Capacitative calcium entry and TRPC channel proteins are expressed in rat distal pulmonary arterial smooth muscle

Jian Wang, L. A. Shimoda, and J. T. Sylvester

Division of Pulmonary & Critical Care Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21224

Submitted 10 September 2003 ; accepted in final form 9 December 2003


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mammalian homologs of transient receptor potential (TRP) genes in Drosophila encode TRPC proteins, which make up cation channels that play several putative roles, including Ca2+ entry triggered by depletion of Ca2+ stores in endoplasmic reticulum (ER). This capacitative calcium entry (CCE) is thought to replenish Ca2+ stores and contribute to signaling in many tissues, including smooth muscle cells from main pulmonary artery (PASMCs); however, the roles of CCE and TRPC proteins in PASMCs from distal pulmonary arteries, which are thought to be the major site of pulmonary vasoreactivity, remain uncertain. As an initial test of the possibility that TRPC channels contribute to CCE and Ca2+ signaling in distal PASMCs, we measured [Ca2+]i by fura-2 fluorescence in primary cultures of myocytes isolated from rat intrapulmonary arteries (>4th generation). In cells perfused with Ca2+-free media containing cyclopiazonic acid (10 µM) and nifedipine (5 µM) to deplete ER Ca2+ stores and block voltage-dependent Ca2+ channels, restoration of extracellular Ca2+ (2.5 mM) caused marked increases in [Ca2+]i whereas MnCl2 (200 µM) quenched fura-2 fluorescence, indicating CCE. SKF-96365, LaCl3, and NiCl2, blocked CCE at concentrations that did not alter Ca2+ responses to 60 mM KCl (IC50 6.3, 40.4, and 191 µM, respectively). RT-PCR and Western blotting performed on RNA and protein isolated from distal intrapulmonary arteries and PASMCs revealed mRNA and protein expression for TRPC1, -4, and -6, but not TRPC2, -3, -5, or -7. Our results suggest that CCE through TRPC-encoded Ca2+ channels could contribute to Ca2+ signaling in myocytes from distal intrapulmonary arteries.

cation channel transient receptor potential; intracellular calcium concentration; fura-2; SKF-96365; LaCl3; NiCl2


IN VASCULAR SMOOTH MUSCLE, global increases in intracellular Ca2+ concentration ([Ca2+]i) trigger contraction via a signaling pathway that includes formation of Ca2+-calmodulin, activation of myosin light chain kinase, and actin-myosin interaction. Increases in [Ca2+]i can be caused by 1) release of Ca2+ from internal storage sites, such as sarcoplasmic reticulum (SR), or 2) influx of Ca2+ from extracellular fluid through L-type voltage-dependent Ca2+ channels (VDCCs), receptor-operated Ca2+ channels (ROCCs), or store-operated Ca2+ channels (SOCCs) (17). SOCCs are activated by depletion of SR Ca2+ stores, and the resulting capacitative calcium entry (CCE) is thought to replenish these stores and contribute to signaling (26).

CCE can be assessed by exposing cells to Ca2+-free media containing inhibitors of VDCCs, such as nifedipine or diltiazem, and inhibitors of sarcoplasmic-endoplasmic Ca2+-ATPase (SERCA) pumps, such as thapsigargin (TG) or cyclopiazonic acid (CPA). Under these conditions, SR Ca2+ stores are depleted, and CCE is estimated by the increases in vasomotor tone or [Ca2+]i induced by restoration of extracellular Ca2+ or by the rate of Mn2+ influx occurring after addition of this Ca2+ surrogate to extracellular fluid. In addition, patch-clamping techniques can be used to measure effects of SR Ca2+ depletion on the relationship between transsarcolemmal ion current and membrane potential under conditions that isolate store-operated Ca2+ currents.

Using these methods, several groups have recently demonstrated that CCE can occur in proximal pulmonary arteries or myocytes from these arteries (8, 12, 22, 25, 36, 50); however, there have been few assessments of CCE in distal pulmonary arteries, which are thought to be the major site of pulmonary vasomotor responses such as hypoxic pulmonary vasoconstriction (1, 19, 31). In cultured myocytes of small muscular intrapulmonary arteries obtained from patients undergoing heart/lung transplantation or treatment for bronchogenic carcinoma, Golovina et al. (11) found that CPA caused a nifedipine-resistant increase in [Ca2+]i on restoration of extracellular Ca2+ and enhanced cation currents measured under conditions that prevented K+ currents and voltage-dependent Na+ or Ca2+ currents. Robertson et al. (28) found that rat distal pulmonary arteries treated with TG in Ca2+-free solution showed sustained constriction on re-exposure to Ca2+ and an increased rate of Mn2+ influx. Recently, Snetkov et al. (32) confirmed that these constrictions were accompanied by increases in [Ca2+]i and that TG caused La3+-sensitive inward currents through nonspecific cation channels in myocytes freshly isolated from these arteries. These findings suggest that CCE can also occur in distal pulmonary arteries.

The SOCCs responsible for CCE are probably composed of mammalian homologs of transient receptor potential (TRP) and transient receptor potential-like (TRPL) proteins, which form light-activated cation channels in photoreceptor cells of Drosophila melanogaster. To date, seven of these homologous TRPC proteins (TRPC1–7) have been cloned and sequenced (23). Surveys of TRPC expression in pulmonary arterial smooth muscle cells (PASMCs) have been reported by three laboratories. Using RT-PCR in freshly isolated myocytes from canine main pulmonary artery, Walker et al. (42) detected mRNA for TRPC4, -6, and -7. Protein expression was not assessed. In freshly isolated myocytes from rat main pulmonary artery, Ng and Gurney (25) detected mRNA for TRPC1, -3, -4, -5, and -6. Protein expression was confirmed by immunocytochemistry for TRPC1, -3, -4, and -6. Expression of mRNA for TRPC2 and -7 and protein for TRPC2, -5, and -7 was not determined. In cultured myocytes from rat right main pulmonary artery, McDaniel et al. (22) measured mRNA for TRPC1–7 by RT-PCR and detected TRPC1, -2, -4, -5, and -6. Protein expression was not evaluated. None of these studies compared TRPC expression in cells to that in vascular tissue. To our knowledge, a comprehensive survey of TRPC expression in distal pulmonary arteries has not been performed.

