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Capacitative Ca2+ entry in agonist-induced pulmonary vasoconstriction

Sharon S. McDaniel, Oleksandr Platoshyn, Jian Wang, Ying Yu, Michele Sweeney, Stefanie Krick, Lewis J. Rubin, and Jason X.-J. Yuan

Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of California School of Medicine, San Diego, California 92103


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 MOmega ) 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.

The bath solution for recording optimal ISOC contained (in mM) 120 sodium methane sulfonate, 20 calcium aspartate, 0.5 3,4-diaminopyridine, 10 glucose, and 10 HEPES (pH 7.4 with methane sulfonic acid). The pipette solution contained (in mM) 138 cesium aspartate, 1.15 EGTA, 1 Ca(OH)2, 2 Na2-ATP, and 10 HEPES (pH 7.2). These ionic conditions eliminated the currents through the K+ and Cl- channels. In the Ca2+-free bath solution, calcium aspartate was replaced by equimolar sodium aspartate to maintain osmolarity.

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 beta -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 beta -actin signals. The normalized values are expressed as arbitrary units for quantitative comparison.

                              
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Table 1.   Oligonucleotide sequences of the primers used for RT-PCR

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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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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Fig. 1.   Extracellular Ca2+ is required for pulmonary vasoconstriction induced by 40 mM K+ (40 K) and phenylephrine (PE). A, top: Ca2+-free solution was applied to the vessels when 40 mM K+- or PE-mediated contraction reached a plateau. A, bottom: summarized data showing active tension induced by 40 mM K+ (n = 8 rings) or 2 µM PE (n = 6 rings) before (control), during (0 Ca), and after (recovery) application of Ca2+-free solution. Values are means ± SE. *** P < 0.001 vs. control (1.8 mM Ca2+). B, top: EGTA was applied to the vessels when 40 mM K+- or PE-mediated contraction, respectively, reached a plateau. B, bottom: active tension induced by 40 mM K+ (n = 8 rings) or PE (n = 4 rings) in the presence of EGTA. Values are means ± SE. Significant difference from 0 mM EGTA: *** P < 0.001; * P < 0.05.



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Fig. 2.   Effects of the voltage-dependent Ca2+ channel blocker nifedipine (Nif) on pulmonary artery (PA) contraction induced by 40 mM K+ and PE. A, top: nifedipine (10 µM) was applied to the vessels when 40 mM K+- or PE-induced contraction reached a plateau. A, bottom: summarized data showing active tension in the absence (control) and presence of 10 µM nifedipine in vessels preconstricted with 40 mM K+ (n = 4 rings) or PE (n = 4 rings). Values are means ± SE. B: summarized data showing active tension in the absence (control) and presence of 0.5 µM verapamil (Vp) in PA rings preconstricted with 40 mM K+ (n = 8 rings) or PE (n = 8 rings). Values are means ± SE. Significant difference from control: *** P < 0.001; * P < 0.05.

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 alpha -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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Fig. 3.   Contribution of Ca2+ mobilization from intracellular stores and Ca2+ influx due to PE-induced PA contraction. A: capacitative Ca2+ entry (CCE)-mediated PA contraction. PE (2 µM) was first applied to the vessel in the presence (a), and absence (b) of extracellular Ca2+. The alpha -receptor blocker phentolamine (Phen; 1 µM) was applied to the vessel when the PE-induced transient contraction returned to baseline. In the presence of Phen, restoration of extracellular Ca2+ induced a contraction most likely due to Ca2+ influx via CCE. Removal of Phen further constricted the vessel, probably due to Ca2+ influx via receptor-operated Ca2+ channels (ROCs). The Ca2+ channel blocker verapamil (0.5 µM) negligibly affected the CCE-mediated contraction in the presence of Phen (c). B: summarized data showing the PE-induced active tension in the presence (total) and absence (Ca release) of extracellular Ca2+. Values are means ± SE; nos. in parentheses, no. of PA rings tested. The active tension induced by restoration of extracellular Ca2+ in the presence of Phen and Vp (CCE) accounted for ~23% of the total active tension induced by PE.

