2+ concentration ([Ca2+]) to ~0.5 µM in
MKS containing 1.8 mM CaCl2 (10), had the same inhibitory effects on contractions induced by 40 mM K+ or
PE (Fig. 1B). Furthermore, extracellular application of the L-type VDCC blockers nifedipine (10 µM) and verapamil (0.5 µM) almost abolished the 40 mM K+-induced active tension but
only inhibited the PE-induced contraction by 35-45% (Fig.
2). These observations indicate that PE
induces PA contraction by activating multiple Ca2+ entry
pathways, whereas 40 mM K+-induced PA contraction is solely
dependent on the membrane depolarization-mediated opening of VDCCs
(8, 24).
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CCE-mediated pulmonary vasoconstriction.
In the absence of extracellular Ca2+, PE
induced a transient contraction that was apparently due to
Ca2+ release from the SR (Fig.
3Ab). Restoration of
extracellular Ca2+ in the presence of the -receptor
blocker phentolamine (1 µM) and the VDCC blocker verapamil (0.5 µM)
caused a similar contraction that was apparently due to CCE triggered
by PE-induced store depletion (Fig. 3Ac). The CCE-mediated
PA contraction in the presence of phentolamine and verapamil accounted
for ~23% of the total peak contraction induced by PE in the presence
of extracellular Ca2+ (Fig. 3, Aa and
B). Removal of phentolamine further constricted the vessel
in the presence of extracellular Ca2+, which presumably
resulted from Ca2+ influx through ROCs (Fig.
3Ab). These results indicate that Ca2+ release
from the SR and CCE through SOCs both contribute to agonist-mediated pulmonary vasoconstriction.
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Enhancement of CCE-induced contraction in endothelium-denuded PA
rings.
CCE is also a pathway for raising [Ca2+]cyt
in PAECs (23). An increase in
[Ca2+]cyt in PAECs stimulates nitric oxide
(NO) synthesis and release, thereby causing PA relaxation
(2). Accordingly, functional removal of the endothelium in
isolated rat PA rings, confirmed by the loss of acetylcholine-mediated
vasodilation (Fig. 4A,
left), significantly enhanced the CCE-mediated PA
contraction but negligibly affected the Ca2+
release-mediated PA contraction (Fig. 4B). The active
tension due to CCE was 189 ± 36 mg/mg in endothelium-intact PA
rings (n = 11) and 637 ± 28 mg/mg in
endothelium-denuded rings (P < 0.001; n = 6), whereas the tension due to Ca2+
release was 206 ± 52 (n = 27 rings) and 251 ± 26 mg/mg (P = 0.1; n = 6 rings),
respectively (Fig. 4B).
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Inhibitory effects of Ni2+ and
La3+ on CCE-mediated PA contraction.
CCE is largely determined by the conductance of SOCs when the
Ca2+ transmembrane electrochemical gradient is constant.
Ni2+ and La3+ have been demonstrated to block
SOCs in many cell types (18, 19). Indeed, extracellular
application of Ni2+ (1 mM) or La3+ (50 µM)
significantly and reversibly attenuated the CCE-mediated PA contraction
(Fig. 5).
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CCE-induced [Ca2+]cyt
increases in single PASMCs.
In the absence of extracellular Ca2+, CPA, by blocking
Ca2+ sequestration into the SR, induced a transient
[Ca2+]cyt rise due to leakage of
Ca2+ from the SR to the cytosol. The CPA-induced
[Ca2+]cyt transient returned to the original
baseline level after 5-7 min as the SR Ca2+ was
depleted. Under these conditions, restoration of extracellular Ca2+ induced a second rise in
[Ca2+]cyt, which was probably due to CCE
(Fig. 6A).
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Whole cell ISOC in single PASMCs.
Cells were superfused with solutions containing 120 mM Na+
and 20 mM Ca2+ and dialyzed with solutions containing 138 mM Cs+ and ~100 nM free Ca2+ (Fig.
7A). The holding potential was
set at 0 mV to inactivate voltage-gated Na2+ and
Ca2+ channels. Whole cell currents were elicited by 300-ms
voltage steps from 120 to +20 mV before and after application of CPA (10 µM; Fig. 7Ba) or PE (10 µM; Fig.
8A). Subtraction of the
current recorded under control conditions from the current recorded
during application of CPA revealed that ISOC was
activated by the CPA-induced depletion of SR Ca2+ (Fig.
7Bb). The inward current was carried by Ca2+
(and Na+), and the outward current was carried by Cs (Cs
permeability = Na permeability for SOCs) (18,
19).