As an initial step toward elucidating the roles of CCE, SOCCs, and TRPC proteins in pulmonary vasoreactivity, we performed the present study to confirm the presence of CCE in distal PASMCs, to determine how different concentrations of known SOCC inhibitors affect CCE and to identify which TRPC proteins are expressed in distal PASMCs and freshly isolated pulmonary arterial tissue.


    METHODS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PASMC isolation and culture. As previously described (30, 43), distal (>4th generation) intrapulmonary arteries were dissected from lungs of male Wistar rats (body wt 300–500 g). We removed the endothelium by rubbing the luminal surface with a cotton swab. The arteries were allowed to recover for 30 min in cold (4°C) physiological salt solution (PSS) that contained (in mM) 130 NaCl, 5 KCl, 1.2 Mg Cl2, 10 HEPES, and 10 glucose. This was followed by 20 min in reduced-Ca2+ PSS (20 µM CaCl2) at room temperature. The tissue was then digested at 37°C for 20 min in reduced-Ca2+ PSS containing collagenase (type I, 1,750 U/ml), papain (9.5 U/ml), bovine serum albumin (2 mg/ml), and dithiothreitol (1 mM). After resuspension and trituration of the digested tissue, cells were plated onto 25-mm coverslips (for fluorescent microscopy) or 10-cm petri dishes (for molecular biological measurements) and incubated for 3–6 days in smooth muscle growth medium 2 (Clonetics, Walkersville, MD) containing 5% serum in a humidified atmosphere of 5% CO2-95% air at 37°C. Twenty-four hours before an experiment, the concentration of serum in culture media was decreased to 0.3% to stop cell growth.

Cellular purity of the cultures was assessed by morphological appearance under phase-contrast microscopy and immunofluorescence staining for {alpha}-actin under fluorescence microscopy. For the latter, we used a primary monoclonal antibody raised against smooth muscle {alpha}-actin (Sigma, St. Louis, MO), Cy3-conjugated secondary antibody (excitation {lambda} = 550 nm, emission {lambda} = 570 nm; Jackson ImmunoResearch, West Grove, PA), and a nuclear stain (YO-PRO-1, excitation {lambda} = 488 nm, emission {lambda} = 509 nm; Molecular Probes, Eugene, OR) (43). Cells were examined under a Zeiss LSM-510 inverted laser-scanning confocal fluorescence microscope with a Zeiss Plan-Neofluor x40 oil immersion objective (Atlanta, GA). For each determination, we inspected at least 1,000 cells in at least 40 randomly selected fields. Coverslips not exposed to the primary antibody, but otherwise treated similarly, served as controls. Finally, after loading with fura-2 to permit measurement of [Ca2+]i by fluorescence microscopy (see below), we determined the effect of 60 mM KCl on [Ca2+]i in each isolate on the day of the experiment to confirm the presence of functional VDCCs.

Measurement of intracellular Ca2+. After incubation with 7.5 µM fura-2 (Molecular Probes) for 60 min at 37°C under an atmosphere of 5% CO2-95% air, coverslips with PASMCs were mounted in a closed polycarbonate chamber clamped in a heated aluminum platform (PH-2; Warner Instrument, Hamden, CT) on the stage of a Nikon TSE 100 Ellipse inverted microscope (Melville, NY). The chamber was perfused at 0.5 ml/min with Krebs-Ringer bicarbonate (KRB) solution, which contained (in mM) 118 NaCl, 4.7 KCl, 0.57 MgSO4, 1.18 KH2PO4, 25 NaHCO3, 2.5 CaCl2, and 10 glucose. The KRB solution was equilibrated with 16% O2-5% CO2 at 38°C in heated reservoirs and led via stainless steel tubing and a manifold to an in-line heat exchanger (SF-28, Warner Instrument), which rewarmed the perfusate just before it entered the cell chamber. The temperature of the heat exchanger and chamber platform was controlled by a dual-channel heater controller (TC-344B, Warner Instrument). This system maintained temperature at 37°C and oxygen tension at 112 ± 2.0 mmHg at the coverslip. In most of the experiments with LaCl3, we used a HEPES-buffered salt solution that did not contain bicarbonate, phosphate, or EGTA, to avoid precipitation and chelation of La3+ (28). This perfusate was equilibrated with 16% O2-balance N2 and contained (in mM) 130 NaCl, 5 KCl, 1.2 MgCl2, 2.5 CaCl2, 10 HEPES, and 10 glucose. After perfusing the chamber for 10 min to remove extracellular dye, we performed ratiometric measurement of fura-2 fluorescence at 60-s intervals using a collimated light beam from a xenon arc lamp filtered by interference filters at 340 and 380 nm and focused onto PASMCs visualized with a x20 fluorescence objective (Super Fluor 20; Nikon, Torrance, CA). Light emitted from the cells at 510 nm was returned through the objective and detected by a cooled charge-coupled device imaging camera. An electronic shutter (Vincent Associates, Rochester, NY) was used to minimize photobleaching. Protocols were executed, and data were collected on-line with InCyte software (Intracellular Imaging, Cincinnati, OH). We calculated [Ca2+]i from fura-2 fluorescence ratios (F340/F380) using linear regression between adjacent points on a calibration curve generated by measuring F340/F380 in at least seven calibration solutions with [Ca2+] between 0 and 610 nM (Molecular Probes). Because the behavior of fura-2 in solution may differ from that in cells, calculated [Ca2+]i should be considered an estimate of true [Ca2+]i.