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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Fig. 4.   Removal of endothelium enhanced CCE-mediated PA contraction. A, left: acetylcholine (ACh; 10 µM) was applied to the vessel preconstricted with 40 mM K+; the loss of ACh-mediated relaxation confirmed the functional removal of the endothelium. A, right: PE (2 µM) was first applied to the vessel in the absence of extracellular Ca2+ (0 Ca). Phen (1 µM) was then applied to the vessel when the PE-induced transient contraction returned to baseline. In the presence of Phen, restoration of extracellular Ca2+ induced a contraction in the endothelium-denuded PA ring. B: comparison of active tension induced by 40 mM K+, PE-mediated Ca2+ release, and restoration of extracellular Ca2+ in the presence of Phen (CCE) between PA rings with [Endo(+)] and without [Endo(-)] endothelium. Values are means ± SE; nos. in parentheses, no. of PA rings tested. *** P < 0.001 vs. Endo(+).

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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Fig. 5.   Inhibitory effects of Ni2+ and La3+ on CCE-mediated PA contraction. A, top: Ni2+ (1 mM) was applied to the vessels for 6 min when CCE-induced contraction was maximized. A, bottom: summarized data showing CCE-mediated contraction before (control), during (Ni2+), and after (recovery) exposure to Ni2+. Values are means ± SE; n = 12 rings. B, top: La3+ (50 µM) was applied to the vessels for 6 min when CCE-induced contraction was maximized. B, bottom: summarized data showing CCE-mediated contraction before (control), during (La3+), and after (recovery) exposure to La3+. Values are means ± SE; n = 8 rings. Significantly different from control: *** P < 0.001; ** P < 0.01.

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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Fig. 6.   Inhibitory effects of Ni2+ on the CCE-induced increase in cytosolic Ca2+ concentration ([Ca2+]cyt) in PA smooth muscle cells (SMCs). A, top: representative record of the time course of [Ca2+]cyt changes in response to cyclopiazonic acid (CPA; 10 µM) and Ni2+ (0.5 mM) in the absence (0 Ca) and presence of extracellular Ca2+. F340/F380, 340- to 380-nm fluorescence ratio. B: summarized data showing the amplitudes of CCE-mediated increases in [Ca2+]cyt (n = 30 rings) before (Cont), during (Ni), and after (Wash) application of 0.5 mM Ni2+. Values are means ± SE. *** P < 0.001 vs. Cont and Wash.

The amplitude of the CCE-induced increase in [Ca2+]cyt determined the magnitude of PA contraction induced by CCE. Consistent with the results that Ni2+, a SOC blocker (3, 18, 19), reversibly inhibited the CCE-mediated contraction in isolated PAs (Fig. 5), extracellular application of 1 mM Ni2+ almost abolished the increase in [Ca2+]cyt due to CCE in single PASMCs (Fig. 6).

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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Fig. 7.   Inhibitory effect of Ni2+ on whole cell store-operated Ca2+ channel current (ISOC) activated by CPA-induced passive depletion of the sarcoplasmic reticulum (SR) Ca2+ in PASMCs. A: ionic compositions of the extracellular (bath) and intracellular (pipette) solutions. HP, holding potential. Ba: currents elicited by 300-ms voltage steps from -120 to +20 mV in 20-mV increments before (Cont) and after 10-min application of CPA (10 µM) in the absence (CPA) and presence (CPA+Ni) of 1 mM Ni2+. The currents recorded after washout of Ni2+ in the presence of CPA [Wash (CPA)] are also shown. Bb: subtraction currents (ISOC) between the currents recorded before and after application of CPA. The cells were held at 0 mV to minimize voltage-gated Ca2+ and Na2+ currents. Bc: current-voltage (I-V) relationship of ISOC in PASMCs before (Cont) and during (CPA) application of CPA in the absence and presence (CPA+Ni) of Ni2+. Bd: I-V curve of the Ni-sensitive components of ISOC. Data are means ± SE; n = 16 rings. C: summarized data showing the currents elicited by a test potential of -80 mV (holding potential, 0 mV) before (Cont) and after (CPA) application of CPA in the absence (-Ni) and presence (+Ni) of 1 mM Ni2+. Values are means ± SE; n = 16 rings. *** P < 0.001 vs. +Ni.