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Expression of TRP channels in PASMCs and PAECs.
The SOCs involved in CCE are believed to be encoded by TRP genes
(4, 5). Two TRP transcripts, TRP1 and TRP5, were detected in both PASMCs and PAECs (Fig.
9A). Interestingly, expression of TRP2, TRP3, TRP4, and TRP6 was quite different between PASMCs and
PAECs. TRP4 and TRP6 were detected only in PASMCs, whereas TRP3 was detectable only in PAECs (Fig. 9B). The mRNA
expression of TRP2 appeared to be much greater in PAECs than in PASMCs
(Fig. 9B). Furthermore, TRP7 was not detected in PASMCs and
PAECs but was strongly expressed in the rat brain (Fig. 9C).
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DISCUSSION |
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Pulmonary vasoconstriction greatly contributes to the elevated
pulmonary vascular resistance in patients with pulmonary hypertension (32). A rise in [Ca2+]cyt in
PASMCs is a major trigger for pulmonary vasoconstriction (1, 33,
34). The results from this study demonstrate that 1)
CCE contributes to the PE-induced contraction in isolated PA rings in
the presence of the -receptor blocker phentolamine and the
VDCC blocker verapamil; 2) removal of the endothelium
enhances the CCE-mediated PA contraction with little effect on the
Ca2+ release-mediated PA contraction; 3) the
CPA-mediated passive store depletion induces CCE by eliciting
ISOC in single PASMCs; 4) the SOC
blocker Ni2+ (and La3+) reduces the
ISOC, attenuates CCE, and inhibits the
CCE-induced PA contraction; and 5) the TRP genes that are
believed to encode SOCs responsible for ISOC and
CCE are heterogeneously expressed in PASMCs (TRP1, TRP2, TRP4, TRP5,
and TRP6) and PAECs (TRP1, TRP3, and TRP5). These results suggest that
CCE, putatively through TRP-encoded SOCs in PASMCs, is involved in
agonist-mediated pulmonary vasoconstriction. Pathophysiologically,
increases in CCE as a result of upregulated TRP expression and/or
augmented SOC activity may play a role in the development of excessive
pulmonary vasoconstriction in patients with pulmonary hypertension.
Role of Ca2+ in vasoconstriction. A rise in [Ca2+]cyt induced by vasoactive agonists in vascular SMCs has been well documented to initiate and maintain smooth muscle contraction (8, 33, 34). When [Ca2+]cyt rises, Ca2+ binds to calmodulin, which activates myosin light chain kinase to phosphorylate myosin light chain. This phosphorylation increases the activity of myosin ATPase that hydrolyzes ATP, thereby releasing energy. Subsequent cycling of the myosin cross bridges produces displacement of the myosin filament in relation to the actin filament causing contraction (33, 34).
In the absence of extracellular Ca2+, PE induced a transient (5-7 min) PA contraction and a small steady-state active tension. That phentolamine blocked the contraction in Ca2+-free solution suggests that 1) Ca2+ release from intracellular stores and 2) Ca2+ sensitization of contractile proteins are both involved in PE-mediated PA contraction (1). After the PE-mediated transient contraction in the absence of extracellular Ca2+ returned to baseline, restoration of extracellular Ca2+ induced a sustained contraction in the presence of theRegulation of [Ca2+]cyt in PASMCs. The agonist-mediated increases in [Ca2+]cyt usually consist of an initial Ca2+ release from intracellular stores followed by a sustained Ca2+ entry through sarcolemmal Ca2+ channels (9). The SR has been described as the most important intracellular Ca2+ store in many cell types (5, 37). At least two functionally and spatially distinguishable SRs have been identified in vascular SMCs: 1) an Ins(1,4,5)P3-sensitive SR that is involved in Ca2+ mobilization during agonist stimulation and is sensitive to the SR Ca2+ pump inhibitors CPA and thapsigargin and 2) a ryanodine-sensitive SR that is activated by caffeine and is responsible for Ca2+-induced Ca2+ release (36). Most of the intracellular Ca2+ in unstimulated cells is sequestered into the SR by the SR Ca2+ pump to maintain a very low [Ca2+]cyt (50-100 nM). Extracellular [Ca2+] and intracellularly stored [Ca2+] in the SR are ~1-2 mM, which is 10,000- to 20,000-fold greater than [Ca2+]cyt (5). Passive Ca2+ influx (from the extracellular milieu) and release (from the SR) to the cytoplasm are, therefore, driven by the electrochemical gradient that causes [Ca2+]cyt to rise without an expenditure of energy.