To assess CCE, we perfused PASMCs for at least 10 min with Ca2+-free PSS containing 5 µM nifedipine to prevent calcium entry through L-type VDCCs and 10 µM CPA to deplete SR calcium stores. KRB perfusate also contained 0.5 mM EGTA to chelate any residual Ca2+; however, as noted above, EGTA was not included in the HEPES-buffered salt solution used in most La3+ experiments. When used, La3+ and other CCE antagonists were added to the perfusate at the same time as CPA. CCE was assessed in two ways. First, we measured [Ca2+]i at 1-min intervals before and after restoration of extracellular [Ca2+] to 2.5 mM. CCE was evaluated from the increase in [Ca2+]i caused by restoration of extracellular [Ca2+] in the continued presence of CPA and nifedipine. Second, we monitored fura-2 fluorescence excited at 360 nm at 30-s intervals before and after addition of MnCl2 (200 µM) to the cell perfusate. CCE was evaluated from the rate at which fura-2 fluorescence was quenched by Mn2+, which enters the cell as a Ca2+ surrogate and reduces fura-2 fluorescence on binding to the dye. Fluorescence excited at 360 nm is the same for Ca2+-bound and Ca2+-free fura-2; therefore, changes in fluorescence can be assumed to be caused by Mn2+ alone.

RNA isolation and measurement by RT-PCR. Total RNA was prepared from de-endothelialized distal pulmonary arteries or primary cultures of PASMCs, as previously described (43). Briefly, samples were placed in cold TRIzol reagent (1 ml/50–100 mg tissue) and homogenized at 20,000 rpm (Ultra-Turrax T-25). After incubation at 30°C for 5 min and addition of chloroform (0.2 ml/ml TRIzol), samples were centrifuged at 8,000 g and 4°C for 15 min. The upper aqueous phase of centrifugates was mixed with 100% isopropanol (0.5 ml/ml TRIzol), incubated at 30°C for 10 min, and recentrifuged at 4°C and 12,000 g for 10 min. The clear gel-like RNA precipitate was washed with 1 ml of 70% alcohol, dissolved in diethyl pyrocarbonate water (1 µg/µl), and stored at -70°C. The quality of the RNA isolate was determined from the ratio of absorbance at 260 nm to that at 280 nm (>1.7) and the integrity of the 28S and 18S ribosomal RNA bands appearing on electrophoresis of denatured RNA samples through a 1% agarose formaldehyde gel.

RT was performed with the First-Strand cDNA Synthesis kit (Pharmacia Biotech, Austin, TX), as previously described (43). We subjected the resultant cDNA to PCR by adding 4 µl of the First Strand cDNA reaction mixture to a 50-µl PCR reaction mixture, which consisted of 1 pM of each PCR primer, 10 mM Tris·HCl (pH 8.3), 50 mM KCl, 2 mM MgCl2, 200 µM each dNTP, and 2 units Taq DNA polymerase. The sense and antisense PCR oligonucleotide primers chosen to amplify the cDNA (Table 1) were specifically designed from coding regions of the various TRPC channels. The fidelity and specificity of these oligonucleotides were confirmed with use of the Basic Local Alignment Search Tool program. The cDNA was amplified in a DNA thermal cycler (model 2400 GeneAmp PCR System; Perkin-Elmer, Foster City, CA). The mixture was annealed at 52–60°C (1 min), extended at 72°C (2 min), and denatured at 94°C (1 min) for 28–33 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 1% agarose gel, and the amplified cDNA bands were visualized by ethidium bromide staining.


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Table 1. TRPC primer sequence for RT-PCR

 

Protein isolation and measurement by Western blotting. De-endothelialized distal pulmonary arteries from rats or primary cultures of PASMCs were washed with phosphate-buffered saline and homogenized in HEPES buffer at 20,000 rpm (Ultra-Turrax T-25). As previously described (43), total protein concentration in the homogenate was determined by the BCA protein assay (Lowry method). Homogenate proteins were separated by SDS-PAGE calibrated with prestained protein molecular weight markers (Bio-Rad, Hercules, CA). Separated proteins were transferred to nitrocellulose membranes (Hybond-C, Bio-Rad). After being blocked with 5% nonfat dry milk in Tris-buffered saline and 0.1% Tween 20, membranes were incubated with affinity-purified polyclonal antibodies specific for various TRPC channels (Alomone Laboratories, Jerusalem, Israel). The membranes were then washed and incubated with anti-rabbit or anti-mouse horseradish peroxidase-conjugated IgG for 1 h. An enhanced luminol-linked chemiluminescence detection system (ECL; Amersham, Piscataway, NJ) was used to detect bound antibody.

Drugs and materials. Fura-2-AM (Molecular Probes) was prepared on the day of the experiment as a 2.5 mM stock solution in dimethyl sulfoxide (DMSO). Stock solutions of SKF-96365, LaCl3, and NiCl2 (Sigma) were made daily in water (10, 100, and 500 mM, respectively), and 30 mM stock solutions of CPA and nifedipine (Sigma) were made in DMSO.

Statistical analysis. Data are expressed as means ± SE, where n is the number of experiments performed, and the number of cells in each experiment ranged between 20 and 30, as indicated in RESULTS and the figure legends. Statistical comparisons were performed using Student's t-test. Differences were considered to be significant when P < 0.05.


    RESULTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Characteristics of distal PASMCs. Cells cultured from rat distal pulmonary arteries were phase bright and assumed a spindle-shaped appearance with cytoplasmic projections extending from a larger central area that contained the nucleus (Fig. 1A). Immunostaining for smooth muscle {alpha}-actin was positive in >95% of cells (Fig. 1B). None of the cells were positive when staining was performed without the primary anti-{alpha}-actin antibody. All cells increased [Ca2+]i in response to 60 mM KCl, indicating the presence of VDCCs (Fig. 2A). These findings confirmed that the cells were pulmonary arterial myocytes.



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Fig. 1. A: phase-contrast microscopy of rat distal pulmonary arterial smooth muscle cells (PASMCs) cultured for 3–5 days. B: fluorescence immunocytochemical identification of {alpha}-actin (shown in red) in PASMCs grown in primary culture for 3–5 days. The nuclear marker YO-PRO-1 is shown in green.

 


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Fig. 2. Effects of 0.5–5.0 µM nifedipine (A, n = 14 experiments in 390 cells), 50 µM SKF-96365 (B, n = 7 experiments in 192 cells), 500 µM NiCl2 (C, n = 4 experiments in 105 cells), and 100 µM LaCl3 (D, n = 8 experiments in 195 cells) on Ca2+ responses of rat distal PASMCs to KCl. PASMCs exposed to LaCl3 were perfused with HEPES-buffered salt solution not containing bicarbonate or phosphate (see METHODS).

 

Assessment of CCE in distal PASMCs. We assessed CCE by giving extracellular calcium or Mn2+ to PASMCs perfused with Ca2+-free KRB solution containing 10 µM CPA and 5 µM nifedipine. This nifedipine concentration was used because it abolished the increase in [Ca2+]i caused by 60 mM KCl (Fig. 2A), indicating complete blockade of Ca2+ entry through L-type VDCCs. As shown in Fig. 3A, CPA given in the absence of extracellular Ca2+ and the presence of nifedipine caused a small increase in [Ca2+]i, which returned to baseline after 5–10 min. Subsequent restoration of extracellular Ca2+ induced a second large increase in [Ca2+]i, which rose quickly to a peak ({Delta}[Ca2+]i = 511 ± 70 nM; n = 8, P < 0.0001) before falling to a lower but still elevated final value ({Delta}[Ca2+]i = 209 ± 39 nM; n = 9, P < 0.001) (Fig. 3, A and B). When CPA was omitted, these changes in [Ca2+]i did not occur (Fig. 3).



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Fig. 3. A: time course of {Delta}[Ca2+]i before and after restoration of extracellular Ca2+ in distal PASMCs perfused with Ca2+-free Krebs-Ringer bicarbonate (KRB) solution containing 10 µM cyclopiazonic acid (CPA), 0.5 mM EGTA, and 5 µM nifedipine (Nifed, n = 8 experiments in 227 cells) and in control cells, which did not receive CPA but were otherwise treated similarly (n = 9 experiments in 231 cells). B: average peak {Delta}[Ca2+]i obtained from cells shown in A.

 

To confirm that the response to restoration of extracellular Ca2+ reflected Ca2+ influx, we measured the effect of extracellular Mn2+ on fura-2 fluorescence in PASMCs perfused with Ca2+-free KRB solution containing nifedipine. As shown in Fig. 4, fura-2 fluorescence decreased 39 ± 4% 10 min after administration of Mn2+ in the presence of CPA (n = 6), but only 16 ± 2% in the absence of CPA (n = 8, P < 0.0001).



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Fig. 4. A: fura-2 fluorescence at 360 nm before and after administration of MnCl2 (200 µM) to distal PASMCs perfused with Ca2+-free KRB solution containing 10 µM CPA, 0.5 mM EGTA, and 5 µM nifedipine (n = 6 experiments in 166 cells) and in control cells, which did not receive CPA but were otherwise treated similarly (n = 8 experiments in 223 cells). B: average decrease in fura-2 fluorescence 10 min after administration of MnCl2 to in cells shown in A.

 

Effects of CCE antagonists. SKF-96365, Ni2+, and La3+ have been demonstrated to block CCE in many cell types (22, 25). To assess the effects of these agents in distal PASMCs, we determined how different inhibitor concentrations altered [Ca2+]i responses to restoration of extracellular Ca2+ after store depletion (Figs. 5 and 6). SKF-96365 caused a concentration-dependent decrease in this response. For example, peak {Delta}[Ca2+]i at [SKF-96365] of 1, 10, and 50 µM (n = 4–9) averaged 414 ± 69, 212 ± 18, and 70 ± 12 nM, respectively, compared with 511 ± 70 nM in untreated cells (n = 8), yielding an estimated IC50 of 6.3 µM (Fig. 6). NiCl2 also reduced the response to Ca2+ restoration, but higher concentrations were required. Peak {Delta}[Ca2+]i at 50, 200, and 500 µM (n = 4–8) averaged 412 ± 41, 271 ± 107, and 96 ± 12 nM, respectively, yielding an estimated IC50 of 191 µM. LaCl3 (500 and 1,000 µM; n = 4 and 8, respectively) had little effect in PASMCs perfused with KRB solution (peak {Delta}[Ca2+]i = 415 ± 84 and 372 ± 66 nM, respectively; Figs. 5 and 6); however, in distal PASMCs perfused with bicarbonate-, phosphate-, and EGTA-free HEPES-buffered salt solution, LaCl3 caused a concentration-dependent decrease in CCE. Peak {Delta}[Ca2+]i at [LaCl3] of 10, 30, and 100 µM (n = 5–6) averaged 339 ± 20, 271 ± 23, and 35 ± 8 nM, respectively, compared with 393 ± 30 nM in untreated PASMCs perfused with the same solution (n = 7). The estimated IC50 was 40.4 µM (Fig. 6). The marked overshoot in {Delta}[Ca2+]i caused by restoration of extracellular Ca2+ in cells perfused with KRB solution was not seen in cells perfused with the HEPES-buffered salt solution (Fig. 5).