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Fig. 8.   Inhibitory effect of La3+ on whole cell ISOC activated by PE-induced active depletion of SR Ca2+ in PASMCs. A: currents elicited by 300-ms voltage steps from -120 to +20 mV in 20-mV increments before (Cont) and after 10-min application of PE (10 µM) in the absence (PE) and presence (PE+La) of 50 µM La3+. The currents recorded after washout of Ni2+ in the presence of CPA [Wash (CPA)] are also shown. B: I-V relationship of ISOC in PASMCs before (PE) and after (PE+La) treatment of the cells with 50 µM La3+. Data are means ± SE; n = 8 rings.

Activity of SOCs is the major determinant of the CCE amplitude, which, in turn, governs the magnitude of the PA contraction induced by CCE. The SOC blocker Ni2+ (1 mM) caused a marked reduction in whole cell ISOC elicited by passive depletion of Ca2+ from the SR with CPA (10 µM for 15 min; Fig. 7, B and C). Extracellular application of La3+, a selective blocker of SOCs, also significantly and reversibly reduced the whole cell ISOC induced by active depletion of SR Ca2+ by PE (10 µM for 10-12 min; Fig. 8). The similarity of the blockade effects of Ni2+ (and La3+) on CCE-induced PA contraction (Fig. 5), CPA-induced increase in [Ca2+]cyt due to CCE (Fig. 6), and whole cell ISOC (Figs. 7 and 8) further suggests that CCE participates in pulmonary vasoconstriction.

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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Fig. 9.   RT-PCR analysis of transient receptor potential (TRP) channel mRNAs in PASMCs and PA endothelial cells (PAECs). A: PCR-amplified products displayed in agarose gels stained with ethidium bromide for TRP1 (372 bp), TRP2 (487 bp), TRP3 (318 bp), TRP4 (415 bp), TRP5 (340 bp), TRP6 (327 bp), TRP7 (400 bp), and beta -actin (244 bp) in PASMCs (S) and PAECs (E). M, 100-bp DNA ladder. B: summarized data showing TRP mRNA levels that were normalized to the amount of beta -actin (ratio of optical density of TRP mRNA to optical density of beta -actin mRNA). Values are means ± SE; n = 4 rings. C: PCR-amplified products for TRP1-7 and beta -actin in rat brain.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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 the alpha -receptor blocker phentolamine and the VDCC blocker verapamil. This contraction was independent of 1) activation of the alpha -receptor, 2) opening of VDCCs, and 3) the intact endothelium. In the absence of extracellular Ca2+, PE can actively deplete Ca2+ from the SR by increasing Ins(1,4,5)P3 production (9); therefore, activation of SOCs and induction of CCE may be the trigger for this contraction, which accounts for ~23% of the total active tension induced by PE.

These results indicate that the PE-induced PA contraction is composed of at least four components: 1) a transient contraction due to Ca2+ release from the SR, 2) a sustained contraction due to Ca2+ influx through ROCs and VDCCs, 3) a gradually decreasing contraction due to CCE, and 4) a small, sustained Ca2+-independent contraction due to an increased Ca2+ sensitivity of contractile proteins.

Regulation 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 from -85 to -31 mV and caused membrane depolarization. Therefore, the 40 mM K+-induced contraction, which is mainly due to membrane depolarization-mediated opening of VDCCs, was completely eliminated by the blockade of VDCCs.

In the absence of extracellular Ca2+, the time courses of the CPA-induced transient [Ca2+]cyt rise (Fig. 6A) and the PE-induced transient PA contraction (Fig. 3A, b and c) are similar: each lasted for 5-7 min. After SR Ca2+ was depleted, restoration of extracellular Ca2+ induced a second increase in [Ca2+]cyt due to CCE in PASMCs and a second PA contraction in the presence of the alpha -receptor blocker phentolamine and the VDCC blocker verapamil. The CPA- and PE-induced depletion of the SR Ca2+ also induced ISOC. In this study, ISOC was isolated in single PASMCs held at 0 mV (to inactivate voltage-gated Ca2+ and Na+ channels), bathed in Cl--free solution, and dialyzed with Cs+-containing, Cl--free solution (to eliminate K+ and Cl- currents). Inhibition of CCE and ISOC by the SOC blocker Ni2+ significantly attenuated PA contraction induced by SR depletion. These results suggest that store depletion-activated CCE directly increases [Ca2+]cyt to cause contraction in addition to indirectly regulating agonist-induced contraction by refilling the emptied SR with Ca2+.

Role 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

We thank Drs. Judy Creighton and T. Stevens for providing the endothelial cells.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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