The Ca2+ influx through sarcolemmal VDCCs is an important mechanism involved in agonist-mediated vasoconstriction. An important component of the PE-induced PA contraction is the sustained contraction due to Ca2+ influx through L-type VDCCs. To determine the contribution of VDCC-independent mechanisms to the PE-induced PA contraction, we used both nifedipine and verapamil to block the L-type VDCC in PA rings. Blockade of VDCCs by nifedipine or verapamil (44) abolished the PA contraction induced by 40 mM K+ but only decreased the PE-mediated contraction by 35-45%. Because nifedipine and verapamil block L-type VDCCs by different mechanisms, these results clearly show that 55-65% of the PE-induced PA contraction are due to mechanisms other than Ca2+ influx through VDCCs. In addition to opening VDCCs, PE triggers Ca2+ release from intracellular stores and induces Ca2+ influx through ROCs and SOCs. This is why nifedipine or verapamil only partially inhibited PE-mediated pulmonary contraction. Increasing the extracellular K+ concentration from 4.7 to 40 mM shifted the K+ equilibrium potential fromRole of CCE in PAECs. It has been demonstrated that endothelial cells do not express VDCCs; thus CCE may be an important pathway for the agonist-mediated increases in [Ca2+]cyt in PAECs (23). A rise in [Ca2+]cyt in PAECs would stimulate NO synthase activity, increase NO production, and induce pulmonary vasodilation. Indeed, the Ca2+ ionophore A-23187 causes an elevation in [Ca2+]cyt in PAECs, NO production, and pulmonary vasodilation (2). Our observations indicate that removal of the endothelium significantly enhances the CCE-mediated PA contraction and that multiple TRPs (TRP1, TRP2, TRP3, and TRP5) are expressed in PAECs. These results suggest that CCE through TRP-encoded SOCs is also an important mechanism responsible for the agonist-mediated increases in [Ca2+]cyt in PAECs (8, 23). The resultant vasorelaxation may play a negative feedback role in controlling agonist-induced vasomotor tone.
Molecular identity of CCE channels in PASMCs. Electrophysiological studies on endogenous ISOC indicate that there are at least five types of SOCs based on the single-channel conductance: a <0.2-pS channel (17), a 3- to 5-pS channel (14, 35), a 16-pS channel (21), a 36- to 40-pS channel (19), and an 89- to 110-pS channel (20). Because of the diversity and variability of biophysical and pharmacological properties of ISOC, SOCs are believed to be complex and heterogeneous in molecular composition and cellular regulation.
In excised membrane patches of mouse aortic smooth muscle cells, Trepakova et al. (35) identified a 3.4-pS SOC that was activated by 1,2-bis(2-aminophenoxy)- ethane-N,N,N,'N'-tetraacetic acid- or thapsigargin-induced passive depletion of intracellular Ca2+ stores. The 3.4-pS channel was also activated by a Ca2+ influx factor released from platelets when intracellularly stored Ca2+ was depleted by thapsigargin (35). In cell-attached membrane patches of human PASMCs, Golovina et al. (14) previously observed a 5.34-pS Ca2+ channel that was activated by CPA- or thapsigargin-induced passive depletion of intracellular Ca2+ stores (14). These observations suggest that this 3- to 5-pS Ca2+-permeable SOC is a native store-operated channel in systemic and pulmonary vascular smooth muscle cells. It remains to be investigated whether active depletion of intracellular Ca2+ stores with PE would activate this 3- to 5-pS channel in rat PASMCs. Heterologous expression of TRP genes in Xenopus oocytes and mammalian cells causes the appearance of store depletion-activated Ca2+ currents (25, 38, 46) and enhances the increase in [Ca2+]cyt due to CCE (6, 39, 45). These observations provide strong evidence that SOCs are made up of subunits encoded in TRP genes; the diversity and heterogeneity of ISOC and CCE may result from the homo- and heteromultimeric SOCs formed by multiple TRP subunits (4, 5). It has been proposed in mammalian cells that TRP1, TRP2, TRP4, and TRP5 may form endogenous SOCs that are activated solely by store depletion, whereas TRP3, TRP6, and TRP7 may form the channels that are activated by both store depletion and the production of Ins(1,4,5)P3 and diacylglycerol (4). In this study, we observed that 1) TRP1, TRP2, and TRP5 were ubiquitously expressed in both PASMCs and PAECs, 2) TRP3 was specifically expressed in PAECs, and 3) TRP4 and TRP6 were selectively expressed in PASMCs. The exclusive expression of TRP4 and TRP6 in PASMCs and of TRP3 in PAECs suggests that different cell types may use different TRP genes to express SOCs. Because PAECs lack VDCCs, TRP3-encoded Ca2+-permeable channels may also serve as an important ROC that can be activated by receptor-induced Ins(1,4,5)P3 activation and diacylglycerol production (4, 5, 22). In summary, CCE is an important mechanism for triggering PA contraction by maintaining the sustained increase in [Ca2+]cyt and refilling Ca2+ into the SR in PASMCs. The endogenous SOCs responsible for ISOC and CCE in PASMCs may be formed heteromerically by multiple TRP gene products such as TRP1, TRP4, and TRP5. Upregulation of TRP gene expression and augmentation of ISOC and CCE may be involved in the excessive pulmonary vasoconstriction implicated in pulmonary hypertension. Drugs directed at inhibiting TRP expression or blocking SOCs may be helpful for the treatment of patients with pulmonary hypertension. ![]() |
ACKNOWLEDGEMENTS |
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We thank Drs. Judy Creighton and T. Stevens for providing the endothelial cells.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-54043 and HL-64945 (to J. X.-J. Yuan).