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Fig. 5. Effects of different concentrations of SKF-96365 (SKF, A, n = 4–9 experiments in 112–251 cells), NiCl2 (B, n = 4–8 experiments in 105–215 cells), and LaCl3 (C, n = 4–8 experiments in 95–203 cells) on the time course of {Delta}[Ca2+]i before and after restoration of extracellular Ca2+ in distal PASMCs perfused with Ca2+-free KRB solution containing 10 µM CPA, 0.5 mM EGTA, and 5 µM nifedipine. D: effects of LaCl3 in distal PASMCs perfused with Ca2+-free HEPES-buffered salt solution not containing bicarbonate, phosphate, or EGTA (n = 5–7 experiments in 106–179 cells)

 


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Fig. 6. Concentration-response relations for inhibition of capacitative Ca2+ entry (CCE, Fig. 5) by SKF-96365, NiCl2, and LaCl3. The sigmoid line represents the least-squares fit of the Hill equation to the data. Concentrations which inhibited responses by 50% (IC50) are indicated.

 

To confirm that SKF-96365, NiCl2, and LaCl3 acted by blocking calcium entry, we assessed their effects on Mn2+ quenching of fura-2 fluorescence. As shown in Fig. 8, fura-2 fluorescence decreased only 20 ± 1, 18 ± 2, and 19 ± 1% 10 min after administration of Mn2+ in PASMCs treated with 50 µM SKF-96365, 500 µM NiCl2, and 100 µM LaCl3, respectively (n = 6–8). These values were significantly less than the average decreases observed in untreated cells perfused with KRB solution (39 ± 4%, n = 8, P <= 0.001; Fig. 7A) or HEPES-buffered salt solution (36 ± 4%, n = 6, P <= 0.001; Fig. 7B).



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Fig. 8. mRNA detected by RT-PCR for transient receptor potential channel (TRPC) proteins 1–7 in rat distal pulmonary arterial tissue (PA, A, n = 6 experiments in 4 animals), distal PASMCs (B, n = 5 experiments in 3 animals), and brain (C, n = 3 experiments in 3 animals). RT-PCR was carried out on cDNA using specific primers shown in Table 1.

 


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Fig. 7. A: inhibitory effects of SKF-96365 (n = 6 experiments in 161 cells) and NiCl2 (n = 8 experiments in 211 cells) on Mn2+ quenching of fura-2 fluorescence in distal PASMCs perfused with Ca2+-free KRB solution containing 10 µM CPA, 0.5 mM EGTA, and 5 µM nifedipine. Also shown are results obtained in control cells not exposed to inhibitors of CCE (n = 8 experiments in 223 cells). B: average decreases in fura-2 fluorescence 10 min after administration of MnCl2 to in cells shown in A. C: inhibitory effects of LaCl3 (n = 7 experiments in 183 cells) on Mn2+ quenching of fura-2 fluorescence in distal PASMCs perfused with Ca2+-free HEPES-buffered salt solution containing 10 µM CPA and 5 µM nifedipine, but no bicarbonate, phosphate, or EGTA. Also shown are results obtained in control cells not exposed to LaCl3 (n = 6 experiments in 136 cells). D: average decreases in fura-2 fluorescence 10 min after administration of MnCl2 to in cells shown in C.

 

To test the specificity of CCE antagonists, we measured the effects of the highest concentrations of SKF-96365, NiCl2, and LaCl3 (50, 500, and 100 µM, respectively) on the [Ca2+]i response to depolarization with 60 mM KCl. As shown in Fig. 2, B–D, none of the agents significantly altered this response (n = 4–8, P > 0.9). In contrast, the response to KCl could be abolished by nifedipine (Fig. 2A, n = 14). Control KCl responses were greater in untreated distal PASMCs perfused with KRB solution than in cells perfused with HEPES-buffered salt solution (peak {Delta}[Ca2+]i = 363 ± 53 vs. 147 ± 17 nM, P < 0.01).

Expression of TRPC mRNA and protein in distal pulmonary arteries and PASMCs. Messenger RNAs for TRPC1, TRPC4, and TRPC6 were detected by RT-PCR in both distal pulmonary arteries (Fig. 8A) and smooth muscle cells from these vessels (Fig. 8B). The PCR products were of the expected size. Messenger RNAs for TRPC2, TRPC3, TRPC5, and TRPC7 were not expressed in distal pulmonary arteries or PASMCs but were readily detected in rat brain (Fig. 8C).

Western blotting revealed expression of TRPC1, TRPC4, and TRPC6 proteins in distal pulmonary arteries and PASMCs (Fig. 9). The anti-TRPC1 antibody recognized a single band at 170 kDa (Fig. 9A). This size is larger than the 87 kDa expected for TRPC1 (7, 36) and may be due to glycosylation of native TRPC1 proteins (35, 49). The anti-TRPC4 antibody recognized a single band at 90 kDa in distal pulmonary arteries (Fig. 9C). The size of this band is in good agreement with the predicted molecular mass of the TRPC4 channel protein (6). In distal pulmonary arteries and PASMCs, the anti-TRPC6 antibody recognized two bands at 55 and 117 kDa (Fig. 9D), as expected for this protein (37). Bands for TRPC1, TRPC4, and TRPC6 did not appear when antibodies were pretreated with their respective antigens. TRPC3 protein was detected in brain, but not in distal pulmonary arteries or PASMCs (Fig. 9B). We did not test protein expression for TRPC2 and -7 because mRNAs were not expressed and antibodies were not available.



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Fig. 9. Western blots for TRPC1 (A), TRPC3 (B), TRPC4 (C), and TRPC6 (D) in rat distal pulmonary arterial tissue (PA, n = 7 experiments in 5 animals), distal PASMCs (n = 4 experiments in 3 animals), and brain (Br, n = 3 experiments in 3 animals). Ag-, primary antibody without associated competitive antigen; Ag+, primary antibody with associated competitive antigen. Arrows indicate TRPC proteins.

 


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To activate CCE in distal PASMCs, we exposed the cells to Ca2+-free perfusate containing the SERCA pump inhibitor, CPA, and the L-type VDCC antagonist nifedipine. When SERCA pumps are blocked, leakage of Ca2+ down a 10,000–20,000-fold concentration gradient from the SR to the cytoplasm is unopposed. The leaked Ca2+ is taken up by intracellular buffers, exchanged for extracellular Na+, or pumped out of the cell by plasma membrane Ca2+-ATPases (PMCA), which are not blocked by CPA. Thus in the absence of Ca2+ influx, [Ca2+]i remains low, and SR Ca2+ continues to leak until the store is depleted (39). In agreement with this sequence of events, we observed a small transient rise in [Ca2+]i immediately after exposure of PASMCs to Ca2+-free perfusate containing CPA and nifedipine (Fig. 3A). This did not occur in PASMCs exposed to Ca2+-free perfusate containing nifedipine but not CPA, consistent with CPA-induced depletion of SR Ca2+.

SERCA inhibitors have been widely used to stimulate CCE independently of receptor activation (26), but it remains unclear which SR Ca2+ stores are depleted by these agents. Normally, Ca2+ is released from SR by activation of ryanodine or inositol 1,4,5-trisphosphate receptors (RyRs, IP3Rs) in the SR membrane. In canine pulmonary arterial smooth muscle, CPA and TG prevented contractions and increases in [Ca2+]i caused by phenylephrine or angiotensin II, which activate IP3Rs; however, CPA and TG did not block responses to caffeine, which activates RyRs (14, 15). In aortic smooth muscle, TG depleted SR Ca2+ stores that were functionally and structurally distinct from those depleted by caffeine (40). These results suggest that SR Ca2+ stores accessed by RyRs and IP3Rs were independent and that CPA and TG depleted only stores accessed by IP3Rs. In contrast, caffeine prevented responses to norepinephrine and vice versa in rat PASMCs, suggesting that stores accessed by RyRs and IP3Rs were coupled rather than independent (44). Further investigation will be required to determine which SR Ca2+ stores in distal PASMCs are depleted by CPA.

To measure CCE, we first determined the effects of restoring extracellular Ca2+ in PASMCs perfused with Ca2+-free media containing CPA and nifedipine. As in other cell types (18, 21, 33), Ca2+ restoration caused a large rapid increase in [Ca2+]i followed by a gradual decline toward an elevated plateau (Fig. 3A). Because Ca2+ influx through L-type VDCCs was blocked by nifedipine and Ca2+ restoration had no effect in PASMCs not exposed to CPA (Fig. 3A), it is likely that this response resulted from CCE through SOCCs; however, other mechanisms may have contributed. For example, it has been proposed that the gradual decline in [Ca2+]i after its peak is caused by enhanced Ca2+ efflux secondary to increased PMCA pump activity and reduced Ca2+ influx secondary to inhibition of SOCCs (18, 21, 33). In addition, activation of Ca2+-dependent sarcolemmal K+ or Cl- channels may have altered membrane potential and the electrochemical gradient for Ca2+ influx through SOCCs or other pathways (26). It is also possible that changes in membrane potential or Na+ and Ca2+ concentrations altered the magnitude and/or direction of Na+/Ca2+ exchange (4). The gradual decline in [Ca2+]i after its peak was not seen in PASMCs perfused with HEPES-buffered salt solution without phosphate, bicarbonate, or EGTA (Fig. 5D). The mechanism of this effect remains to be determined.

To confirm that depletion of SR Ca2+ stores caused Ca2+ entry, we determined the effects of Mn2+ on fura-2 fluorescence excited at 360 nm in PASMCs exposed to Ca2+-free perfusate containing CPA and nifedipine (26). Mn2+ is a surrogate for Ca2+ and reduces fura-2 fluorescence when it binds to the dye. The intensity of fluorescence excited at 360 nm is the same (isosbestic) for Ca2+-bound and Ca2+-free fura-2; therefore, changes in fluorescence induced by Mn2+ can be assumed to be due to Mn2+ alone. As shown in Fig. 4, there was a slow decrease in fura-2 fluorescence before exposure to Mn2+, presumably due to photobleaching of the dye. Mn2+ increased the rate of this decline slightly in cells not exposed to CPA, suggesting the presence of Ca2+ entry pathways other than SOCCs or L-type VDCCs. In contrast, Mn2+ caused marked quenching of fura-2 fluorescence in cells treated with CPA, suggesting enhanced entry through SOCCs. Together, the effects of Mn2+ and restoration of extracellular Ca2+ provide strong evidence for CCE through SOCCs in rat distal PASMCs.