J. X.-J. Yuan is an Established Investigator of the American Heart Association (974009N).
Address for reprint requests and other correspondence: J. X.-J. Yuan, Dept. of Medicine, UCSD Medical Center (8382), 200 West Arbor Dr., San Diego, CA 92103-8382 (E-mail: xiyuan{at}ucsd.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 11 July 2000; accepted in final form 21 September 2000.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Aburto, TK,
Lajoie C,
and
Morgan KG.
Mechanisms of signal transduction during 2-adrenergic receptor-mediated contraction of vascular smooth muscle.
Circ Res
72:
778-785,
1993[Abstract].
2.
Archer, SL,
and
Cowan NJ.
Measurement of endothelial cytosolic calcium concentration and nitric oxide production reveals discrete mechanisms of endothelium-dependent pulmonary vasodilatation.
Circ Res
68:
1569-1581,
1991[Abstract].
3.
Arnon, A,
Hamlyn JM,
and
Blaustein MP.
Na+ entry via store-operated channels modulates Ca2+ signaling in arterial myocytes.
Am J Physiol Cell Physiol
278:
C163-C173,
2000
4.
Berridge, MJ,
Lipp P,
and
Bootman MD.
The calcium entry pas de deux.
Science
287:
1604-1605,
2000
5.
Birnbaumer, L,
Zhu X,
Jiang M,
Boulay G,
Peyton M,
Vannier B,
Brown D,
Platano D,
Sadeghi H,
Stefani E,
and
Birnbaumer M.
On the molecular basis and regulation of cellular capacitative calcium entry: roles for Trp proteins.
Proc Natl Acad Sci USA
93:
15195-15202,
1996
6.
Boulay, G,
Brown DM,
Qin N,
Jiang M,
Dietrich A,
Zhu MX,
Chen Z,
Birnbaumer M,
Mikoshiba K,
and
Birnbaumer L.
Modulation of Ca2+ entry by polypeptides of the inositol 1,4,5-trisphosphate receptor (IP3R) that bind transient receptor potential (TRP): evidence for roles of TRP and IP3R in store depletion-activated Ca2+ entry.
Proc Natl Acad Sci USA
96:
14955-14960,
1999
7.
Christman, BW,
McPherson CD,
Newman JH,
King GA,
Bernard GR,
Groves BM,
and
Loyd JE.
An imbalance between the excretion of thromboxane and prostacyclin metabolites in pulmonary hypertension.
N Engl J Med
327:
70-75,
1992[Abstract].
8.
Davis, MJ,
and
Hill MA.
Signaling mechanisms underlying the vascular myogenic response.
Physiol Rev
79:
387-423,
1999
9.
Doi, S,
Damron DS,
Horibe M,
and
Murray PA.
Capacitative Ca2+ entry and tyrosine kinase activation in canine pulmonary arterial smooth muscle cells.
Am J Physiol Lung Cell Mol Physiol
278:
L118-L130,
2000
10.
Fabiato, A,
and
Fabiato F.
Calculator programs for computing the composition of the solutions containing multiple metals and ligands used for experiments in skinned muscle cells.
J Physiol (Paris)
75:
463-505,
1979[Medline].