Our results are consistent with previous reports that CCE can occur in PASMCs. Proximal pulmonary arteries treated with TG or CPA in Ca2+-free media exhibited constriction when extracellular Ca2+ was restored (12, 22, 25). These contractions were blocked by agents known to inhibit SOCCs (La3+, Cd2+, Ni2+, and SKF-96365), but not nifedipine. In proximal PASMCs exposed to the same conditions, Mn2+ quenching was increased and Ca2+ restoration caused a sustained increase in [Ca2+]i (8, 22, 25). Both responses were insensitive to VDCC antagonists but blocked by Ni2+ and SKF-96365. In proliferating human PASMCs or myocytes from rat proximal pulmonary arteries, CPA activated inward cation currents that had reversal potentials near 0 mV and could be inhibited by Cd2+, Ni2+, La3+, or SKF-96365 (11, 22, 25, 36, 50). Quenching of fura-2 fluorescence by extracellular Mn2+ was increased in distal pulmonary arteries exposed to TG or CPA in Ca2+-free media (16, 28, 32). In addition, extracellular Ca2+ restoration caused constriction and increased [Ca2+]i in these vessels, and TG induced currents through nonspecific cation channels in distal PASMCs. These effects were blocked by La3+ (16, 28, 32).

CCE pathways have been characterized pharmacologically by the relative potencies of inorganic antagonists such as La3+, Cd2+, and Ni2+ and organic antagonists such as SKF-96365 (20). Ng and Gurney (25) found that contractile responses of rat main pulmonary artery to CPA were 50% inhibited by 6 µM Cd2+, 7 µM SKF-96365, 10 µM Ni2+, or 600 µM La3+. In myocytes from these arteries, the nitrendipine-resistant increase in [Ca2+]i induced by restoration of extracellular Ca2+ after SR Ca2+ depletion was inhibited 49% by 10 µM Ni2+ and 69% by 7 µM SKF-96365. These inhibitory potencies were similar to those observed for CCE in mouse anococcygeus (45) but different from CCE in rat main and rabbit distal pulmonary arteries, where 50–100 µM La3+ caused >70% inhibition of contraction (16, 22). They were also different from rat distal pulmonary arteries and PASMCs, where 1 µM La3+ abolished the increase in [Ca2+]i after extracellular Ca2+ restoration, quenching of fura-2 fluorescence by Mn2+, and induction of nonspecific cation currents by TG (28, 32). Similarly, the apparent IC50 for Ni2+ has ranged widely from 10 to ~300 µM in vascular smooth muscle (11, 22, 25, 38). This variability could be due to differences in SOCC structure or function among different cell preparations, species, or experimental conditions. Conversely, the apparent IC50 for SKF-96365 has been quite consistent, with values close to 10 µM obtained in a variety of smooth muscles (8, 9, 11, 25, 46). In agreement with these findings, we estimated that the increase in [Ca2+]i induced by restoration of extracellular Ca2+ after SR Ca2+ depletion was inhibited 50% by 6.3 µM SKF-96365 or 191 µM Ni2+ (Figs. 5 and 6). Furthermore, we found that SKF-96365 and Ni2+ could block the increase in Mn2+ influx elicited by depletion of SR Ca2+ stores (Fig. 7). These results indicate that SKF-96365 and Ni2+ were effective antagonists of CCE in rat distal PASMCs.

In contrast, La3+ concentrations as high as 1,000 µM inhibited the response to Ca2+ restoration by only 27% in PASMCs perfused with KRB solution (Figs. 5C and 6). This ineffectiveness could reflect either the inherent characteristics of SOCCs in distal PASMCs or decreased bioavailabilty of La3+ due to precipitation or chelation in the cell perfusate, which contained bicarbonate, phosphate, and EGTA (28). To assess these possibilities, we repeated the La3+ experiments in PASMCs perfused with HEPES-buffered salt solution that did not contain bicarbonate, phosphate, or EGTA. As shown in Figs. 5D, 6, and 7, C and D, La3+ was an effective antagonist of CCE under these conditions (IC50 = 40 µM), suggesting that bioavailability was diminished in PASMCs perfused with KRB solution. Decreased bioavailability may have contributed to the relative inability of La3+ to inhibit contractile responses to CPA in rat main pulmonary arteries that were exposed to phosphate (25).

Despite the improved efficacy provided by HEPES-buffered salt solution, our estimated IC50 for La3+ (40 µM) was still substantially higher than the 0.1 µM value estimated by Robertson and colleagues (28, 32). The reasons for this discrepancy are unclear. One possibility is the use of freshly isolated vs. cultured distal PASMCs. Exposure to growth media can enhance TRPC expression in PASMCs (11), which might alter La3+ sensitivity. Another possibility is the assessment of CCE by different methods. Comparing effects of La3+ on contractile vs. [Ca2+]i responses to restoration of Ca2+ after CPA or TG could be complicated by alinearities and modulation of the [Ca2+]i-tension relationship. Similarly, comparing changes in [Ca2+]i after CPA and Ca2+ restoration to changes in cation currents after TG might be difficult, because these currents were carried largely by cations other than Ca2+ (25, 32). Furthermore, it has been suggested that CPA and TG deplete different Ca2+ stores in vascular smooth muscle (14, 15, 40). Although Mn2+ quenching was measured in both studies, methodological differences such as temperature (25 vs. 37°C) and perfusate composition (no VDCC antagonist vs. nifedipine; extracellular [Ca2+] = 1.8 vs. 0 mM) could again complicate comparisons. Further studies are needed to resolve these differences.

Depending on concentration and tissue, CCE antagonists may have actions other than inhibition of SOCCs. For example, SKF-96365 has been shown to prevent influx of Ca2+ through VDCCs and ROCCs, promote influx of Ca2+ through nonspecific cation channels or other pathways, and inhibit SERCA pumps (20); however, it is unlikely that these actions played a role in our experiments, because the cells were not exposed to vasomotor agonists, Ca2+ entry was reduced rather than promoted by CCE antagonists, and SERCA pumps and VDCCs were already blocked by CPA and nifedipine. Nevertheless, given the importance of VDCCs as a Ca2+ entry pathway in vascular smooth muscle, we felt it was important to determine whether CCE antagonists inhibited VDCCs in our preparation. As shown in Fig. 2, 50 µM SKF-96365, 500 µM NiCl2, or 100 µM LaCl3 did not alter the increase in [Ca2+]i elicited by depolarization with 60 mM KCl, whereas 5 µM nifedipine inhibited this response completely. These results indicate that SKF-96365, NiCl2, and LaCl3 did not block VDCCs in distal PASMCs and increase the likelihood that their actions were due to inhibition of CCE through SOCCs.