11.
Fasolato, C,
Innocenti B,
and
Pozzan T.
Receptor-activated Ca2+ influx; how many mechanisms for how many channels?
Trends Pharmacol Sci
15:
77-83,
1994[ISI][Medline].
12.
Giaid, A,
Yanagisawa M,
Langleben D,
Michel RP,
Levy R,
Shennib H,
Kimura S,
Masaki T,
Duguid WP,
and
Stewart DJ.
Expression of endothelin-1 in the lungs of patients with pulmonary hypertension.
N Engl J Med
328:
1732-1739,
1993
13.
Gibson, A,
McFadzean I,
Wallace P,
and
Wayman CP.
Capacitative Ca2+ entry and the regulation of smooth muscle tone.
Trends Pharmacol Sci
19:
266-269,
1998[ISI][Medline].
14.
Golovina, VA,
Platoshyn O,
Bailey CL,
Wang J,
Limsuwan A,
Sweeney M,
Rubin LJ,
and
Yuan JX-J.
Upregulated TRP and enhanced capacitative Ca2+ entry in human pulmonary artery myocytes during proliferation.
Am J Physiol Heart Circ Physiol
280:
H746-H755,
2001
15.
Grynkiewicz, G,
Poenie M,
and
Tsien RY.
A new generation of Ca2+ indicators with greatly improved fluorescence properties.
J Biol Chem
260:
3440-3450,
1985[Abstract].
16.
Hamill, OP,
Marty A,
Neher E,
Sakmann B,
and
Sigworth FJ.
Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.
Pflügers Arch
391:
85-100,
1981[ISI][Medline].
17.
Hoth, M,
and
Penner R.
Calcium release-activated calcium current in rat mast cells.
J Physiol (Lond)
465:
359-386,
1993[Abstract].
18.
Kerschbaum, HH,
and
Cahalan MD.
Monovalent permeability, rectification, and ionic block of store-operated calcium channels in Jurkat T lymphocytes.
J Gen Physiol
111:
521-537,
1998
19.
Kerschbaum, HH,
and
Cahalan MD.
Single-channel recording of a store-operated Ca2+ channel in Jurkat T lymphocytes.
Science
283:
836-839,
1999
20.
Kunze, DL,
Sinkins WG,
Vaca L,
and
Schilling WP.
Properties of single Drosophila Trpl channels expressed in Sf9 insect cells.
Am J Physiol Cell Physiol
272:
C27-C34,
1997
21.
Luckhoff, A,
and
Clapham DE.
Calcium channels activated by depletion of internal calcium stores in A431 cells.
Biophys J
67:
177-182,
1994[Abstract].
22.
Ma, H-T,
Patterson RL,
van Rossum DB,
Birnbaumer L,
Mikoshiba K,
and
Gill DL.
Requirement of the inositol trisphosphate receptor for activation of store-operated Ca2+ channels.
Science
287:
1647-1651,
2000
23.
Moore, TM,
Brough GH,
Babal P,
Kelly JJ,
Li M,
and
Stevens T.
Store-operated calcium entry promotes shape change in pulmonary endothelial cells expressing Trp1.
Am J Physiol Lung Cell Mol Physiol
275:
L203-L222,
1998
24.
Nelson, MT,
Patlak JB,
Worley JF,
and
Standen NB.
Calcium channels, potassium channels, and voltage dependence of arterial smooth muscle tone.
Am J Physiol Cell Physiol
259:
C3-C18,
1990
25.
Niemeyer, B,
Suzuki E,
Scott K,
Jalinik K,
and
Zuker CS.
The Drosophila light-activated conductance is composed of the two channels TRP and TRPL.
Cell
83:
651-659,
1996.
26.
Okada, T,
Inoue R,
Yamazaki K,
Maeda A,
Kurosaki T,
Yamakuni T,
Tanaka I,
Shimizu S,
Ikenaka K,
Imoto K,
and
Mori Y.
Molecular and functional characterization of a novel mouse transient receptor potential protein homologue TRP7. Ca2+-permeable cation channel that is constitutively activated and enhanced by stimulation of G protein-coupled receptor.
J Biol Chem
274:
27359-27370,
1999
27.
Pacaud, P,
Loirand G,
Gregoire G,
Mironneau C,
and
Mironneau J.
Noradrenaline-activated heparin-sensitive Ca2+ entry after depletion of intracellular Ca2+ store in portal vein smooth muscle cells.
J Biol Chem
268:
3866-3872,
1993
28.