Evidence is accumulating that CCE channels are composed of mammalian homologs of TRP and TPRL proteins, which form plasmalemmal cation channels in photoreceptor cells of the Drosophila eye (24, 27). To date, seven homologs of TRP and TRPL, termed TRPC1–7, have been cloned and sequenced in mammals, including human, mouse, rat, rabbit, and cow (23). Heterologous expression of TRPC genes in Xenopus oocytes and mammalian cells caused the appearance of store-activated Ca2+ currents (22, 51) or enhanced the increase in [Ca2+]i due to CCE (5, 41). In addition, treatment with specific TRPC antisense oligonucleotides depressed expression of both TRPC proteins and CCE (3, 23). These observations indicate that SOCCs are made up of subunits encoded in TRPC genes. Although the exact structural composition of these channels remains unknown, an assembly of four heteromeric TRPC subunits seems likely and could explain the observed diversity with respect to conductance, cation selectivity, and susceptibility to pharmacological inhibition (2, 3).

It is likely that TRPC channels are widely expressed and play important roles in the vascular system (29, 34). Binding of specific antibody to an extracellular component of TRPC1 blocked CCE in systemic vascular myocytes, suggesting that TRPC1 is a CCE channel subunit in these cells (48). In TRPC4-deficient knockout mice, endothelium-dependent relaxation was inhibited, and aortic endothelial cells did not express IP3-induced inward cation currents and exhibited smaller increases in [Ca2+]i after stimulation with ACh (10). In portal vein myocytes, treatment with TRPC6 antisense oligonucleotides inhibited TRPC6 protein expression and currents through nonselective cation channels activated by {alpha}1-adrenoceptor stimulation (13). In rat cerebellar and cerebral resistance arteries, TRPC6 antisense oligonucleotides suppressed both TRPC6 protein expression and inward cation currents, as well as depolarization and constriction induced by increased transmural pressure (47). In proliferating human pulmonary arterial smooth muscle studied at passages 4–6, increases in CCE and expression of TRPC1 mRNA (as well as the rate of proliferation) and protein were reduced by treatment with a TRPC1 antisense oligonucleotide (11, 36). Similarly, treatment of PASMCs from rat main pulmonary arteries with a TRPC6 antisense oligonucleotide decreased TRPC6 expression, CCE, and proliferation mediated by platelet-derived growth factor (50). These observations suggested that TRPC1 and -6 are components of CCE channels that contributed to regulation of growth in PASMCs.

Using RT-PCR and Western blotting, we detected mRNA and protein for three of the seven known mammalian TRPC subtypes in distal PASMCs. As shown in Figs. 8 and 9, TRPC1, -4, and -6 were expressed in both de-endothelialized distal pulmonary arteries and PASMCs from these vessels. This pattern of expression differs qualitatively from that observed in proximal pulmonary arteries of the rat. McDaniel et al. (22) showed that TRPC1, -2, -4, -5, and -6 were the predominant mRNAs expressed in cultured myocytes from rat main pulmonary arteries. Ng and Gurney (25) detected mRNAs for TRPC1, -3, -4, -5, and -6 in freshly isolated myocytes from rat main pulmonary artery and used immunocytochemistry to confirm protein expression for TRPC1, -3, -4, and -6, but not -5. In contrast, we did not detect TRPC2, -3, -5, or -7 in rat distal PASMCs. This qualitative difference cannot be explained by inadequate sensitivity or specificity of our methods, because we were able to detect all TRPCs in rat brain (Figs. 8 and 9) and to block binding of antibodies to TRPC proteins in Western blots by pretreatment of antibodies with their respective antigens (Fig. 9). Nor is the difference likely to be the consequence of primary cell culture, since the TRPC proteins detected in freshly isolated rat distal pulmonary arterial tissue were the same as those detected in myocytes cultured from these arteries (Figs. 8 and 9). Thus expression of TRPC1, -4, and -6 (but not TRPC2, -3, -5, or -7) may be characteristic of rat distal PASMCs. The functional significance of this qualitative difference in TRPC expression between distal and proximal PASMCs remains to be determined.

In summary, we have shown that 1) CCE was present in myocytes from rat distal pulmonary arteries, 2) CCE in these cells was blocked by putative SOCC antagonists at concentrations that did not block Ca2+ influx through L-type VDCCs, and 3) mRNAs and proteins for TRPC1, -4, and -6 (but not TRPC2, -3, -5, or -7) were expressed in both distal PASMCs and distal pulmonary arterial tissue. Because distal pulmonary arteries are thought to be the major site of vasomotor responses in the pulmonary vasculature (1, 19, 31), these results suggest that CCE through sarcolemmal SOCCs composed of TRPC proteins could play an important role in pulmonary vasoreactivity.


    ACKNOWLEDGMENTS
 
We are grateful to Letitia Weigand and Kristina Zavage for excellent technical assistance.

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grants HL-51912 (J. T. Sylvester) and HL-67919 (L. A. Shimoda) and an American Lung Association of Maryland research grant (J. Wang).


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. T. Sylvester, Div. of Pulmonary & Critical Care Medicine, The Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Cir., Baltimore, MD 21224 (E-mail: jsylv{at}jhmi.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.


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