Palevsky, HI,
Schloo BL,
Pietra GG,
Weber KT,
Janicki JS,
Rubin E,
and
Fishman AP.
Primary pulmonary hypertension. Vascular structure, morphometry, and responsiveness to vasodilator agents.
Circulation
80:
1207-1221,
1989[Abstract].
29.
Parekh, AB,
and
Penner R.
Store depletion and calcium influx.
Physiol Rev
77:
901-930,
1997
30.
Pitt, BR,
Weng W,
Steve AR,
Blakely RD,
Reynolds I,
and
Davies P.
Serotonin increases DNA synthesis in rat proximal and distal pulmonary vascular smooth muscle cells in culture.
Am J Physiol Lung Cell Mol Physiol
266:
L178-L186,
1994
31.
Putney, JW, Jr.
A model for receptor-regulated calcium entry.
Cell Calcium
7:
1-12,
1986[ISI][Medline].
32.
Rubin, LJ.
Primary pulmonary hypertension.
N Engl J Med
336:
111-117,
1997
33.
Somlyo, AP,
and
Somlyo AV.
Signal transduction and regulation in smooth muscle.
Nature
372:
231-236,
1994[ISI][Medline].
34.
Stull, JT,
Kamm KE,
and
Taylor DA.
Calcium control of smooth muscle contractility.
Am J Med Sci
296:
241-245,
1988[ISI][Medline].
35.
Trepakova, ES,
Csutora P,
Hunton DL,
Marchase RB,
Cohen RA,
and
Bolotina VM.
Calcium influx factor directly activates store-operated cation channels in vascular smooth muscle cells.
J Biol Chem
275:
26158-26163,
2000
36.
Tribe, RM,
Borin ML,
and
Blaustein MP.
Functionally and spatially distinct Ca2+ stores are revealed in cultured vascular smooth muscle cells.
Proc Natl Acad Sci USA
91:
5908-5912,
1994[Abstract].
37.
Tsien, RW,
and
Tsien RY.
Calcium channels, stores, and oscillations.
Annu Rev Cell Biol
6:
715-760,
1990[ISI].
38.
Vaca, L,
and
Kunze DL.
Depletion of intracellular Ca2+ stores activates a Ca2+-selective channel in vascular endothelium.
Am J Physiol Cell Physiol
267:
C920-C925,
1994
39.
Vannier, B,
Peyton M,
Boulay G,
Brown D,
Qin N,
Jiang M,
Zhu X,
and
Birnbaumer L.
Mouse trp2, the homologue of the human trpc2 pseudogene, encodes mTrp2, a store depletion-activated capacitative Ca2+ entry channel.
Proc Natl Acad Sci USA
96:
2060-2064,
1999
40.
Wagenvoort, CA.
Vasoconstriction and medial hypertrophy in pulmonary hypertension.
Circulation
22:
535-546,
1960[ISI].
41.
Wang, J,
Juhaszova M,
Rubin LJ,
and
Yuan X-J.
Hypoxia inhibits gene expression of voltage-gated K+ channel alpha subunits in pulmonary artery smooth muscle cells.
J Clin Invest
100:
2347-2353,
1997
42.
Yoshimura, M,
Oshima T,
Matsuura H,
Ishida T,
Kambe M,
and
Kajiyama G.
Extracellular Mg2+ inhibits capacitative Ca2+ entry in vascular smooth muscle cells.
Circulation
95:
2567-2572,
1997
43.
Yuan, X-J.
Voltage-gated K+ currents regulate resting membrane potential and [Ca2+]i in pulmonary arterial myocytes.
Circ Res
77:
370-378,
1995
44.
Yuan, X-J,
Tod ML,
Rubin LJ,
and
Blaustein MP.
Contrasting effects of hypoxia on tension in rat pulmonary and mesenteric arteries.
Am J Physiol Heart Circ Physiol
259:
H281-H289,
1990
45.
Zhu, X,
Jiang M,
Peyton M,
Boulay G,
Hurst R,
Stefani E,
and
Birnbaumer L.
trp, a novel mammalian gene family essential for agonist-activated capacitative Ca2+ entry.
Cell
85:
661-671,
1996[ISI][Medline].
46.
Zitt, C,
Zobel A,
Obukhov AG,
Harteneck C,
Kalkbrenner F,
Luckhoff A,
and
Schultz G.
Cloning and functional expression of a human Ca2+-permeable cation channel activated by calcium store depletion.
Neuron
16:
1189-1196,
1996[ISI][Medline].